From U to mnm⁵Se²U: tuning base pairing preferences through 2-chalcogen and 5-methylaminomethyl modifications

From U to mnm⁵Se²U: tuning base pairing preferences through 2-chalcogen and 5-methylaminomethyl modifications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article From U to mnm⁵Se²U: tuning base pairing preferences through 2-chalcogen and 5-methylaminomethyl modifications Paulina Kuwerska, Katarzyna Kulik, Karolina Podskoczyj, Agnieszka Dziergowska, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7980593/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 16 You are reading this latest preprint version Abstract 5-Substituted uridines, 2-thiouridines and 2-selenouridines represent the most common wobble-positioned bacterial tRNA modifications, with the 5-methylaminomethyl (mnm5) substituent being particularly widespread. Their biological role in the precise recognition of synonymous purine-ending codons is still under investigation. Modified uridines are also known to enhance the stability and base-pairing specificity of therapeutic nucleic acids. However, a full understanding of the O2/S2/Se2 chalcogen effect, particularly in combination with the naturally occurring mnm⁵ substituent, remains limited. To address this, a systematic comparative study was conducted on the thermodynamic and structural contributions of mnm⁵ and 2-chalcogen modifications to RNA duplex properties. We found that chalcogens modulate the stability of duplexes containing opposing adenosine in the following order: uridines < Se2-uridines < S2-uridines, with the mnm⁵ substituent exerting a significantly destabilizing effect. In duplexes with opposing guanosine, the influence of chalcogens is less pronounced, whether alone or in combination with mnm5, however, Se2-uridines promote duplex formation more effectively than their 2-thio and 2-oxo counterparts. This effect is likely associated with their high ionization propensity, as we demonstrated by pH-dependent melting studies. Overall, the base-pairing specificity for adenosine over guanosine was found to follow the order: uridines < Se2-uridines < S2-uridines, with the mnm⁵ group significantly reducing this specificity. All studied RNA duplexes exhibited circular dichroism (CD) spectra characteristic of A-RNA double stranded helices. To afford the above data, the first chemical synthesis of an mnm⁵Se²U-modified RNA oligomer was developed. Biological sciences/Biochemistry Biological sciences/Chemical biology Physical sciences/Chemistry 5-substituted uridines 2-thiouridines 2-selenouridines modified oligoribonucleotides phosphoramidite solid-phase synthesis thermal stability circular dichroism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cellular RNAs contain over 170 structurally distinct modified nucleosides that serve diverse biological functions 1 . Most of them have been identified in transfer RNAs (tRNAs), with two major hotspots: the wobble position (position 34), corresponding to the first anticodon letter, and position 37, adjacent to the anticodon from the 3' end. Wobble modifications are known to fine-tune the decoding process by stabilizing codon–anticodon interactions, thereby enhancing the efficiency and accuracy of translation 2,3 . The representative group of the wobble-positioned tRNA modifications are 5-substituted uridines, 2-thiouridines and 2-selenouridines, with the 5-aminomethyl (xnm5) substituent being particularly widespread 1,4 . Among them, 5-methylaminomethyl (mnm, 1 - 3 , Fig. 1) and 5-carboxymethylaminomethyl (cmnm, 4 - 6 ) modifying groups adorn all three types of O 2 -, S 2 - and Se 2 -uridines. The (c)mnm 5 S 2 U and (c)mnm 5 Se 2 U nucleosides have been identified in bacterial tRNAs specific for lysine, glutamate, and glutamine, where they share the wobble position with S -geranyl-2-thiouridines (mnm 5 geS 2 U or cmnm 5 geS 2 U) 5-8 . All 5-aminomethyl-containing uridines, 2-thiouridines and 2-selenouridines recognize not only adenosine but also guanosine as the third codon letter (A- and G-3') 5,9,10 . This dual mode of base pairing with purines has been widely discussed in the literature, particularly in the context of the geometry of the non-Watson-Crick xnm⁵U-G and xnm 5 S(Se) 2 U-G base pairs 10-16 . Beyond their established functions in cellular tRNAs, uridine derivatives have also been applied in the design of therapeutic nucleic acids 17-19 . Among others, the 2-thiouridine (S²U) and 2-selenouridine (Se 2 U) derivatives have been utilized to improve the stability and base pairing specificity of antisense oligonucleotides and small interfering RNAs (siRNAs) with their complementary RNA targets 20-22 . As demonstrated by thermal denaturation experiments, replacing U with S²U or Se²U increases the stability of S(Se)²U-A duplexes and decreases the stability of S(Se)²U-G duplexes, thereby supporting the strong preferential pairing of S(Se)²U with adenosine over guanosine 14,20-24 . The enhanced thermodynamic stability of duplexes containing S(Se)²U-A vs. U-A base pairs arises from three key features of S 2 - and Se 2 -uridines (Table 1) 20,23-25 : (1) their preference for the C3′- endo sugar conformation (20-30% higher than that of U) 11,12 , which improves base stacking interactions with the adjacent nucleobase and supports the rigid A-form RNA duplex; (2) increased N3-H acidity (by 1-2 pKa units relative to U) 11,12 , which promotes stronger hydrogen bonding with the N1-acceptor site of adenosine (Fig. 2a vs. 2b) and (3) poor H-bonding ability of S 2 /Se 2 causing a reduced desolvation during duplex formation. On the other hand, the destabilizing effect of the U→S(Se) 2 U substitution in duplexes with opposite G is attributed to the larger atomic radius and lower electronegativity of sulfur/selenium compared to oxygen which weaken hydrogen bonding (Fig. 2c vs. 2d) and make the interaction with G less favourable. Although the (de)stabilizing effects of U→S²U and U→Se²U substitutions in individual RNA duplexes are well established 14,20-24 , the contribution of S²U vs. Se²U remains unknown due to the lack of studies on the same model RNAs. Based on the steric and electronic properties of the U, S 2 U, and Se 2 U nucleosides described above, the preferential A pairing effect is expected to follow the order: U < S²U < Se²U, however, this trend has not been experimentally confirmed at the oligonucleotide level. Table 1. The physicochemical and structural properties of uridines (U, mnm 5 U), 2-thiouridines (S 2 U, mnm 5 S 2 U) and 2-selenouridines (Se 2 U, mnm 5 Se 2 U) 11, 12 . Properties of uridines U S 2 U Se 2 U mnm 5 U mn m 5 S 2 U mnm 5 Se 2 U p K a for N3- H 9.15 8.09 7.30 8.15 7.27 6.43 pKa for CH 3 NH 2 + CH 2 - - - - 10.02 9.51 9.36 Content of the ionized fraction of nucleoside under physiological conditions [%] a 2 17 58 (34) b 15 57 (34) b >90 (78) b C 3’-endo sugar puckering [%] 53 71 80 57 76 72 a Calculated according to the Henderson–Hasselbalch equation pKa-pH = log [BH]/[B - ], based on the pKa values determined for N3-H (BH and B - are the neutral and ionized forms, respectively). b Recalculated for nucleotides, since the pKa values for N3-H in pyrimidine nucleotides are 0.4 unit higher than for nucleosides. From nucleoside studies, substitution of uridine, 2-thiouridine and 2-selenouridine with a 5-methylaminomethyl (mnm 5 ) group introduces changes in the uracil electron density and sugar puckering which should affect duplex stability and base-pairing specificity¹¹ , ¹². The mnm⁵-induced changes in the electronic properties of uracil arise from protonation of the mnm⁵ group under physiological conditions (the pKa of CH₃NH₂⁺CH₂ ranges from 9.3 to 10.0, Table 1)¹¹ , ¹² making it a strong electron-withdrawing substituent that promotes deprotonation of the N3-H group. The mnm⁵-modified uridines are ~1 pKa unit more acidic than their 5- un substituted analogs (Table 1), whereas the combined presence of mnm⁵ and S²/Se² substituents yields unusually low pKa values: 7.27 for mnm⁵S²U and 6.43 for mnm⁵Se²U¹¹ , ¹². As a result, mnm 5 S(Se) 2 U partially exists in the zwitterionic state with the positive charge on the aminoalkyl side chain and the negative charge dispersed on the S(Se)2-C2-N3-C4-O4 edge of nucleobase, that was confirmed in theoretical, structural and physicochemical studies 10-12,15 . The content of the ionized fraction reaches 57% for mnm 5 S²U and an exceptionally high, >90% for mnm 5 Se²U (Table 1). 11,12 Using thermal denaturation experiments, we recently demonstrated that increased ionization of C5-substituted 2-thiouridines, including mnm 5 S 2 U, correlates with reduced base pairing specificity for A over G 26 . In addition, we confirmed the previously proposed hypothesis that the neutral form of mnm⁵S²U preferentially pairs with adenosine (Fig. 2e), whereas its zwitterionic form favours pairing with guanosine (Fig. 2h) 11,15,26 . Of note, the formation of zwitterionic mnm 5 S 2 U-G base pair requires a slight shift of mnm 5 S 2 U toward the minor groove. Other base-pairing systems, such as zwitterionic mnm⁵S²U interacting with A (Fig. 2f) or neutral mnm⁵S²U pairing with G (Fig. 2g) appear to be energetically unfavourable. Regarding mnm⁵Se²U base pairing with A/G, experimental data on its thermodynamic and structural contributions to RNA duplex properties remain unavailable, primary due to its challenging synthetic accessibility. As was shown by density functional theory (DFT) calculations at the nucleobase level, the zwitterionic form of mnm⁵Se²-uracil binds more effectively to guanine than the corresponding 2-oxo and 2-thio analogs, due to a stronger H-bond between the N3 of mnm 5 Se 2 -uracil and the 2-amino group of guanine, suggesting a U-G base pairing mode analogous to that previously observed for zwitterionic mnm 5 S 2 U (Fig. 2h) 12 . Interestingly, mnm 5 Se 2 U exhibits slightly reduced C3′- endo sugar puckering (and thus potentially weaker stacking interactions) relative to mnm 5 S 2 U (Table 1) 11,12 , which contradicts data obtained for 5- un substituted Se 2 U and S 2 U as well as established knowledge that a larger C2 substituent favours the C3′- endo conformation. Driven by the distinct structural and electronic properties of uridines bearing 2-chalcogen and mnm⁵ functions, and by the incomplete data on the hybridization behaviour of selenium-containing RNAs, we undertook a systematic investigation on the individual and synergistic contributions of these modifications to RNA duplex stability. All oligonucleotides of 5’-GUUGACU(mnm 5 )U*UUAAUCAAC-3’ sequence (U* = U, S 2 U, Se 2 U or mnm 5 U* = mnm 5 U, mnm 5 S 2 U, mnm 5 Se 2 U) were synthesized in house according to the previously developed procedures 6,26-28 , except for mnm 5 Se 2 U-RNA, whose original synthesis is presented in this work. Twelve duplexes containing either adenosine or guanosine opposite the (mnm 5 )U* nucleoside were analyzed using circular dichroism (CD) and UV melting experiments to evaluate their structural and thermodynamic properties. The structural contribution of the modified uridines appears to be minimal, as the CD spectra exhibited a characteristic A-form RNA duplex profile. Thermodynamic analyses indicate that S 2 /Se 2 -chalcogens and mnm⁵ substituent exert a pronounced influence on the stability of duplexes with opposing adenosine, but have minimal impact on those with opposing guanosine. Comparing these data, a base pairing specificity for adenosine over guanosine was established as follows: uridines < Se 2 -uridines < S 2 -uridines, with mnm 5 group significantly reducing this specificity. Overall, S 2 -uridines (contrary to expectations for Se 2 -uridines) exhibited the strongest preference for pairing with adenosine. Material and methods Synthesis and characterization of mnm 5 Se 2 U-RNA oligomer. Synthesis of mnm 5 Se 2 U-RNA oligomer was performed at 2.5-mmol scale by phosphoramidite chemistry using a synthesizer (K&A H-8 DNA/RNA/LNA Synthesizer). We employed the commercially available rC(TAC)-succinyl-CPG (Proligo) support and phosphoramidites of A, C, U and G protected with 5’- O -DMTr-2’- O -TBDMS-NH-TAC, prepared as a 0.1 M solutions in acetonitrile. Incorporation of the canonical monomeric units was performed in an 8 molar excess with a coupling time of 8 min. The mnm 5 Se 2 U phosphoramidite (synthetic protocol and full spectral characteristic are given in ESI, Fig. S1-S11) was coupled twice, each time using an 8-fold molar excess and 10 min coupling time. Condensation steps were carried out in the presence of 0.25 M solution of 5-(3,5-bis(trifluoromethyl)phenyl)-1 H -tetrazole in acetonitrile (Activator 42 ® ). The mixture of Cap A (THF/TAC 2 O, 100:5 v/w), and Cap B (THF/ N -methylimidazole, 84:16 v/v) was used in the capping step for 2 min. In the oxidation step, a solution of I 2 (0.02 M in THF/H 2 O/pyridine, 90.54/9.05/0.41 v/v/v) was applied for 2 min. After synthesis, the support-linked DMTr-off oligomer was cleaved from the beads and deprotected on 0.2 μmol scale. The b -cyanoethyl groups were selectively removed from the phosphate residues using Et 3 N-acetonitrile (272 μL, 1/1 v/v, 30 min, rt.). The supernatant was removed. The resin was washed with acetonitrile (3 × 200 μL) and dried in vacuo for 30 min. Subsequently, the resin was treated with 0.05 M solution of K 2 CO 3 in anhydrous methanol (200 μL) for 10 h at rt. The supernatant was transferred to an Eppendorf tube. The resin was washed with anhydrous MeOH (3 x 200 μL). The combined oligonucleotide-containing fractions were neutralized by 99% AcOH and evaporated to dryness on a Speed-Vac. Oligomer was dissolved in 300 μL anhydrous EtOH and dried using Speed-Vac for 3 h. Then, oligomer was treated with neat Et 3 N·3HF (48 μL, 24 h, rt.) and desalted on a C-18 cartridge (Sep-Pak ® , Waters). After solvent evaporation, the oligomer was briefly incubated with conc. NH 4 OH (100 μL, 30 min, rt), followed by removal of ammonia under reduced pressure using Speed-Vac. The fully deprotected mnm 5 Se 2 U-RNA 17 was purified by anion-exchange high-performance liquid chromatography (IE-HPLC, Fig. S12) and characterized by electrospray ionization mass spectrometry (ESI MS, Fig. S13) and enzymatic digestion analysis (Fig. S14). From a 0.2 µmol-scale synthesis, we obtained 5 OD 260 of the final product 