2’FY-RNA aptamers form metastable multimeric G-quadruplexes that selectively bind pyoverdines

2’FY-RNA aptamers form metastable multimeric G-quadruplexes that selectively bind pyoverdines | 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 2’FY-RNA aptamers form metastable multimeric G-quadruplexes that selectively bind pyoverdines Sharif Anisuzzaman, Joshua Alterman, George Kraus, Marit Nilsen-Hamilton This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7885693/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Two 2’FY-RNA aptamers with distinct sequences were selected for specific binding to pyoverdine-Pf5 (PVD-Pf5), increasing chromophore fluorescence upon binding. They also recognized the peptide portion of pyoverdines, as shown by their differential specificity for related variants. Computational analysis and experimental data (NMM binding, CD spectra) identified G-quadruplex structures that were thermally metastable but reformed in the presence of PVD-Pf5. Further structural studies mainly with one aptamer revealed imino proton peaks in 1D H-NMR and pressure stability up to 2 kbar. Electrophoretic evidence identified dimeric G-quadruplexes formed by the 2’FY-RNA aptamers and their RNA equivalents. While cations were necessary for PVD-Pf5 binding, they were not required for G-quadruplex formation. Given the established role of G-quadruplexes as protein interaction sites, multimeric G-quadruplexes offer a potential framework for structure-based regulatory mechanisms in cellular RNAs. In addition to previously characterized multimeric G-quadruplexes, these aptamers contribute novel sequences that expand the repertoire of known multimeric G-quadruplexes. Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Chemical biology Physical sciences/Chemistry aptamer 2’FY-RNA2 pyoverdine5 G-quadruplex1 metastable Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Aptamers can exquisitely selective for their molecular target and are therefore being developed as possible therapeutic agents 1 , receptors for tissue and cell-specific drug targeting 2 , targeted protein degradation 3 and recognition elements on sensors 4 . The improving technology for introducing RNAs into cells is opening avenues for applying aptamers as drugs to inhibit intracellular functions 5 and for binding metabolites to alter metabolic flow 6 – 8 and drug intake 9 . As short but structured oligonucleotides, aptamers can also be informative for understanding nucleic acid structure and interactions between structural units. Folding patterns and binding strategies of aptamers can reveal principles that will help to decode the architecture and function of natural RNA and DNA systems, including riboswitches, ribozymes, and regulatory complexes. Modification of the ribose sugar with fluorine, as in 2’FY-RNA in which the pyrimidine-linked sugars are 2’F, confers the resulting molecules with more helical stability and nuclease resistance than their RNA equivalents 10 – 12 . These features are important for applications of RNAs in therapeutics or on sensors 13 . Pseudomonads are an important genus of bacteria with species that range from beneficial to deleterious to animal and plant health and that populate many habitats 14 . Fluorescent pseudomonads, produce a characteristic fluorescent pyoverdine, which is a siderophore that usually contains a 2,3-diamino-6,7-dihydroxyquinoline fluorophore to which is attached a polypeptide produced by a non-ribosomal synthetic pathway 15 . The polypeptides bind iron with affinities around 10 32 M 16 and enable these pseudomonad species to survive in hospitals where they are a serious threat to already compromised individuals and in the soil, where the released siderophores can protect plants from pathogens by mechanisms that include iron sequestration 17 . Each species produces unique siderophores with strain-specific peptide chains 18 . Here we describe the selection and structural analysis of 2’FY-RNA aptamers that recognize pyoverdine-Pf5 (PVD-Pf5), a unique product of Pseudomonas protegens , that signals the presence of this microbe. These aptamers bind the pyoverdine chromophore and structural elements of the peptides with different patterns of specificity for a range of pyoverdines and display properties consistent with high structural stability. Although their structural signatures appear independent of cations, pyoverdine binding has a complex dependence on cations. Both aptamers are thermally metastable and have spectral and other characteristics that are consistent with their forming multimeric G-quadruplexes. RESULTS Aptamer selection Aptamer selection was performed as described in Materials and Methods (Fig. 1A, B). This protocol was designed to isolate aptamers with the characteristics of high affinities and specificities for the ligand and for switching structure on binding the ligand. In addition, the presence of complementary oligonucleotides to the PCR primer sequences during selection limited structure selection to the single-stranded central randomized region. Aptamer selection started with a high molar ratio of pool to PVD-Pf5 (1:1) and a harmonic increase of pool to target PVD-Pf5 in later cycles to increase the selection pressure 19 