17 . UV melting experiments. UV absorbance measurements and thermal denaturation experiments were performed in a cell with 1 cm path length on a Jasco V-770 UV-VIS/NIR spectrophotometer equipped with a Peltier thermal cell. Solutions of complementary RNA/RNA oligonucleotide strands were prepared in a phosphate buffer (10 mM sodium phosphate pH 7.4, pH 7.0 or pH 6.4 with 100 mM NaCl) at the final concentration of 2 µM. Samples were then heated to 85 °C and cooled to 15 °C with a temperature gradient of 1.5 °C/min. Melting profiles were recorded from 15 to 85 °C, with a temperature gradient of 0.5 °C/min (Fig. S15-S18, ESI). Thermodynamic parameters (Tm, DG 0 , DH 0 , DS 0 ) were calculated by numerically fitting a given melting curve using a two-state model algorithm by MeltWinv.3.5 software (MeltWin software license was kindly provided by Jeffrey McDowell, www.meltwin.com). Each result was taken as an averaged one from three independent experiments. The resulting UV melting temperatures (T m ) and thermodynamic parameters (DH 0 , DS 0 , DG 0 ) of the U*- and mnm 5 U*-modified duplexes are given in Table 2. CD spectra. Circular dichroism measurements were performed using a J-715 circular dichroism spectrometer (CD) (Jasco, Japan). Duplex samples were prepared at the concentration of 2 µM in a 10 mM sodium phosphate buffer, pH 7.4 containing 100 mM NaCl. The oligonucleotides were mixed in a buffer, heated to 85 °C and slowly cooled to room temperature. CD spectra were recorded using a quartz cell with 0.5 cm slice thickness. The acquisition parameters were as follow: scanning speed 50 nm/min, response time 2 s, band width 1.0 nm and step resolution 0.2 nm. The spectra were recorded at 24 °C in the wavelength range from 200 to 360 nm. The spectrum recorded for the buffer was numerically subtracted from the spectrum for each sample (recorded in triplicate) and the resulting averaged spectra were smoothed using an averaging algorithm (convolution width 25). CD spectra for U*- and mnm 5 U*-modified RNA duplexes with opposite A and G units are given in Fig. 5. Results and discussion Synthesis of mnm 5 Se 2 U-phosphoramidite. The C2-selenocarbonyl group was introduced via selenation of appropriately protected 2- S -methylated 2-thiouridine 8 (Fig. 3a) with NaSeH, following the method originally developed by Klayman and Griffin 29 and adopted by others for Se 2 U phosphoramidite synthesis 14,30 , yielding 82%. Recently, the same protocol was successfully applied to obtain a series of naturally occurring 5-aminoalkyl-2-selenouridines 6,12 . The 2- S -methylated 2-thiouridine 8 was obtained by direct methylation of 2-thiouridine 7 , which was protected with a 4,4'-dimethoxytrityl group (DMTr) at the 5'-hydroxyl and a trifluoroacetyl group (TFA) on the amine function of the mnm 5 side chain. The TFA group was previously successfully employed in the solid-phase synthesis of several RNA oligomers modified with 5-aminomethyluridine derivatives 31-34 . After selenation, the 2'-OH group of 9 was protected with tert -butyldimethylsilyl (TBDMS) via a standard procedure 35 . The obtained regiomers 10 and 11 (1:1 ratio) were purified without separation (Y=62%) and used as a mixture to introduce the b -cyanoethyl protecting group on 2-Se with iodopropionitrile (note that the unprotected Se 2 tends to be oxidized after treatment with an oxidizing agent during RNA synthesis 14 ). The Se-protected isomers 12 and 13 were separated in 48% and 32% yields, respectively, and ubiquitously identified by 1 H- 1 H COSY NMR. The 2'- O -TBDMS isomer 12 was phosphitylated to give the Se-phosphoramidite 14 in 72% yield. The synthetic procedures and spectral characterization of 9 - 14 are shown in ESI (Fig. S1-S11). Chemical synthesis of mnm 5 Se 2 U-oligoribonucleotide. The chemical incorporation of 2-seleno-uridines into RNA remains a significant synthetic challenge. In the past, Huang and co-workers performed a successful chemical synthesis of Se 2 U-RNA oligomers employing b -cyanoethyl protection for Se 2 function 14 . Using 2-selenouridine synthase (SelU), Sierant et al. transformed S 2 U-RNA via its geranylated derivative to Se 2 U-RNA 6 , while the Stadtman group obtained mnm 5 Se 2 U-modified E. coli tRNA Lys through enzymatic reaction of bulk tRNAs 36 . In our work, fully protected amidite 14 (Fig. 3) was incorporated into the 5'- GUUGACU mnm 5 Se 2 U UUAAUCAAC-3’ RNA chain related to the anticodon arm domain of E. coli tRNA Lys . The synthesis was performed via phosphoramidite chemistry on the CPG-C(TAC) support. After synthesis, the support-linked DMTr-off oligomer was cleaved from the beads and deprotected (Fig. 3b). The b -cyanoethyl groups were selectively removed from the phosphate residues using triethylamine in acetonitrile. The resin deprived of b -acrylonitrile was dried and treated with anhydrous 0.05 M K 2 CO 3 methanol solution (10 h, rt.) according to the protocol of Huang, who found these conditions safe for Se-modification during Se 2 U-RNA and Se 2 T-DNA preparation 14,37 . In our studies, methanolic K 2 CO 3 proved effective for CPG cleavage and removal of the Se- b -cyanoethyl and exoamine TAC blockage, however the TFA group remained intact. The 2'-TBDMS groups were removed with neat TEA x 3HF (24 h, rt.). After desalting and evaporation, Se-oligomer was briefly treated with conc. NH 4 OH (30 min, rt.) to remove TFA. The reduced time of TFA-ammonolysis was important to avoid the RNA degradation and/or deselenation. After deprotection, mnm 5 Se 2 U-RNA 17 was purified by IE-HPLC, (Fig. S12) and characterized by ESI MS (Fig. S13) and enzymatic digestion analysis (Fig. S14). To simplify the deprotection protocol and enable simultaneous removal of all base-labile protecting groups in a single step, we tested the use of conc. NH 4 OH-EtOH (3:1 v/v, 16 h, 36 °C) and conc. NH 4 OH (3 h, rt.). However, both conditions led to the formation of an undesirable deselenation product 38 . The oligomers modified with S 2 U, Se 2 U, mnm 5 U, and mnm 5 S 2 U were synthesized in house according to the protocols described in the literature 6,26-28 . Thermodynamic and structural analysis of RNA duplexes. Two sets of RNA duplexes 5'- GUUGACU (mnm 5 )U* UUAAUCAAC-3’/3’-CAACUGA A AAUUAGUUG-5’ (C1-C6; C denotes duplexes containing a canonical U-A base pair) and 5'- GUUGACU (mnm 5 )U* UUAAUCAAC-3’/3’-CAACUGA G AAUUAGUUG-5’ (M1-M6, M denotes duplexes containing a mismatched U-G base pair) were selected for our studies. The U* and mnm 5 U* abbreviations represent U/S 2 U/Se 2 U and mnm 5 U/mnm 5 S 2 U/ mnm 5 Se 2 U, respectively, whereas two uridines e.g. U and mnm 5 U are denoted as (mnm 5 )U. The resulting UV melting temperatures (T m ) and thermodynamic parameters (Gibbs free energy DG 0 , enthalpy DH 0 , entropy DS 0 ) for (mnm 5 )U*-modified duplexes with opposing A and G are summarized in Table 2 with corresponding melting profiles shown in Figs. S15-S18 (ESI). Table 2. UV melting temperature and thermodynamic parameters for 5’-GUUGACU (mnm 5 )U* UUAAUCAA C-3’/3’-CAACUGA A AAUUAGUUG-5’ and 5’-GUUGACU (mnm 5 )U* UUAAUCAAC-3’/3’-CAACUGA G AAUUAGU UG-5’ duplexes, U* =U/S 2 U/Se 2 U and mnm 5 U* =mnm 5 U/mnm 5 S 2 U/mnm 5 Se 2 U. Errors for thermodynamic quantities were assessed based on multiple UV melting experiments. No. Name -ΔG 0 (kcal/mol) -ΔH 0 (kcal/mol) -ΔS 0 (eu) Tm (°C) C1. U-A 14.2 ± 0.1 118.5 ± 2.7 336.5 ± 8.5 52.8 ± 0.3 C2. mnm 5 U-A 12.6 ± 0.1 92.8 ± 0.9 258.6 ± 2.6 51.6 ± 0.4 C3. S 2 U-A 16.1 ± 0.4 128.1 ± 3.7 361.2 ± 10.6 56.9 ± 0.4 C4. mnm 5 S 2 U-A 14.6 ± 0.2 112.3 ± 4.3 315.2 ± 13.3 55.0 ± 0.3 C5. Se 2 U-A 14.8 ± 0.5 108.6 ± 4.7 302.6 ± 13.5 56.6 ± 0.6 C6. mnm 5 Se 2 U-A 13.8 ± 0.2 110.4 ± 2.6 311.3 ± 7.7 53.0 ± 0.2 M1. U-G 12.7 ± 0.1 107.6 ± 2.6 306.1 ± 8.0 49.9 ± 0.3 M2. 8. mnm 5 U-G 11.4 ± 0.1 82.4 ± 4.1 229.0 ± 12.7 48.7 ± 0.2 M3. S 2 U-G 12.2 ± 0.3 99.4 ± 2.5 281.3 ± 7.7 49.3 ± 0.3 M4. mnm 5 S 2 U-G 11.9 ± 0.1 90.7 ± 2.1 254.2 ± 6.4 49.2 ± 0.3 M5. Se 2 U-G 12.8 ± 0.2 104.5 ± 3.8 298.6 ± 16.0 50.5 ± 0.1 M6. mnm 5 Se 2 U-G 12.1 ± 0.2 92.1 ± 2.8 258.0 ± 8.6 49.8 ± 0.4 It is generally observed that all canonical (mnm 5 )U*-A base pairs stabilize RNA duplexes more efficiently than the corresponding (mnm 5 )U*-G mismatched pairs. The magnitude of this stabilizing effect is reflected by the ΔΔG⁰ values (Fig. 4), calculated as the differences between the DG° values determined for (mnm 5 )U*-A duplexes and corresponding (mnm 5 )U*-G duplexes and indicate the preferential pairing of modified uridines with adenosine over guanosine (higher ΔΔG⁰ value reflects a stronger preference for binding with A over G). Analysis of these data shows the same enhanced trend for both 5- un substituted and mnm 5 -substituted uridines arranged as follows (mnm 5 )U < (mnm 5 )Se 2 U < (mnm 5 )S 2 U, indicating the highest preference of 2-thiouridines to bind to A. Comparison of the ΔΔG° values between U/S 2 U/Se 2 U and corresponding mnm 5 -uridines (Fig. 4) reveals that the mnm 5 group markedly reduces base-pairing specificity for A, regardless the type of chalcogen at the C2 position. In other words, the mnm 5 substituent promotes base pairing with G. These findings are consistent with in vivo translation studies demonstrating that the mnm⁵ group is primarily responsible for the enhanced ability of wobble mnm⁵S²U in E. coli tRNA Glu to recognize 3′-G-ending codons, whereas the 2-thio group plays a key role in recognizing 3′-A-ending codons and ensuring efficient tRNA aminoacylation 39 . The global structures of U*- and mnm 5 U*-modified RNA duplexes were examined by CD spectroscopy. As shown in Fig. 5, all duplexes exhibit CD spectra characteristic of the A-type conformation, with a positive band at λ max of 265 nm and a negative band at λ max of 210 nm. Minor differences were observed, such as an increase in the intensity of the positive band at λ max of 265 nm of the S(Se) 2 U-A compared with the S(Se) 2 U-G duplexes, most likely due to their improved π-π base stacking (Figs. 5a, 5b). Similar trends were also observed for duplexes containing mnm 5 -uridines (Figs. 5c, 5d). The individual and synergistic effects of 2-chalcogen and mnm⁵ functions on conformation and thermal stability and of RNA duplexes are discussed in the following sections. Characterization of duplexes containing U*-A and U*-G base pairs (U*=U/S 2 U/Se 2 U). According to our data, the stabilizing effect of 5- un substituted uridines (U*) on duplexes with opposing A increases in the order: U < Se 2 U < S 2 U. While the enhanced stabilizing effect of the O 2 →S 2 substitution (~2 kcal/mol, as indicated by the ΔG 0 difference between C1 and C3) is expected and is well-documented in the literature 20-22 , the O 2 →Se 2 substitution results in only minor changes in duplex stability (C1 vs. C5). In addition, a 1.3 kcal/mol destabilization of the Se²U-A duplex relative to S²U-A duplex (C3 vs. C5) was observed, which is entirely unexpected considering the electronic and structural properties of the Se²U nucleoside - its stronger preference for the C3′- endo sugar conformation, higher N3-H acidity, and enhanced desolvation (Table 1) – all of which would be anticipated to favour Se²U-A duplex formation. Thermodynamic analyses further revealed unfavourable enthalpic and entropic contributions of Se 2 U compared to S 2 U, suggesting weakened Se²U-A base-pairing and reduced duplex rigidity. CD spectra of U*-A duplexes support this interpretation (Fig. 5a), showing diminished stacking interaction for Se 2 U-containing duplex compared to S 2 U counterpart, as evidenced by the lower intensity of the positive band at l max of 265 nm. We propose that the destabilizing effect of the S² → Se² replacement arises from the higher content of the ionized form of Se²U (58%) compared to S²U (17%) (Table 1), resulting from the difference in their acidity. Unlike the neutral form of Se 2 U, which favours the formation of a Watson-Crick Se²U-A base pair by two H-bonds (Fig. 6a), the ionized species (Fig. 6b) likely perturbs the hydrogen-bonding pattern, reducing the ability of Se²U to pair effectively with adenosine and potentially limiting the interaction to a single hydrogen bond. While the neutral diketo tautomer of Se²U facilitates the formation of Se 2 U-A base pair, its presence in Se 2 U-G duplexes should have a destabilizing effect (Fig. 6c). This behaviour can be attributed to the poor ability of selenium to form hydrogen bonds, resulting from its large atomic radius and low electronegativity. Due to these properties, Se 2 U is expected to reduce the base pairing with G more effectively than U and S 2 U. In our thermodynamic experiments, the differences in the stability of U*-G duplexes are insufficient to establish a definitive stability ranking, nevertheless, all thermodynamic parameters consistently indicate that Se 2 U facilitate base pairing with G more efficiently than S 2 U (M2 vs. M5). It is likely, that the same population of ionized Se²U that reduces A-binding efficiency facilitates G-binding through the formation of two hydrogen bonds (Fig. 6d), with Se²U adopting a geometry shifted toward the minor groove. The contribution of chalcogens to the stability of U*–A/G duplexes evaluated in this study is aligned with the findings of Habuchi et al. for LNA-type duplexes 22 ; but it is not fully consistent with the results reported by Sun et al. , who observed a significantly reduced stability of Se 2 U-G duplexes relative to their unmodified counterparts. This discrepancy may be attributed to the thermodenaturation conditions used in Sun’s study, which were performed at pH 6.8 (compared to pH 7.2 employed by Habuchi and pH 7.4 used in our experiments). At the lower pH, the content of the ionized form of Se²U would be reduced, given its N3-H pKₐ of 7.3, thereby affecting base pairing behaviour. To demonstrate this, we performed thermodenaturation experiments at pH 6.4, 7.0 and 7.4 (Table 3) revealing a pH-dependent modulation of Se²U base pairing. All three thermodynamic parameters indicate enhanced stabilization of Se²U–A duplexes at lower pH, where the N3-protonated form of Se²U is favoured, and increased stability of Se²U–G duplexes at higher pH, which promotes the formation of the ionized Se²U form. These findings indicate that even modest pH changes can shift the content of neutral–ionized form of Se-uracil, thereby altering its binding preference for A over G. Table 3. UV melting temperature and thermodynamic parameters for (5’-GUUGACU Se 2 U UUAAUCAAC-3’/3’-CAACUGA A AAUUAGUUG-5’) and (5’-GUUGACU Se 2 U UUAAUCAAC-3’/3’-CAACUGA G AAUUAGUUG-5’) duplexes determined at pH 7.4, 7.0 and 6.4. Errors for thermodynamic quantities were assessed based on multiple UV melting experiments. No. Name -ΔG 0 (kcal/mol) -ΔH 0 (kcal/mol) -ΔS 0 (eu) Tm (°C) C5. Se 2 U-A 14.8 ± 0.5 (pH 7.4) 108.6 ± 4.7 (pH 7.4) 302.6 ± 13.5 (pH 7.4) 56.6 ± 0.6 (pH 7.4) 14.8 ± 0.4 (pH 7.0) 109.2 ± 5.5 (pH 7.0) 304.2 ± 16.4 (pH 7.0) 56.6 ± 0.4 (pH 7.0) 15.4 ± 0.3 (pH 6.4) 121.1 ± 4.0 (pH 6.4) 340.9 ± 11.9 (pH 6.4) 56.0 ± 0.3 (pH 6.4) M5. Se 2 U-G 12.8 ± 0.2 (pH 7.4) 104.5 ± 3.8 (pH 7.4) 298.6 ± 16.0 (pH 7.4) 50.5 ± 0.1 (pH 7.4) 12.3 ± 0.3 (pH 7.0) 97.0 ± 4.1 (pH 7.4) 273.2 ± 12.3 (pH 7.0) 49.8 ± 0.4 (pH 7.0) 11.9 ± 0.2 (pH 6.4) 94.2 ± 3.5 (pH 6.4) 265.2 ± 10.8 (pH 6.4) 49.0 ± 0.2 (pH 6.4) Characterization of duplexes containing mnm 5 U*-A and mnm 5 U*-G base pairs (mnm 5 U*=mnm 5 U/mnm 5 S 2 U/mnm 5 Se 2 U). Thermodynamic analyses of mnm 5 U*-A duplexes revealed that the stabilizing effect of mnm 5 -substituted uridines follows the same trend as their 5- un substituted counterparts: mnm 5 U < mnm 5 Se 2 U < mnm 5 S 2 U. However, the presence of the mnm 5 substituent decreases duplex stability by approximately 1.0 - 1.6 kcal/mol, as indicated by differences between the corresponding duplexes pairs (C1 vs. C2, C3 vs. C4, and C5 vs. C6; Table 2). Previous thermodynamic 26 and structural studies 15 on mnm 5 S 2 U-containing RNAs demonstrated that this effect is attributed to the high content (57%) of ionized (zwitterionic) form of mnm 5 S 2 U at pH 7.4, which is approximately 3-fold greater than that observed for S 2 U. Zwitterionic structure of mnm 5 S 2 U weakens interactions within the mnm⁵S²U-A base pair (Fig. 2e vs. 2f), thereby reducing base-pairing efficiency. Similar increase in the content of zwitterionic form is observed for mnm 5 U (15% vs. 2% for U) and mnm 5 Se 2 U (>90% vs. 58 % for Se 2 U) nucleosides (Table 1) 11,12 what shows the general tendency of mnm 5 substituent to promote the zwitterionic structure and to destabilize the duplexes with opposing A. Comparison of the mnm 5 Se 2 U-A and mnm 5 S 2 U-A duplexes (C4 vs. C6) reveals a 0.8 kcal/mol decrease in stability upon selenium substitution, which is consistent with the increased acidity of Se-nucleosides that limits the formation of a canonical Watson-Crick base pair with adenosine (mnm⁵Se²U exhibits a 0.85 pKₐ-unit greater N3–H acidity than mnm⁵S²U, resulting in an increased zwitterionic fraction by ca 40%, Table 1). 11,12 As indicated by thermodynamic data, mnm⁵U exerts the most pronounced destabilizing effect on duplexes with opposing A (C2 vs. C6 and C2 vs. C4), although the ‘zwitterionic effect’ is unlikely to contribute, since mnm⁵U exists in only a minor ionized fraction (~15%) under physiological conditions. In this case, the destabilization results from markedly reduced stacking interactions (as supported by CD spectra, Fig. 5c), due to the lower C3′- endo sugar pucker preference of mnm⁵U nucleoside (57%) relative to >70% observed for mnm⁵S²U and mnm⁵Se²U 12 . Thermodynamic analysis of mnm⁵U*-G and U*-G duplexes indicates a modest destabilizing effect of the mnm⁵ substituent, ranging from 0.3 to 1.3 kcal/mol, as reflected by the ΔG 0 differences between the corresponding duplexes (M1 vs. M2; M3 vs. M4; M5 vs. M6). The contribution of O 2 /S 2 /Se 2 chalcogens to the thermal stability of mnm 5 U*-G duplexes - similarly to 5- un subtituted U*-G duplexesn- is difficult to evaluate due to the small ΔG 0 differences among M2, M4 and M6 (Table 2). However, analysis of the complete thermodynamic data suggests that both mnm⁵S²U and mnm⁵Se²U increase the binding capacity to guanosine, with a slightly stronger G-affinity observed for mnm 5 Se 2 U. This effect is likely driven by the higher population of the zwitterionic forms of mnm⁵S 2 U (57%) and mnm⁵Se²U (>90%) at pH 7.4 which facilitate base pairing with G (Fig. 2h), as well as by enhanced stacking interactions, as supported by enthalpic and entropic effects (Table 2) and CD spectral data (Fig. 5d). Previously reported electron density analysis of the zwitterionic forms of mnm⁵uracil, mnm⁵-2-thiouracil or mnm⁵-2-selenouracil nucleobases upon guanine binding suggests that the superior G-binding capacity of mnm⁵Se²U arises from the higher polarizability of the selenium atom, which strengthens the N3···H–N2(G) hydrogen bond 12 . Finally, we examined the impact of the ionization state of mnm⁵S(Se)²U on base pairing with A and G through thermal denaturation studies conducted at pH 6.4, 7.0, and 7.4 (Table 4). Our results indicate that acidic conditions (pH 6.4) enhance the formation of mnm⁵S(Se)²U–A duplexes (C4, C6) while decrease the stability of mnm⁵S(Se)²U–G duplexes (M4, M6). The opposite trend was observed at pH 7.4. These findings align with expectations: lower pH reduces the content of the ionized (zwitterionic) form, thereby favouring A pairing, whereas higher pH increases zwitterion abundance, which promotes more efficient pairing with G. Notably, Se²U-containing duplexes exhibited a similar pH-dependent trend (Table 3), reinforcing the conclusion that the content of N3 (de)protonated form of Se-uridines modulates base pairing with adenosine and guanosine, affecting overall duplexes stability. Table 4. UV melting temperature and thermodynamic parameters for (5’-GUUGACU mnm 5 Se 2 U UUAAUCAAC-3’/3’-CAACUGA A AAUUAGUUG-5’) and (5’-GUUGACU mnm 5 Se 2 U UUAAUCAAC-3’/3’-CAACUGA G AAUUAGUUG-5’) duplexes determined at pH 7.4, 7.0 and 6.4. Errors for thermodynamic quantities were assessed based on multiple UV melting experiments. No. Name -ΔG 0 (kcal/mol) -ΔH 0 (kcal/mol) -ΔS 0 (eu) Tm (°C) C4. mnm 5 S 2 U-A 14.6 ± 0.2 (pH 7.4) 112.3 ± 4.3 (pH 7.4) 315.2 ± 13.3 (pH 7.4) 55.0 ± 0.3 (pH 7.4) 14.6 ± 0.1 (pH 7.0) 111.2 ± 0.5 (pH 7.0) 314.7 ± 6.2 (pH 7.0) 55.3 ± 0.4 (pH 7.0) 14.5 ± 0.1 (pH 6.4) 110.8 ± 1.5 (pH 6.4) 310.3 ± 4.5 (pH 6.4) 55.3 ± 0.3 (pH 6.4) C6. mnm 5 Se 2 U-A 13.8 ± 0.2 (pH 7.4) 110.4 ± 2.6 (pH 7.4) 311.3 ± 7.7 (pH 7.4) 53.0 ± 0.2 (pH 7.4) 14.0 ± 0.3 (pH 7.0) 109.8 ± 3.6 (pH 7.0) 309.0 ± 10.7 (pH 7.0) 53.6 ± 0.2 (pH 7.0) 13.9 ± 0.3 (pH 6.4) 106.7 ± 5.0 (pH 6.4) 299.2 ± 15.2 (pH 6.4) 53.6 ± 0.3 (pH 6.4) M4. mnm 5 S 2 U-G 11.9 ± 0.1 (pH 7.4) 90.7 ± 2.1 (pH 7.4) 254.2 ± 6.4 (pH 7.4) 49.2 ± 0.3 (pH 7.4) 11.6 ± 0.2 (pH 7.0) 87.0 ± 3.3 (pH 7.0) 243.1 ± 10.0 (pH 7.0) 48.8 ± 0.4 (pH 7.0) 11.0 ± 0.2 (pH 6.4) 77.8 ± 4.7 (pH 6.4) 215.4 ± 14.5 (pH 6.4) 47.8 ± 0.2 (pH 6.4) M6. mnm 5 Se 2 U-G 12.1 ± 0.2 (pH 7.4) 92.1 ± 2.8 (pH 7.4) 258.0 ± 8.6 (pH 7.4) 49.8 ± 0.4 (pH 7.4) 12.0 ± 0.2 (pH 7.0) 90.3 ± 2.3 (pH 7.0) 252.4 ± 6.9 (pH 7.0) 49.6 ± 0.3 (pH 7.0) 11.4 ± 0.1 (pH 6.4) 83.1 ± 2.4 (pH 6.4) 230.9 ± 7.4 (pH 6.4) 48.7 ± 0.5 (pH 6.4) Conclusion A series of 17-nt oligonucleotides containing mnm 5 U, mnm 5 S 2 U, mnm 5 Se 2 U (mnm 5 U*), and corresponding 5-un substituted uridines U, S 2 U, Se 2 U (U*), was synthesized and hybridized with complementary RNA strands to form duplexes with (mnm 5 )U*-A and (mnm 5 )U*-G pairs. The synthesis of mnm 5 Se 2 U-RNA was carried out via phosphoramidite chemistry according to the protocol developed in this study. The effects of mnm⁵ substitution and 2-chalcogen modifications were evaluated with respect to RNA duplex structure, thermodynamic stability and base-pairing specificity for adenosine over guanosine. It was observed that all canonical U*-A and mnm 5 U*-A base pairs stabilize RNA duplexes more efficiently than the corresponding U*-G and mnm 5 U*-G mismatched pairs. The stabilizing contribution of chalcogens in duplexes containing (mnm 5 )U*-A pairs increases in the order: uridines < Se 2 -uridines < S 2 -uridines, with the mnm 5 substituent significantly reducing this stability. The effect of O 2 /S²/Se² chalcogens on the stability of (mnm⁵)U*-G duplexes is less pronounced, with the mnm⁵ substituent exerting a slight destabilizing effect. Despite the small differences in the ΔG 0 values, the comparative analysis of all thermodynamic parameters indicates that selenouridines (Se 2 U and mnm 5 Se 2 U) facilitate the formation of duplexes with opposing G more effectively than their O 2 - and S 2 - counterparts. This effect is likely attributed to the higher content of the ionized form of Se 2 -uridines, which preferentially recognize guanosine, while restrict interactions with A. In this aspect, pH-dependent thermal denaturation studies demonstrated the improved stability of duplexes containing Se 2 U-G and mnm 5 Se 2 U-G pairs at higher pH, where the content of the ionized state of selenouridines is elevated. Next, we determined that the base-pairing specificity for adenosine over guanosine follows the order: uridines < Se²-uridines < S²-uridines. The presence of the mnm⁵ substituent reduces this specificity, reflecting a reduced affinity of mnm⁵U* for adenosine relative to their 5- un substituted (U*) counterparts. Overall, S²-uridines exhibit a stronger affinity for adenosine than corresponding Se²-uridines, indicating that Se² substitution increases the propensity for guanosine pairing. In the biological context, our findings may suggest that the dynamic post-transcriptional conversion of wobble mnm⁵S²U to mnm⁵Se²U in bacterial tRNAs Lys,Glu may serve as a regulatory mechanism of gene expression. Given that mnm⁵Se²U displays a marked shift in base-pairing specificity toward guanosine, this modification could promote translation of mRNAs enriched in 3′-G-ending codons, thereby modulating protein synthesis under specific cellular conditions. This hypothesis is supported by in vitro biological experiments on globin translation in rabbits, which demonstrated that 3'-G-ending codons are preferentially recognized by aminoacylated mnm 5 Se 2 U-tRNAs Lys,Glu from E. coli compared to their 2-thio counterparts 40 . Considering our data in the context of designing therapeutic nucleic acids, 2-thiouridine (S²U) has the greatest potential for imparting drug-like properties, as it demonstrates the highest base-pairing specificity for adenosine over guanosine and forms the most thermodynamically stable S²U–A duplex among all systems examined in our study. Declarations Data availability The data presented in this study are available on request from the corresponding author. Acknowledgements Authors thank Dr Ewelina Wielgus (CMMS, PAS, Lodz) and Zofia Gdaniec’ group (IChB, PAS, Poznan) for the mass spectrometry analysis and the access to the synthesizer, respectively. Funding This work was supported by grants UMO-2018/29/B/ST5/02509 from National Science Centre to B.N. and W-3D/FMN/17G/2021 from Young Scientists’ Fund at LUT to P.K. Author Contributions The authors confirm their contribution to the paper as follows: study conception and design: GL., BN., ES.; data collection: PK., KK., AD., KP., MS., TB., analysis and interpretation of results: GL., BN., PK., KK.; draft manuscript: GL., PK. All authors reviewed the results and approved the final version of the manuscript. Additional Information Supplementary information accompanies this paper at …. Competing Interests: The authors declare no competing interests. References Cappannini, A. et al. J. M. MODOMICS: a database of RNA modifications and related information. 2023 update. Nucleic Acids Res. 52 (D1), D239-D244 (2024). Duchler, M., Leszczynska, G., Sochacka, E. & Nawrot, B. Nucleoside modifications in the regulation of gene expression: Focus on tRNA. Cell. Mol. Life Sci. 73 , 3075–3095 (2016). Lei, L. & Burton, Z. F. “Superwobbling” and tRNA-34 wobble and tRNA-37 anticodon loop modifications in evolution and devolution of the genetic code. 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1","display":"","copyAsset":false,"role":"figure","size":52498,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of 5-methylaminomethyluridine (mnm\u003csup\u003e5\u003c/sup\u003eU, \u003cstrong\u003e1\u003c/strong\u003e), 5-methylaminomethyl-2-thiouridine (mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU, \u003cstrong\u003e2\u003c/strong\u003e), 5-methylaminomethyl-2-selenouridine (mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU, \u003cstrong\u003e3\u003c/strong\u003e), 5-carboxymethylaminomethyluridine (cmnm\u003csup\u003e5\u003c/sup\u003eU, \u003cstrong\u003e4\u003c/strong\u003e), 5-carboxymethylaminomethyl-2-thiouridine (cmnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU, \u003cstrong\u003e5\u003c/strong\u003e), 5-carboxymethylaminomethyl-2-selenouridine (cmnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU, \u003cstrong\u003e6\u003c/strong\u003e), uridine (U, \u003cstrong\u003e7\u003c/strong\u003e), 2-thiouridine (S\u003csup\u003e2\u003c/sup\u003eU, \u003cstrong\u003e8\u003c/strong\u003e), and 2-selenouridine (Se\u003csup\u003e2\u003c/sup\u003eU, \u003cstrong\u003e9\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/0297ca326f5812ae2ffc882f.png"},{"id":96249808,"identity":"b3d515f3-e7cf-41e7-a2fb-386e2abe7e8e","added_by":"auto","created_at":"2025-11-19 07:36:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112881,"visible":true,"origin":"","legend":"\u003cp\u003ePossible systems of hydrogen bonding within U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU-A Watson−Crick base pairs (a,b); U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU-G mismatched base pairs (c,d); mnm\u003csup\u003e5\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-A Watson−Crick pairs (e,f) and mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-G mismatched base pairs (g,h).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/8c73a300179d950d81518bc1.png"},{"id":96249839,"identity":"9aaaad48-abe0-4297-8fe1-bde4bdf184c6","added_by":"auto","created_at":"2025-11-19 07:36:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131412,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Synthetic route of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-phosphoramidite (\u003cstrong\u003e14\u003c/strong\u003e). (\u003cstrong\u003eb\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSynthesis of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA (\u003cstrong\u003e17\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/b7e2d95c1a1ab543d2428c40.png"},{"id":96088708,"identity":"cb1795fd-a158-4fbe-8076-12c8c93f63d7","added_by":"auto","created_at":"2025-11-17 12:59:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46856,"visible":true,"origin":"","legend":"\u003cp\u003eThe differences between the DG\u003csup\u003e0\u003c/sup\u003e values (Table 2) determined for U*-A and mnm\u003csup\u003e5\u003c/sup\u003eU*-A duplexes and appropriate U*-G and mnm\u003csup\u003e5\u003c/sup\u003eU*-G duplexes. The arrows indicate an increased trend in base pairing specificity of uridines for A over G (higher ΔΔG\u003csup\u003e0\u003c/sup\u003e value reflects a stronger preference for pairing with A than G).\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/0c0e52919c3c5f0fa155c853.png"},{"id":96246918,"identity":"79a51b03-ac39-4c69-9f7e-2d84f4be928e","added_by":"auto","created_at":"2025-11-19 07:26:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":106667,"visible":true,"origin":"","legend":"\u003cp\u003eCD spectra of RNA duplexes containing: (a) U*-A base pairs; (b) U*-G pairs; (c) mnm\u003csup\u003e5\u003c/sup\u003eU*-A pairs; (b) mnm\u003csup\u003e5\u003c/sup\u003eU*-G pairs (U* = U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eU* = mnm\u003csup\u003e5\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/c1172c843dc460b0e76e67a6.png"},{"id":96248609,"identity":"bb35a184-2a28-41ad-844d-5523e0529437","added_by":"auto","created_at":"2025-11-19 07:28:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63373,"visible":true,"origin":"","legend":"\u003cp\u003eThe hydrogen bonds system within Se\u003csup\u003e2\u003c/sup\u003eU-A and Se\u003csup\u003e2\u003c/sup\u003eU-G base pairs, including ionized form of Se\u003csup\u003e2\u003c/sup\u003eU.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/46d8ce391fcbca691e70aff0.png"},{"id":99545456,"identity":"75b4d57b-af5c-45c1-8c27-620c587f2d52","added_by":"auto","created_at":"2026-01-05 16:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1744935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/f34da27d-6473-480b-a2e4-521f5fdc0266.pdf"},{"id":96249973,"identity":"672a8c5d-6fd6-470e-8f5d-6de48ed76739","added_by":"auto","created_at":"2025-11-19 07:36:58","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1739264,"visible":true,"origin":"","legend":"","description":"","filename":"P.Kuwerskaetal.ESI.doc","url":"https://assets-eu.researchsquare.com/files/rs-7980593/v1/b5b9a584c8cfda1b950e8d9c.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFrom U to mnm⁵Se²U: tuning base pairing preferences through 2-chalcogen and 5-methylaminomethyl modifications\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCellular RNAs contain over 170 structurally distinct modified nucleosides that serve diverse biological functions\u003csup\u003e1\u003c/sup\u003e. Most of them have been identified in transfer RNAs (tRNAs), with two major hotspots: the wobble position (position 34), corresponding to the first anticodon letter, and position 37, adjacent to the anticodon from the 3\u0026apos; end. Wobble modifications are known to fine-tune the decoding process by stabilizing codon\u0026ndash;anticodon interactions, thereby enhancing the efficiency and accuracy of translation\u003csup\u003e2,3\u003c/sup\u003e. The representative group of the wobble-positioned tRNA modifications are 5-substituted uridines, 2-thiouridines and 2-selenouridines, with the 5-aminomethyl (xnm5) substituent being particularly widespread\u003csup\u003e1,4\u003c/sup\u003e. Among them, 5-methylaminomethyl (mnm, \u003cstrong\u003e1\u003c/strong\u003e-\u003cstrong\u003e3\u003c/strong\u003e, Fig. 1) and 5-carboxymethylaminomethyl (cmnm, \u003cstrong\u003e4\u003c/strong\u003e-\u003cstrong\u003e6\u003c/strong\u003e) modifying groups adorn all three types of O\u003csup\u003e2\u003c/sup\u003e-, S\u003csup\u003e2\u003c/sup\u003e- and Se\u003csup\u003e2\u003c/sup\u003e-uridines. The (c)mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU and (c)mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU nucleosides have been identified in bacterial tRNAs specific for lysine, glutamate, and glutamine, where they share the wobble position with \u003cem\u003eS\u003c/em\u003e-geranyl-2-thiouridines (mnm\u003csup\u003e5\u003c/sup\u003egeS\u003csup\u003e2\u003c/sup\u003eU or cmnm\u003csup\u003e5\u003c/sup\u003egeS\u003csup\u003e2\u003c/sup\u003eU)\u003csup\u003e5-8\u003c/sup\u003e. All 5-aminomethyl-containing uridines, 2-thiouridines and 2-selenouridines recognize not only adenosine but also guanosine as the third codon letter (A- and G-3\u0026apos;)\u003csup\u003e5,9,10\u003c/sup\u003e.\u0026nbsp;This dual mode of base pairing with purines has been widely discussed in the literature, particularly in the context of the geometry of the non-Watson-Crick xnm⁵U-G and xnm\u003csup\u003e5\u003c/sup\u003eS(Se)\u003csup\u003e2\u003c/sup\u003eU-G base pairs\u003csup\u003e10-16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBeyond their established functions in cellular tRNAs, uridine derivatives have also been applied in the design of therapeutic nucleic acids\u003csup\u003e17-19\u003c/sup\u003e. Among others, the 2-thiouridine (S\u0026sup2;U) and 2-selenouridine (Se\u003csup\u003e2\u003c/sup\u003eU) derivatives have been utilized to improve the stability and base pairing specificity of antisense oligonucleotides and small interfering RNAs (siRNAs) with their complementary RNA targets\u003csup\u003e20-22\u003c/sup\u003e. As demonstrated by thermal denaturation experiments, replacing U with S\u0026sup2;U or Se\u0026sup2;U increases the stability of S(Se)\u0026sup2;U-A duplexes and decreases the stability of S(Se)\u0026sup2;U-G duplexes, thereby supporting the strong preferential pairing of S(Se)\u0026sup2;U with adenosine over guanosine\u003csup\u003e14,20-24\u003c/sup\u003e. The enhanced thermodynamic stability of duplexes containing S(Se)\u0026sup2;U-A vs. U-A base pairs arises from three key features of S\u003csup\u003e2\u003c/sup\u003e- and\u0026nbsp;\u003cbr\u003eSe\u003csup\u003e2\u003c/sup\u003e-uridines (Table 1)\u003csup\u003e20,23-25\u003c/sup\u003e: (1) their preference for the C3\u0026prime;-\u003cem\u003eendo\u003c/em\u003e sugar conformation (20-30% higher than that of U)\u003csup\u003e11,12\u003c/sup\u003e, which improves base stacking interactions with the adjacent nucleobase and supports the rigid A-form RNA duplex; (2) increased N3-H acidity (by 1-2 pKa units relative to U)\u003csup\u003e11,12\u003c/sup\u003e, which promotes stronger hydrogen bonding with the N1-acceptor site of adenosine (Fig. 2a vs. 2b) and (3) poor H-bonding ability of S\u003csup\u003e2\u003c/sup\u003e/Se\u003csup\u003e2\u003c/sup\u003e causing a reduced desolvation during duplex formation. On the other hand, the destabilizing effect of the U\u0026rarr;S(Se)\u003csup\u003e2\u003c/sup\u003eU substitution in duplexes with opposite G is attributed to the larger atomic radius and lower electronegativity of sulfur/selenium compared to oxygen which weaken hydrogen bonding (Fig. 2c vs. 2d) and make the interaction with G less favourable.\u003c/p\u003e\n\u003cp\u003eAlthough the (de)stabilizing effects of U\u0026rarr;S\u0026sup2;U and U\u0026rarr;Se\u0026sup2;U substitutions in individual RNA duplexes are well established\u003csup\u003e14,20-24\u003c/sup\u003e, the contribution of S\u0026sup2;U vs. Se\u0026sup2;U remains unknown due to the lack of studies on the same model RNAs. Based on the steric and electronic properties of the U, S\u003csup\u003e2\u003c/sup\u003eU, and Se\u003csup\u003e2\u003c/sup\u003eU nucleosides described above, the preferential A pairing effect is expected to follow the order: U \u0026lt; S\u0026sup2;U \u0026lt; Se\u0026sup2;U, however, this trend has not been experimentally confirmed at the oligonucleotide level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe physicochemical and structural properties of uridines (U, mnm\u003csup\u003e5\u003c/sup\u003eU), 2-thiouridines (S\u003csup\u003e2\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU) and 2-selenouridines (Se\u003csup\u003e2\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU)\u003csup\u003e11, 12\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.7308%;\"\u003e\n \u003cp\u003eProperties of uridines\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e\u003cstrong\u003emn\u003c/strong\u003e\u003cstrong\u003em\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.7308%;\"\u003e\n \u003cp\u003ep\u003cem\u003eK\u003c/em\u003ea for\u0026nbsp;N3-\u003cu\u003eH\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e9.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e8.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e7.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e8.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e7.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e6.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.7308%;\"\u003e\n \u003cp\u003epKa for CH\u003csub\u003e3\u003c/sub\u003eNH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003eCH\u003csub\u003e2\u003c/sub\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e10.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e9.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e9.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.7308%;\"\u003e\n \u003cp\u003eContent of the ionized fraction of nucleoside under physiological conditions [%]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003cp\u003e(34)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003cp\u003e(34)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e\u0026gt;90\u003c/p\u003e\n \u003cp\u003e(78)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 31.7308%;\"\u003e\n \u003cp\u003eC\u003cem\u003e3\u0026rsquo;-endo\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003esugar puckering \u0026nbsp;[%]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.61538%;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.141%;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eCalculated according to the Henderson\u0026ndash;Hasselbalch equation pKa-pH = log [BH]/[B\u003csup\u003e-\u003c/sup\u003e], based on the pKa values determined for N3-H (BH and B\u003csup\u003e-\u003c/sup\u003e are the neutral and ionized forms, respectively). \u003csup\u003eb\u003c/sup\u003e Recalculated for nucleotides, since the pKa values for N3-H in pyrimidine nucleotides are 0.4 unit higher than for nucleosides.\u003c/p\u003e\n\u003cp\u003eFrom nucleoside studies, substitution of uridine, 2-thiouridine and 2-selenouridine with a 5-methylaminomethyl (mnm\u003csup\u003e5\u003c/sup\u003e) group introduces changes in the uracil electron density and sugar puckering which should affect duplex stability and base-pairing specificity\u0026sup1;\u0026sup1;\u003csup\u003e,\u003c/sup\u003e\u0026sup1;\u0026sup2;. The mnm⁵-induced changes in the electronic properties of uracil arise from protonation of the mnm⁵ group under physiological conditions (the pKa of CH₃NH₂⁺CH₂ ranges from 9.3 to 10.0, Table 1)\u0026sup1;\u0026sup1;\u003csup\u003e,\u003c/sup\u003e\u0026sup1;\u0026sup2; making it a strong electron-withdrawing substituent that promotes deprotonation of the N3-H group. The mnm⁵-modified uridines are ~1 pKa unit more acidic than their 5-\u003cem\u003eun\u003c/em\u003esubstituted analogs (Table 1), whereas the combined presence of mnm⁵ and S\u0026sup2;/Se\u0026sup2; substituents yields unusually low pKa values: 7.27 for mnm⁵S\u0026sup2;U and 6.43 for mnm⁵Se\u0026sup2;U\u0026sup1;\u0026sup1;\u003csup\u003e,\u003c/sup\u003e\u0026sup1;\u0026sup2;. As a result, mnm\u003csup\u003e5\u003c/sup\u003eS(Se)\u003csup\u003e2\u003c/sup\u003eU partially exists in the zwitterionic state with the positive charge on the aminoalkyl side chain and the negative charge dispersed on the S(Se)2-C2-N3-C4-O4 edge of nucleobase, that was confirmed in theoretical, structural and physicochemical studies\u003csup\u003e10-12,15\u003c/sup\u003e. The content of the ionized fraction reaches 57% for mnm\u003csup\u003e5\u003c/sup\u003eS\u0026sup2;U and an exceptionally high, \u0026gt;90% for mnm\u003csup\u003e5\u003c/sup\u003eSe\u0026sup2;U (Table 1).\u003csup\u003e11,12\u003c/sup\u003e Using thermal denaturation experiments, we recently demonstrated that increased ionization of C5-substituted 2-thiouridines, including mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU, correlates with reduced base pairing specificity for A over G\u003csup\u003e26\u003c/sup\u003e. In addition, we confirmed the previously proposed hypothesis that the neutral form of mnm⁵S\u0026sup2;U preferentially pairs with adenosine (Fig. 2e), whereas its zwitterionic form favours pairing with guanosine (Fig. 2h)\u003csup\u003e11,15,26\u003c/sup\u003e. Of note, the formation of zwitterionic mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-G base pair requires a slight shift of mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU toward the minor groove. Other base-pairing systems, such as zwitterionic mnm⁵S\u0026sup2;U interacting with A (Fig. 2f) or neutral mnm⁵S\u0026sup2;U pairing with G (Fig. 2g) appear to be energetically unfavourable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding mnm⁵Se\u0026sup2;U base pairing with A/G, experimental data on its thermodynamic and structural contributions to RNA duplex properties remain unavailable, primary due to its challenging synthetic accessibility. As was shown by density functional theory (DFT) calculations at the nucleobase level, the zwitterionic form of mnm⁵Se\u0026sup2;-uracil binds more effectively to guanine than the corresponding 2-oxo and 2-thio analogs, due to a stronger H-bond between the N3 of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003e-uracil and the 2-amino group of guanine, suggesting a U-G base pairing mode analogous to that previously observed for zwitterionic mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU (Fig. 2h)\u003csup\u003e12\u003c/sup\u003e. Interestingly, mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU exhibits slightly reduced C3\u0026prime;-\u003cem\u003eendo\u003c/em\u003e sugar puckering (and thus potentially weaker stacking interactions) relative to mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU (Table 1)\u003csup\u003e11,12\u003c/sup\u003e, which contradicts data obtained for 5-\u003cem\u003eun\u003c/em\u003esubstituted Se\u003csup\u003e2\u003c/sup\u003eU and S\u003csup\u003e2\u003c/sup\u003eU as well as established knowledge that a larger C2 substituent favours the C3\u0026prime;-\u003cem\u003e\u0026nbsp;endo\u003c/em\u003e conformation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDriven by the distinct structural and electronic properties of uridines bearing 2-chalcogen and mnm⁵ functions, and by the incomplete data on the hybridization behaviour of selenium-containing RNAs, we undertook a systematic investigation on the individual and synergistic contributions of these modifications to RNA duplex stability. All oligonucleotides of 5\u0026rsquo;-GUUGACU(mnm\u003csup\u003e5\u003c/sup\u003e)U*UUAAUCAAC-3\u0026rsquo; sequence (U* = U, S\u003csup\u003e2\u003c/sup\u003eU, Se\u003csup\u003e2\u003c/sup\u003eU or mnm\u003csup\u003e5\u003c/sup\u003eU* = mnm\u003csup\u003e5\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU) were synthesized \u003cem\u003ein house\u003c/em\u003e according to the previously developed procedures\u003csup\u003e6,26-28\u003c/sup\u003e, except for mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA, whose original synthesis is presented in this work. Twelve duplexes containing either adenosine or guanosine opposite the (mnm\u003csup\u003e5\u003c/sup\u003e)U* nucleoside were analyzed using circular dichroism (CD) and UV melting experiments to evaluate their structural and thermodynamic properties. The structural contribution of the modified uridines appears to be minimal, as the CD spectra exhibited a characteristic A-form RNA duplex profile. Thermodynamic analyses indicate that S\u003csup\u003e2\u003c/sup\u003e/Se\u003csup\u003e2\u003c/sup\u003e-chalcogens and mnm⁵ substituent exert a pronounced influence on the stability of duplexes with opposing adenosine, but have minimal impact on those with opposing guanosine. Comparing these data, a base pairing specificity for adenosine over guanosine was established as follows: uridines \u0026lt; Se\u003csup\u003e2\u003c/sup\u003e-uridines \u0026lt; S\u003csup\u003e2\u003c/sup\u003e-uridines, with mnm\u003csup\u003e5\u003c/sup\u003e group significantly reducing this specificity. Overall, S\u003csup\u003e2\u003c/sup\u003e-uridines (contrary to expectations for Se\u003csup\u003e2\u003c/sup\u003e-uridines) exhibited the strongest preference for pairing with adenosine.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003eSynthesis and characterization of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA oligomer.\u0026nbsp;\u003c/strong\u003eSynthesis of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA oligomer was performed at 2.5-mmol scale by phosphoramidite chemistry using a synthesizer (K\u0026amp;A H-8 DNA/RNA/LNA Synthesizer). We employed the commercially available rC(TAC)-succinyl-CPG (Proligo) support and phosphoramidites of A, C, U and G protected with 5\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-DMTr-2\u0026rsquo;-\u003cem\u003eO\u003c/em\u003e-TBDMS-NH-TAC, prepared as a 0.1 M solutions in acetonitrile. Incorporation of the canonical monomeric units was performed in an 8 molar excess with a coupling time of 8 min. The mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU phosphoramidite (synthetic protocol and full spectral characteristic are given in ESI, Fig. S1-S11) was coupled twice, each time using an 8-fold molar excess and 10 min coupling time. Condensation steps were carried out in the presence of 0.25 M solution of 5-(3,5-bis(trifluoromethyl)phenyl)-1\u003cem\u003eH\u003c/em\u003e-tetrazole in acetonitrile (Activator 42\u003csup\u003e\u0026reg;\u003c/sup\u003e). The mixture of Cap A (THF/TAC\u003csub\u003e2\u003c/sub\u003eO, 100:5 v/w), and Cap B (THF/\u003cem\u003eN\u003c/em\u003e-methylimidazole, 84:16 v/v) was used in the capping step for 2 min. In the oxidation step, a solution of I\u003csub\u003e2\u003c/sub\u003e (0.02 M in THF/H\u003csub\u003e2\u003c/sub\u003eO/pyridine, 90.54/9.05/0.41 v/v/v) was applied for 2 min. After synthesis, the support-linked DMTr-off oligomer was cleaved from the beads and deprotected on 0.2 \u0026mu;mol scale. The \u003cem\u003eb\u003c/em\u003e-cyanoethyl groups were selectively removed from the phosphate residues using Et\u003csub\u003e3\u003c/sub\u003eN-acetonitrile (272\u0026nbsp;\u0026mu;L, 1/1 v/v, 30 min, rt.). The supernatant was removed. The resin was washed with acetonitrile (3 \u0026times; 200\u0026nbsp;\u0026mu;L) and dried in vacuo for 30 min. Subsequently, the resin was treated with 0.05 M solution of K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e in anhydrous methanol (200 \u0026mu;L) for 10 h at rt. The supernatant was transferred to an Eppendorf tube. The resin was washed with anhydrous MeOH (3 x 200 \u0026mu;L). The combined oligonucleotide-containing fractions were neutralized by 99% AcOH and evaporated to dryness on a Speed-Vac. Oligomer was dissolved in 300 \u0026mu;L anhydrous EtOH and dried using Speed-Vac for 3 h. Then, oligomer was treated with neat Et\u003csub\u003e3\u003c/sub\u003eN\u0026middot;3HF (48 \u0026mu;L, 24 h, rt.) and desalted on a C-18 cartridge (Sep-Pak\u003csup\u003e\u0026reg;\u003c/sup\u003e, Waters). After solvent evaporation, the oligomer was briefly incubated with conc. NH\u003csub\u003e4\u003c/sub\u003eOH (100 \u0026mu;L, 30 min, rt), followed by removal of ammonia under reduced pressure using Speed-Vac. The fully deprotected mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA \u003cstrong\u003e17\u003c/strong\u003e was purified by anion-exchange high-performance liquid chromatography (IE-HPLC, Fig. S12) and characterized by electrospray ionization mass spectrometry (ESI MS, Fig. S13) and enzymatic digestion analysis (Fig. S14). From a 0.2 \u0026micro;mol-scale synthesis, we obtained 5 OD\u003csub\u003e260\u003c/sub\u003e of the final product \u003cstrong\u003e17\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUV melting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexperiments.\u0026nbsp;\u003c/strong\u003eUV absorbance measurements and thermal denaturation experiments were performed in a cell with 1 cm path length on a Jasco V-770 UV-VIS/NIR spectrophotometer equipped with a Peltier thermal cell. Solutions of complementary RNA/RNA oligonucleotide strands were prepared in a phosphate buffer (10 mM sodium phosphate pH 7.4, pH 7.0 or pH 6.4 with 100 mM NaCl) at the final concentration of 2 \u0026micro;M. Samples were then heated to 85\u0026nbsp;\u0026deg;C and cooled to 15\u0026nbsp;\u0026deg;C with a temperature gradient of 1.5\u0026nbsp;\u0026deg;C/min. Melting profiles were recorded from 15 to 85\u0026nbsp;\u0026deg;C, with a temperature gradient of 0.5\u0026nbsp;\u0026deg;C/min (Fig. S15-S18, ESI). Thermodynamic parameters (Tm,\u0026nbsp;DG\u003csup\u003e0\u003c/sup\u003e,\u0026nbsp;DH\u003csup\u003e0\u003c/sup\u003e,\u0026nbsp;DS\u003csup\u003e0\u003c/sup\u003e) were calculated by numerically fitting a given melting curve using a two-state model algorithm by MeltWinv.3.5 software (MeltWin software license was kindly provided by Jeffrey McDowell, www.meltwin.com). Each result was taken as an averaged one from three independent experiments. The resulting UV melting temperatures (T\u003cem\u003e\u003csub\u003em\u003c/sub\u003e\u003c/em\u003e) and thermodynamic parameters (DH\u003csup\u003e0\u003c/sup\u003e,\u0026nbsp;DS\u003csup\u003e0\u003c/sup\u003e,\u0026nbsp;DG\u003csup\u003e0\u003c/sup\u003e) of the U*- and mnm\u003csup\u003e5\u003c/sup\u003eU*-modified duplexes are given in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD spectra.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCircular dichroism measurements were performed using a J-715 circular dichroism spectrometer (CD) (Jasco, Japan). Duplex samples were prepared at the concentration of 2 \u0026micro;M in a 10 mM sodium phosphate buffer, pH 7.4 containing 100 mM NaCl. The oligonucleotides were mixed in a buffer, heated to 85\u0026nbsp;\u0026deg;C and slowly cooled to room temperature. CD spectra were recorded using a quartz cell with 0.5 cm slice thickness. The acquisition parameters were as follow: scanning speed 50 nm/min, response time 2 s, band width 1.0 nm and step resolution 0.2 nm. The spectra were recorded at 24\u0026nbsp;\u0026deg;C in the wavelength range from 200 to 360 nm. The spectrum recorded for the buffer was numerically subtracted from the spectrum for each sample (recorded in triplicate) and the resulting averaged spectra were smoothed using an averaging algorithm (convolution width 25). CD spectra for U*- and mnm\u003csup\u003e5\u003c/sup\u003eU*-modified RNA duplexes with opposite A and G units are given in Fig. 5.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eSynthesis of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-phosphoramidite.\u0026nbsp;\u003c/strong\u003eThe C2-selenocarbonyl group was introduced \u003cem\u003evia\u003c/em\u003e selenation of appropriately protected 2-\u003cem\u003eS\u003c/em\u003e-methylated 2-thiouridine \u003cstrong\u003e8\u003c/strong\u003e (Fig. 3a) with NaSeH, following the method originally developed by Klayman and Griffin\u003csup\u003e29\u003c/sup\u003e and adopted by others for Se\u003csup\u003e2\u003c/sup\u003eU phosphoramidite synthesis\u003csup\u003e14,30\u003c/sup\u003e, yielding 82%. Recently, the same protocol was successfully applied to obtain a series of naturally occurring 5-aminoalkyl-2-selenouridines\u003csup\u003e6,12\u003c/sup\u003e. The 2-\u003cem\u003eS\u003c/em\u003e-methylated 2-thiouridine \u003cstrong\u003e8\u003c/strong\u003e was obtained by direct methylation of 2-thiouridine \u003cstrong\u003e7\u003c/strong\u003e, which was protected with a 4,4\u0026apos;-dimethoxytrityl group (DMTr) at the 5\u0026apos;-hydroxyl and a trifluoroacetyl group (TFA) on the amine function of the mnm\u003csup\u003e5\u003c/sup\u003e side chain. The TFA group was previously successfully employed in the solid-phase synthesis of several RNA oligomers modified with 5-aminomethyluridine derivatives\u003csup\u003e31-34\u003c/sup\u003e. After selenation, the 2\u0026apos;-OH group of \u003cstrong\u003e9\u003c/strong\u003e was protected with \u003cem\u003etert\u003c/em\u003e-butyldimethylsilyl\u003cem\u003e\u0026nbsp;\u003c/em\u003e(TBDMS) \u003cem\u003evia\u003c/em\u003e a standard procedure\u003csup\u003e35\u003c/sup\u003e. The obtained regiomers \u003cstrong\u003e10\u003c/strong\u003e and \u003cstrong\u003e11\u003c/strong\u003e (1:1 ratio) were purified without separation (Y=62%) and used as a mixture to introduce the \u003cem\u003eb\u003c/em\u003e-cyanoethyl protecting group on 2-Se with iodopropionitrile (note that the unprotected Se\u003csup\u003e2\u003c/sup\u003e tends to be oxidized after treatment with an oxidizing agent during RNA synthesis\u003csup\u003e14\u003c/sup\u003e). The Se-protected isomers \u003cstrong\u003e12\u003c/strong\u003e and \u003cstrong\u003e13\u003c/strong\u003e were separated in 48% and 32% yields, respectively, and ubiquitously identified by \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY NMR. The 2\u0026apos;-\u003cem\u003eO\u003c/em\u003e-TBDMS isomer \u003cstrong\u003e12\u003c/strong\u003e was phosphitylated to give the Se-phosphoramidite \u003cstrong\u003e14\u003c/strong\u003e in 72% yield. The synthetic procedures and spectral characterization of \u003cstrong\u003e9\u003c/strong\u003e-\u003cstrong\u003e14\u003c/strong\u003e are shown in ESI (Fig. S1-S11).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical synthesis of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-oligoribonucleotide.\u0026nbsp;\u003c/strong\u003eThe chemical incorporation of 2-seleno-uridines into RNA remains a significant synthetic challenge. In the past, Huang and co-workers performed a successful chemical synthesis of Se\u003csup\u003e2\u003c/sup\u003eU-RNA oligomers employing \u003cem\u003eb\u003c/em\u003e-cyanoethyl protection for Se\u003csup\u003e2\u003c/sup\u003e function\u003csup\u003e14\u003c/sup\u003e. Using 2-selenouridine synthase (SelU), Sierant et al. transformed\u0026nbsp;S\u003csup\u003e2\u003c/sup\u003eU-RNA \u003cem\u003evia\u003c/em\u003e its geranylated derivative to Se\u003csup\u003e2\u003c/sup\u003eU-RNA\u003csup\u003e6\u003c/sup\u003e,\u0026nbsp;while the Stadtman group obtained mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-modified \u003cem\u003eE. coli\u003c/em\u003e tRNA\u003csup\u003eLys\u003c/sup\u003e through enzymatic reaction of bulk tRNAs\u003csup\u003e36\u003c/sup\u003e. In our work, fully protected amidite \u003cstrong\u003e14\u003c/strong\u003e (Fig. 3) was incorporated into the 5\u0026apos;- GUUGACU\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo; \u0026nbsp;RNA chain related to the anticodon arm domain of \u003cem\u003eE.\u0026nbsp;coli\u003c/em\u003e tRNA\u003csup\u003eLys\u003c/sup\u003e. The synthesis was performed \u003cem\u003evia\u003c/em\u003e phosphoramidite chemistry on the CPG-C(TAC) support. After synthesis, the support-linked DMTr-off oligomer was cleaved from the beads and deprotected (Fig. 3b). The \u003cem\u003eb\u003c/em\u003e-cyanoethyl groups were selectively removed from the phosphate residues using triethylamine in acetonitrile. The resin deprived of \u003cem\u003eb\u003c/em\u003e-acrylonitrile was dried and treated with anhydrous 0.05 M K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e methanol solution (10 h, rt.) according to the protocol of Huang, who found these conditions safe for Se-modification during Se\u003csup\u003e2\u003c/sup\u003eU-RNA and Se\u003csup\u003e2\u003c/sup\u003eT-DNA preparation\u003csup\u003e14,37\u003c/sup\u003e. In our studies, methanolic K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e proved effective for CPG cleavage and removal of the Se-\u003cem\u003eb\u003c/em\u003e-cyanoethyl and exoamine TAC blockage, however the TFA group remained intact. The 2\u0026apos;-TBDMS groups were removed with neat TEA x 3HF (24 h, rt.). After desalting and evaporation, Se-oligomer was briefly treated with conc. NH\u003csub\u003e4\u003c/sub\u003eOH (30 min, rt.) to remove TFA. The reduced time of TFA-ammonolysis was important to avoid the RNA degradation and/or deselenation. After deprotection, mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA \u003cstrong\u003e17\u003c/strong\u003e was purified by IE-HPLC, (Fig. S12) and characterized by ESI MS (Fig. S13) and enzymatic digestion analysis (Fig. S14). To simplify the deprotection protocol and enable simultaneous removal of all base-labile protecting groups in a single step, we tested the use of conc. NH\u003csub\u003e4\u003c/sub\u003eOH-EtOH (3:1 v/v, 16 h, 36\u0026nbsp;\u0026deg;C) and conc. NH\u003csub\u003e4\u003c/sub\u003eOH (3 h, rt.). However, both conditions led to the formation of an undesirable deselenation product\u003csup\u003e38\u003c/sup\u003e. The oligomers modified with S\u003csup\u003e2\u003c/sup\u003eU, Se\u003csup\u003e2\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eU, and mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU were synthesized \u003cem\u003ein house\u0026nbsp;\u003c/em\u003eaccording to the protocols described in the literature\u003csup\u003e6,26-28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThermodynamic and structural analysis of RNA duplexes.\u003c/strong\u003e Two sets of RNA duplexes 5\u0026apos;- GUUGACU \u003cstrong\u003e(mnm\u003csup\u003e5\u003c/sup\u003e)U*\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eA\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo; (C1-C6; C denotes duplexes containing a canonical U-A base pair) and 5\u0026apos;-\u0026nbsp;GUUGACU\u003cstrong\u003e(mnm\u003csup\u003e5\u003c/sup\u003e)U*\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eG\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo; (M1-M6, M denotes duplexes containing a mismatched U-G base pair) were selected for our studies. The U* and mnm\u003csup\u003e5\u003c/sup\u003eU* abbreviations represent U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU and mnm\u003csup\u003e5\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU/ mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU, respectively, whereas two uridines e.g. U and mnm\u003csup\u003e5\u003c/sup\u003eU are denoted as (mnm\u003csup\u003e5\u003c/sup\u003e)U. The resulting UV melting temperatures (T\u003cem\u003e\u003csub\u003em\u003c/sub\u003e\u003c/em\u003e) and thermodynamic parameters (Gibbs free energy\u0026nbsp;DG\u003csup\u003e0\u003c/sup\u003e, enthalpy\u0026nbsp;DH\u003csup\u003e0\u003c/sup\u003e,\u0026nbsp;entropy\u0026nbsp;DS\u003csup\u003e0\u003c/sup\u003e)\u0026nbsp;for (mnm\u003csup\u003e5\u003c/sup\u003e)U*-modified duplexes with opposing A and G are summarized in Table 2 with corresponding melting profiles shown in Figs. S15-S18 (ESI).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e UV melting temperature and thermodynamic parameters for 5\u0026rsquo;-GUUGACU\u003cstrong\u003e(mnm\u003csup\u003e5\u003c/sup\u003e)U*\u003c/strong\u003eUUAAUCAA C-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eA\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo; and 5\u0026rsquo;-GUUGACU\u003cstrong\u003e(mnm\u003csup\u003e5\u003c/sup\u003e)U*\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eG\u003c/strong\u003eAAUUAGU UG-5\u0026rsquo; duplexes, \u003cstrong\u003eU*\u003c/strong\u003e=U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU and \u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eU*\u003c/strong\u003e=mnm\u003csup\u003e5\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU. Errors for thermodynamic quantities were assessed based on multiple UV melting experiments.\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\u003cbr\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003eNo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;G\u003csup\u003e0\u003c/sup\u003e (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;H\u003csup\u003e0\u003c/sup\u003e (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;S\u003csup\u003e0\u003c/sup\u003e (eu)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTm (\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eC1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003eU-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e14.2 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e118.5 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e336.5 \u0026plusmn; 8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e52.8 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eC2.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003emnm\u003csup\u003e5\u003c/sup\u003eU-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e12.6 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e92.8 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e258.6 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e51.6 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eC3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003eS\u003csup\u003e2\u003c/sup\u003eU-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e16.1 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e128.1 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e361.2 \u0026plusmn; 10.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e56.9 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eC4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003emnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e14.6 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e112.3 \u0026plusmn; 4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e315.2 \u0026plusmn; 13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e55.0 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eC5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003eSe\u003csup\u003e2\u003c/sup\u003eU-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e14.8 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e108.6 \u0026plusmn; 4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e302.6 \u0026plusmn; 13.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e56.6 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eC6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e13.8 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e110.4 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e311.3 \u0026plusmn; 7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e53.0 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eM1.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003eU-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e12.7 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e107.6 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e306.1 \u0026plusmn; 8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e49.9 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eM2.\u003c/p\u003e\n \u003cp\u003e8.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003emnm\u003csup\u003e5\u003c/sup\u003eU-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e11.4 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e82.4 \u0026plusmn; 4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e229.0 \u0026plusmn; 12.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e48.7 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eM3.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003eS\u003csup\u003e2\u003c/sup\u003eU-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e12.2 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e99.4 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e281.3 \u0026plusmn; 7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e49.3 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eM4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003emnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e11.9 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e90.7 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e254.2 \u0026plusmn; 6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e49.2 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eM5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003eSe\u003csup\u003e2\u003c/sup\u003eU-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e12.8 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e104.5 \u0026plusmn; 3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e298.6 \u0026plusmn; 16.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e50.5 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.33333%;\"\u003e\n \u003cp\u003eM6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6667%;\"\u003e\n \u003cp\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e12.1 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e92.1 \u0026plusmn; 2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e258.0 \u0026plusmn; 8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.75%;\"\u003e\n \u003cp\u003e49.8 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIt is generally observed that all canonical (mnm\u003csup\u003e5\u003c/sup\u003e)U*-A base pairs stabilize RNA duplexes more efficiently than the corresponding (mnm\u003csup\u003e5\u003c/sup\u003e)U*-G mismatched pairs. The magnitude of this stabilizing effect is reflected by the \u0026Delta;\u0026Delta;G⁰ values (Fig. 4), calculated as the differences between the\u0026nbsp;DG\u0026deg; values determined for (mnm\u003csup\u003e5\u003c/sup\u003e)U*-A duplexes and corresponding (mnm\u003csup\u003e5\u003c/sup\u003e)U*-G duplexes and indicate the preferential pairing of modified uridines with adenosine over guanosine (higher\u0026nbsp;\u0026Delta;\u0026Delta;G⁰ value reflects a stronger preference for binding with A over G). Analysis of these data shows the same enhanced trend for both 5-\u003cem\u003eun\u003c/em\u003esubstituted and mnm\u003csup\u003e5\u003c/sup\u003e-substituted uridines arranged as follows (mnm\u003csup\u003e5\u003c/sup\u003e)U \u0026lt; (mnm\u003csup\u003e5\u003c/sup\u003e)Se\u003csup\u003e2\u003c/sup\u003eU \u0026lt; (mnm\u003csup\u003e5\u003c/sup\u003e)S\u003csup\u003e2\u003c/sup\u003eU, indicating the highest preference of 2-thiouridines to bind to A. Comparison of the\u0026nbsp;\u0026Delta;\u0026Delta;G\u0026deg; values between U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU and corresponding mnm\u003csup\u003e5\u003c/sup\u003e-uridines (Fig. 4) reveals that the mnm\u003csup\u003e5\u003c/sup\u003e group markedly reduces base-pairing specificity for A, regardless the type of chalcogen at the C2 position. In other words, the mnm\u003csup\u003e5\u003c/sup\u003e substituent promotes base pairing with G. These findings are consistent with \u003cem\u003ein vivo\u003c/em\u003e translation studies demonstrating that the mnm⁵ group is primarily responsible for the enhanced ability of wobble mnm⁵S\u0026sup2;U in \u003cem\u003eE. coli\u003c/em\u003e tRNA\u003csup\u003eGlu\u003c/sup\u003e to recognize 3\u0026prime;-G-ending codons, whereas the 2-thio group plays a key role in recognizing 3\u0026prime;-A-ending codons and ensuring efficient tRNA aminoacylation\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe global structures of U*- and mnm\u003csup\u003e5\u003c/sup\u003eU*-modified RNA duplexes were examined by CD spectroscopy. As shown in Fig. 5, all duplexes exhibit CD spectra characteristic of the A-type conformation, with a positive band at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e of 265 nm and a negative band at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e of 210 nm. Minor differences were observed, such as an increase in the intensity of the positive band at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e of 265 nm of the S(Se)\u003csup\u003e2\u003c/sup\u003eU-A compared with the S(Se)\u003csup\u003e2\u003c/sup\u003eU-G duplexes, most likely due to their improved\u0026nbsp;\u0026pi;-\u0026pi;\u0026nbsp;base stacking (Figs. 5a, 5b).\u0026nbsp;Similar trends were also observed for duplexes containing mnm\u003csup\u003e5\u003c/sup\u003e-uridines (Figs. 5c, 5d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe individual and synergistic effects of 2-chalcogen and mnm⁵ functions on conformation and thermal stability and of RNA duplexes are discussed in the following sections.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCharacterization of duplexes containing U*-A and U*-G base pairs (U*=U/S\u003csup\u003e2\u003c/sup\u003eU/Se\u003csup\u003e2\u003c/sup\u003eU).\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAccording to our data, the stabilizing effect of 5-\u003cem\u003eun\u003c/em\u003esubstituted\u003cem\u003e\u0026nbsp;\u003c/em\u003euridines (U*) on duplexes with opposing A increases in the order: U \u0026lt; Se\u003csup\u003e2\u003c/sup\u003eU \u0026lt; S\u003csup\u003e2\u003c/sup\u003eU. While the enhanced stabilizing effect of the O\u003csup\u003e2\u003c/sup\u003e\u0026rarr;S\u003csup\u003e2\u003c/sup\u003e substitution (~2 kcal/mol, as indicated by the \u0026Delta;G\u003csup\u003e0\u003c/sup\u003e difference between C1 and C3) is expected and is well-documented in the literature\u003csup\u003e20-22\u003c/sup\u003e, the O\u003csup\u003e2\u003c/sup\u003e\u0026rarr;Se\u003csup\u003e2\u003c/sup\u003e substitution results in only minor changes in duplex stability (C1 vs. C5). In addition, a 1.3 kcal/mol destabilization of the Se\u0026sup2;U-A duplex relative to S\u0026sup2;U-A duplex (C3 vs. C5) was observed, which is entirely unexpected considering the electronic and structural properties of the Se\u0026sup2;U nucleoside - its stronger preference for the C3\u0026prime;-\u003cem\u003eendo\u003c/em\u003e sugar conformation, higher N3-H acidity, and enhanced desolvation (Table 1) \u0026ndash; all of which would be anticipated to favour Se\u0026sup2;U-A duplex formation. Thermodynamic analyses further revealed unfavourable enthalpic and entropic contributions of Se\u003csup\u003e2\u003c/sup\u003eU compared to S\u003csup\u003e2\u003c/sup\u003eU, suggesting weakened Se\u0026sup2;U-A base-pairing and reduced duplex rigidity. CD spectra of U*-A duplexes support this interpretation (Fig. 5a), showing diminished stacking interaction for Se\u003csup\u003e2\u003c/sup\u003eU-containing duplex compared to\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS\u003csup\u003e2\u003c/sup\u003eU counterpart, as evidenced by the lower intensity of the positive band at\u0026nbsp;l\u003csub\u003emax\u003c/sub\u003e of 265 nm. We propose that the destabilizing effect of the S\u0026sup2; \u0026rarr; Se\u0026sup2; replacement arises from the higher content of the ionized form of Se\u0026sup2;U (58%) compared to S\u0026sup2;U (17%) (Table 1), resulting from the difference in their acidity. Unlike the neutral form of Se\u003csup\u003e2\u003c/sup\u003eU, which favours the formation of a Watson-Crick Se\u0026sup2;U-A base pair by two H-bonds (Fig. 6a), the ionized species (Fig. 6b) likely perturbs the hydrogen-bonding pattern, reducing the ability of Se\u0026sup2;U to pair effectively with adenosine and potentially limiting the interaction to a single hydrogen bond.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile the neutral diketo tautomer of Se\u0026sup2;U facilitates the formation of Se\u003csup\u003e2\u003c/sup\u003eU-A base pair, its presence in Se\u003csup\u003e2\u003c/sup\u003eU-G duplexes should have a destabilizing effect (Fig. 6c). This behaviour can be attributed to the poor ability of selenium to form hydrogen bonds, resulting from its large atomic radius and low electronegativity. Due to these properties, Se\u003csup\u003e2\u003c/sup\u003eU is expected to reduce the base pairing with G more effectively than U and S\u003csup\u003e2\u003c/sup\u003eU. In our thermodynamic experiments, the differences in the stability of U*-G duplexes are insufficient to establish a definitive stability ranking, nevertheless, all thermodynamic parameters consistently indicate that Se\u003csup\u003e2\u003c/sup\u003eU facilitate base pairing with G more efficiently than S\u003csup\u003e2\u003c/sup\u003eU (M2 vs. M5). It is likely, that the same population of ionized Se\u0026sup2;U that reduces A-binding efficiency facilitates G-binding through the formation of two hydrogen bonds (Fig. 6d), with Se\u0026sup2;U adopting a geometry shifted toward the minor groove.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe contribution of chalcogens to the stability of U*\u0026ndash;A/G duplexes evaluated in this study is aligned with the findings of Habuchi \u003cem\u003eet al.\u003c/em\u003e for LNA-type duplexes\u003csup\u003e22\u003c/sup\u003e; but it is not fully consistent with the results reported by Sun \u003cem\u003eet al.\u003c/em\u003e, who observed a significantly reduced stability of Se\u003csup\u003e2\u003c/sup\u003eU-G duplexes relative to their unmodified counterparts. This discrepancy may be attributed to the thermodenaturation conditions used in Sun\u0026rsquo;s study, which were performed at pH 6.8 (compared to pH 7.2 employed by Habuchi and pH 7.4 used in our experiments). At the lower pH, the content of the ionized form of Se\u0026sup2;U would be reduced, given its N3-H pKₐ of 7.3, thereby affecting base pairing behaviour. To demonstrate this, we performed thermodenaturation experiments at pH 6.4, 7.0 and 7.4 (Table 3) revealing a pH-dependent modulation of Se\u0026sup2;U base pairing. All three thermodynamic parameters indicate enhanced stabilization of Se\u0026sup2;U\u0026ndash;A duplexes at lower pH, where the N3-protonated form of Se\u0026sup2;U is favoured, and increased stability of Se\u0026sup2;U\u0026ndash;G duplexes at higher pH, which promotes the formation of the ionized Se\u0026sup2;U form. These findings indicate that even modest pH changes can shift the content of neutral\u0026ndash;ionized form of Se-uracil, thereby altering its binding preference for A over G.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e UV melting temperature and thermodynamic parameters for (5\u0026rsquo;-GUUGACU\u003cstrong\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eA\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo;) and (5\u0026rsquo;-GUUGACU\u003cstrong\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eG\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo;) duplexes determined at pH 7.4, 7.0 and 6.4. Errors for thermodynamic quantities were assessed based on multiple UV melting experiments.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003eNo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;G\u003csup\u003e0\u0026nbsp;\u003c/sup\u003e(kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;H\u003csup\u003e0\u003c/sup\u003e (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;S\u003csup\u003e0\u003c/sup\u003e (eu)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTm (\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003eC5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSe\u003csup\u003e2\u003c/sup\u003eU-A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e14.8 \u0026plusmn; 0.5 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e108.6 \u0026plusmn; 4.7 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e302.6 \u0026plusmn; 13.5 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e56.6 \u0026plusmn; 0.6 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e14.8 \u0026plusmn; 0.4 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e109.2 \u0026plusmn; 5.5 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e304.2 \u0026plusmn; 16.4 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e56.6 \u0026plusmn; 0.4 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e15.4 \u0026plusmn; 0.3 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e121.1 \u0026plusmn; 4.0 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e340.9 \u0026plusmn; 11.9 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e56.0 \u0026plusmn; 0.3 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003eM5.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSe\u003csup\u003e2\u003c/sup\u003eU-G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e12.8 \u0026plusmn; 0.2 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e104.5 \u0026plusmn; 3.8 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e298.6 \u0026plusmn; 16.0 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e50.5 \u0026plusmn; 0.1 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e12.3 \u0026plusmn; 0.3 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e97.0 \u0026plusmn; 4.1 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e273.2 \u0026plusmn; 12.3 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e49.8 \u0026plusmn; 0.4 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e11.9 \u0026plusmn; 0.2 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e94.2 \u0026plusmn; 3.5 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e265.2 \u0026plusmn; 10.8 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e49.0 \u0026plusmn; 0.2 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCharacterization of duplexes containing mnm\u003csup\u003e5\u003c/sup\u003eU*-A and mnm\u003csup\u003e5\u003c/sup\u003eU*-G base pairs (mnm\u003csup\u003e5\u003c/sup\u003eU*=mnm\u003csup\u003e5\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU/mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU).\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThermodynamic analyses of mnm\u003csup\u003e5\u003c/sup\u003eU*-A duplexes revealed that the stabilizing effect of mnm\u003csup\u003e5\u003c/sup\u003e-substituted uridines follows the same trend as their 5-\u003cem\u003eun\u003c/em\u003esubstituted counterparts: mnm\u003csup\u003e5\u003c/sup\u003eU \u0026lt; mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU \u0026lt; mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU. However, the presence of the mnm\u003csup\u003e5\u003c/sup\u003e substituent decreases duplex stability by approximately 1.0 - 1.6 kcal/mol, as indicated by differences between the corresponding duplexes pairs (C1 vs. C2, C3 vs. C4, and C5 vs. C6; Table 2). Previous thermodynamic\u003csup\u003e26\u0026nbsp;\u003c/sup\u003eand structural studies\u003csup\u003e15\u003c/sup\u003e on mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-containing RNAs demonstrated that this effect is attributed to the high content (57%) of ionized (zwitterionic) form of mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU at pH 7.4, which is approximately 3-fold greater than that observed for S\u003csup\u003e2\u003c/sup\u003eU. Zwitterionic structure of mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU weakens interactions within the mnm⁵S\u0026sup2;U-A base pair (Fig. 2e vs. 2f), thereby reducing base-pairing efficiency. Similar increase in the content of zwitterionic form is observed for mnm\u003csup\u003e5\u003c/sup\u003eU (15% vs. 2% for U) and mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU (\u0026gt;90% vs. 58 % for Se\u003csup\u003e2\u003c/sup\u003eU) nucleosides (Table 1)\u003csup\u003e11,12\u003c/sup\u003e what shows the general tendency of mnm\u003csup\u003e5\u003c/sup\u003e substituent to promote the zwitterionic structure and to destabilize the duplexes with opposing A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComparison of the mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-A and mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-A duplexes (C4 vs. C6) reveals a 0.8 kcal/mol decrease in stability upon selenium substitution, which is consistent with the increased acidity of Se-nucleosides that limits the formation of a canonical Watson-Crick base pair with adenosine (mnm⁵Se\u0026sup2;U exhibits a 0.85 pKₐ-unit greater N3\u0026ndash;H acidity than mnm⁵S\u0026sup2;U, resulting in an increased zwitterionic fraction by ca 40%, Table 1).\u003csup\u003e11,12\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAs indicated by thermodynamic data, mnm⁵U exerts the most pronounced destabilizing effect on duplexes with opposing A (C2 vs. C6 and C2 vs. C4), although the \u0026lsquo;zwitterionic effect\u0026rsquo; is unlikely to contribute, since mnm⁵U exists in only a minor ionized fraction (~15%) under physiological conditions. In this case, the destabilization results from markedly reduced stacking interactions (as supported by CD spectra, Fig. 5c), due to the lower C3\u0026prime;-\u003cem\u003eendo\u003c/em\u003e sugar pucker preference of mnm⁵U nucleoside (57%) relative to \u0026gt;70% observed for mnm⁵S\u0026sup2;U and mnm⁵Se\u0026sup2;U\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThermodynamic analysis of mnm⁵U*-G and U*-G duplexes indicates a modest destabilizing effect of the mnm⁵ substituent, ranging from 0.3 to 1.3 kcal/mol, as reflected by the \u0026Delta;G\u003csup\u003e0\u003c/sup\u003e differences between the corresponding duplexes (M1 vs. M2; M3 vs. M4; M5 vs. M6). The contribution of O\u003csup\u003e2\u003c/sup\u003e/S\u003csup\u003e2\u003c/sup\u003e/Se\u003csup\u003e2\u003c/sup\u003e chalcogens to the thermal stability of mnm\u003csup\u003e5\u003c/sup\u003eU*-G duplexes - similarly to 5-\u003cem\u003eun\u003c/em\u003esubtituted U*-G duplexesn- is difficult to evaluate due to the small \u0026Delta;G\u003csup\u003e0\u003c/sup\u003e differences among M2, M4 and M6 (Table 2). However, analysis of the complete thermodynamic data suggests that both mnm⁵S\u0026sup2;U and mnm⁵Se\u0026sup2;U increase the binding capacity to guanosine, with a slightly stronger G-affinity observed for mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU. This effect is likely driven by the higher population of the zwitterionic forms of mnm⁵S\u003csup\u003e2\u003c/sup\u003eU (57%) and mnm⁵Se\u0026sup2;U (\u0026gt;90%) at pH 7.4 which facilitate base pairing with G (Fig. 2h), as well as by enhanced stacking interactions, as supported by enthalpic and entropic effects (Table 2) and CD spectral data (Fig. 5d). Previously reported electron density analysis of the zwitterionic forms of mnm⁵uracil, mnm⁵-2-thiouracil or mnm⁵-2-selenouracil nucleobases upon guanine binding suggests that the superior G-binding capacity of mnm⁵Se\u0026sup2;U arises from the higher polarizability of the selenium atom, which strengthens the N3\u0026middot;\u0026middot;\u0026middot;H\u0026ndash;N2(G) hydrogen bond\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFinally, we examined the impact of the ionization state of mnm⁵S(Se)\u0026sup2;U on base pairing with A and G through thermal denaturation studies conducted at pH 6.4, 7.0, and 7.4 (Table 4). Our results indicate that acidic conditions (pH 6.4) enhance the formation of mnm⁵S(Se)\u0026sup2;U\u0026ndash;A duplexes (C4, C6) while decrease the stability of mnm⁵S(Se)\u0026sup2;U\u0026ndash;G duplexes (M4, M6). The opposite trend was observed at pH 7.4. These findings align with expectations: lower pH reduces the content of the ionized (zwitterionic) form, thereby favouring A pairing, whereas higher pH increases zwitterion abundance, which promotes more efficient pairing with G. Notably, Se\u0026sup2;U-containing duplexes exhibited a similar pH-dependent trend (Table 3), reinforcing the conclusion that the content of N3 (de)protonated form of Se-uridines modulates base pairing with adenosine and guanosine, affecting overall duplexes stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e UV melting temperature and thermodynamic parameters for (5\u0026rsquo;-GUUGACU\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eA\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo;) and (5\u0026rsquo;-GUUGACU\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU\u0026nbsp;\u003c/strong\u003eUUAAUCAAC-3\u0026rsquo;/3\u0026rsquo;-CAACUGA\u003cstrong\u003eG\u003c/strong\u003eAAUUAGUUG-5\u0026rsquo;) duplexes determined at pH 7.4, 7.0 and 6.4. Errors for thermodynamic quantities were assessed based on multiple UV melting experiments.\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003eNo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;G\u003csup\u003e0\u003c/sup\u003e (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;H\u003csup\u003e0\u003c/sup\u003e (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u0026Delta;S\u003csup\u003e0\u003c/sup\u003e (eu)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTm (\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003eC4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e14.6 \u0026plusmn; 0.2 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e112.3 \u0026plusmn; 4.3 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e315.2 \u0026plusmn; 13.3 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e55.0 \u0026plusmn; 0.3 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e14.6 \u0026nbsp;\u0026plusmn; 0.1 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e111.2 \u0026plusmn; 0.5 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e314.7 \u0026plusmn; 6.2 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e55.3 \u0026plusmn; 0.4 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e14.5 \u0026plusmn; 0.1 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e110.8 \u0026plusmn; 1.5 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e310.3 \u0026plusmn; 4.5 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e55.3 \u0026plusmn; 0.3 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003eC6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e13.8 \u0026plusmn; 0.2 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e110.4 \u0026plusmn; 2.6 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e311.3 \u0026plusmn; 7.7 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e53.0 \u0026plusmn; 0.2 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e14.0 \u0026plusmn; 0.3 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e109.8 \u0026plusmn; 3.6 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e309.0 \u0026plusmn; 10.7 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e53.6 \u0026plusmn; 0.2 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e13.9 \u0026plusmn; 0.3 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e106.7 \u0026plusmn; 5.0 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e299.2 \u0026plusmn; 15.2 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e53.6 \u0026plusmn; 0.3 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003eM4.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU-G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e11.9 \u0026plusmn; 0.1 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e90.7 \u0026plusmn; 2.1 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e254.2 \u0026plusmn; 6.4 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e49.2 \u0026plusmn; 0.3 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e11.6 \u0026plusmn; 0.2 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e87.0 \u0026plusmn; 3.3 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e243.1 \u0026plusmn; 10.0 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e48.8 \u0026plusmn; 0.4 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e11.0 \u0026plusmn; 0.2 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e77.8 \u0026plusmn; 4.7 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e215.4 \u0026plusmn; 14.5 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e47.8 \u0026plusmn; 0.2 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9px;\"\u003e\n \u003cp\u003eM6.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e12.1 \u0026plusmn; 0.2 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e92.1 \u0026plusmn; 2.8 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e258.0 \u0026plusmn; 8.6 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e49.8 \u0026plusmn; 0.4 (pH 7.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e12.0 \u0026plusmn; 0.2 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e90.3 \u0026plusmn; 2.3 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e252.4 \u0026plusmn; 6.9 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e49.6 \u0026plusmn; 0.3 (pH 7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e11.4 \u0026plusmn; 0.1 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e83.1 \u0026plusmn; 2.4 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e230.9 \u0026plusmn; 7.4 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e48.7 \u0026plusmn; 0.5 (pH 6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA series of 17-nt oligonucleotides containing mnm\u003csup\u003e5\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eS\u003csup\u003e2\u003c/sup\u003eU, mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU (mnm\u003csup\u003e5\u003c/sup\u003eU*), and corresponding \u003cem\u003e5-un\u003c/em\u003esubstituted uridines U, S\u003csup\u003e2\u003c/sup\u003eU, Se\u003csup\u003e2\u003c/sup\u003eU (U*), was synthesized and hybridized with complementary RNA strands to form duplexes with (mnm\u003csup\u003e5\u003c/sup\u003e)U*-A and (mnm\u003csup\u003e5\u003c/sup\u003e)U*-G pairs. The synthesis of mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-RNA was carried out \u003cem\u003evia\u003c/em\u003e phosphoramidite chemistry according to the protocol developed in this study. The effects of mnm⁵ substitution and 2-chalcogen modifications were evaluated with respect to RNA duplex structure, thermodynamic stability and base-pairing specificity for adenosine over guanosine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was observed that all canonical U*-A and mnm\u003csup\u003e5\u003c/sup\u003eU*-A base pairs stabilize RNA duplexes more efficiently than the corresponding U*-G and mnm\u003csup\u003e5\u003c/sup\u003eU*-G mismatched pairs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe stabilizing contribution of chalcogens in duplexes containing (mnm\u003csup\u003e5\u003c/sup\u003e)U*-A pairs increases in the order: uridines \u0026lt; Se\u003csup\u003e2\u003c/sup\u003e-uridines \u0026lt; S\u003csup\u003e2\u003c/sup\u003e-uridines, with the mnm\u003csup\u003e5\u003c/sup\u003e substituent significantly reducing this stability. The effect of O\u003csup\u003e2\u003c/sup\u003e/S\u0026sup2;/Se\u0026sup2; chalcogens on the stability of (mnm⁵)U*-G duplexes is less pronounced, with the mnm⁵ substituent exerting a slight destabilizing effect. Despite the small differences in the \u0026Delta;G\u003csup\u003e0\u003c/sup\u003e values, the comparative analysis of all thermodynamic parameters indicates that selenouridines (Se\u003csup\u003e2\u003c/sup\u003eU and mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU) facilitate the formation of duplexes with opposing G more effectively than their O\u003csup\u003e2\u003c/sup\u003e- and S\u003csup\u003e2\u003c/sup\u003e- counterparts. This effect is likely attributed to the higher content of the ionized form of Se\u003csup\u003e2\u003c/sup\u003e-uridines, which preferentially recognize guanosine, while restrict interactions with A. In this aspect, pH-dependent thermal denaturation studies demonstrated the improved stability of duplexes containing Se\u003csup\u003e2\u003c/sup\u003eU-G and mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-G pairs at higher pH, where the content of the ionized state of selenouridines is elevated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we determined that the base-pairing specificity for adenosine over guanosine follows the order: uridines \u0026lt; Se\u0026sup2;-uridines \u0026lt; S\u0026sup2;-uridines. The presence of the mnm⁵ substituent reduces this specificity, reflecting a reduced affinity of mnm⁵U* for adenosine relative to their 5-\u003cem\u003eun\u003c/em\u003esubstituted (U*) counterparts. Overall, S\u0026sup2;-uridines exhibit a stronger affinity for adenosine than corresponding Se\u0026sup2;-uridines, indicating that Se\u0026sup2; substitution increases the propensity for guanosine pairing. In the biological context, our findings may suggest that the dynamic post-transcriptional conversion of wobble mnm⁵S\u0026sup2;U to mnm⁵Se\u0026sup2;U in bacterial tRNAs\u003csup\u003eLys,Glu\u003c/sup\u003e may serve as a regulatory mechanism of gene expression. Given that mnm⁵Se\u0026sup2;U displays a marked shift in base-pairing specificity toward guanosine, this modification could promote translation of mRNAs enriched in 3\u0026prime;-G-ending codons, thereby modulating protein synthesis under specific cellular conditions. This hypothesis is supported by \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003ebiological experiments on globin translation in rabbits, which demonstrated that 3\u0026apos;-G-ending codons are preferentially recognized by aminoacylated mnm\u003csup\u003e5\u003c/sup\u003eSe\u003csup\u003e2\u003c/sup\u003eU-tRNAs\u003csup\u003eLys,Glu\u003c/sup\u003e from \u003cem\u003eE. coli\u003c/em\u003e compared to their 2-thio counterparts\u003csup\u003e40\u003c/sup\u003e. Considering our data in the context of designing therapeutic nucleic acids, 2-thiouridine (S\u0026sup2;U) has the greatest potential for imparting drug-like properties, as it demonstrates the highest base-pairing specificity for adenosine over guanosine and forms the most thermodynamically stable S\u0026sup2;U\u0026ndash;A duplex among all systems examined in our study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank Dr Ewelina Wielgus (CMMS, PAS, Lodz) and Zofia Gdaniec\u0026rsquo; group (IChB, PAS, Poznan) for the mass spectrometry analysis and the access to the synthesizer, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants UMO-2018/29/B/ST5/02509 from National Science Centre to B.N. and W-3D/FMN/17G/2021 from Young Scientists\u0026rsquo; Fund at LUT to P.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm their contribution to the paper as follows: study conception and design: GL., BN., ES.; data collection: PK., KK., AD., KP., MS., TB., analysis and interpretation of results: GL., BN., PK., KK.; draft manuscript: GL., PK. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e accompanies this paper at \u0026hellip;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCappannini, A. et al. J. M. MODOMICS: a database of RNA modifications and related information. 2023 update. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e(D1), D239-D244 (2024). \u003c/li\u003e\n\u003cli\u003eDuchler, M., Leszczynska, G., Sochacka, E. \u0026amp; Nawrot, B. Nucleoside modifications in the regulation of gene expression: Focus on tRNA. \u003cem\u003eCell. Mol. 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Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity. \u003cem\u003eBiofactors\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 27\u0026ndash;34 (1989). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"5-substituted uridines, 2-thiouridines, 2-selenouridines, modified oligoribonucleotides, phosphoramidite solid-phase synthesis, thermal stability, circular dichroism","lastPublishedDoi":"10.21203/rs.3.rs-7980593/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7980593/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"5-Substituted uridines, 2-thiouridines and 2-selenouridines represent the most common wobble-positioned bacterial tRNA modifications, with the 5-methylaminomethyl (mnm5) substituent being particularly widespread. Their biological role in the precise recognition of synonymous purine-ending codons is still under investigation. Modified uridines are also known to enhance the stability and base-pairing specificity of therapeutic nucleic acids. However, a full understanding of the O2/S2/Se2 chalcogen effect, particularly in combination with the naturally occurring mnm⁵ substituent, remains limited. To address this, a systematic comparative study was conducted on the thermodynamic and structural contributions of mnm⁵ and 2-chalcogen modifications to RNA duplex properties. We found that chalcogens modulate the stability of duplexes containing opposing adenosine in the following order: uridines \u003c Se2-uridines \u003c S2-uridines, with the mnm⁵ substituent exerting a significantly destabilizing effect. In duplexes with opposing guanosine, the influence of chalcogens is less pronounced, whether alone or in combination with mnm5, however, Se2-uridines promote duplex formation more effectively than their 2-thio and 2-oxo counterparts. This effect is likely associated with their high ionization propensity, as we demonstrated by pH-dependent melting studies. Overall, the base-pairing specificity for adenosine over guanosine was found to follow the order: uridines \u003c Se2-uridines \u003c S2-uridines, with the mnm⁵ group significantly reducing this specificity. All studied RNA duplexes exhibited circular dichroism (CD) spectra characteristic of A-RNA double stranded helices. To afford the above data, the first chemical synthesis of an mnm⁵Se²U-modified RNA oligomer was developed.","manuscriptTitle":"From U to mnm⁵Se²U: tuning base pairing preferences through 2-chalcogen and 5-methylaminomethyl modifications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 12:59:48","doi":"10.21203/rs.3.rs-7980593/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-19T14:00:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T23:25:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T06:32:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-08T04:49:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T09:25:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T04:21:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322812365698936106409387360294270643485","date":"2025-11-06T01:24:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28413662054177347493992756242569738687","date":"2025-11-06T00:24:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148411484631696759194522039098853035218","date":"2025-11-05T23:38:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30696479626039990696060610006347765328","date":"2025-11-05T23:16:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61385848990321633085325920527167427324","date":"2025-11-05T20:10:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T16:57:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-03T11:24:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-31T01:44:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-31T01:43:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-29T13:45:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f5de599b-7362-4a8c-8249-a9f3e7b2f312","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58113035,"name":"Biological sciences/Biochemistry"},{"id":58113036,"name":"Biological sciences/Chemical biology"},{"id":58113037,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2026-01-05T16:04:22+00:00","versionOfRecord":{"articleIdentity":"rs-7980593","link":"https://doi.org/10.1038/s41598-025-34018-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-29 15:57:27","publishedOnDateReadable":"December 29th, 2025"},"versionCreatedAt":"2025-11-17 12:59:48","video":"","vorDoi":"10.1038/s41598-025-34018-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34018-y","workflowStages":[]},"version":"v1","identity":"rs-7980593","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7980593","identity":"rs-7980593","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}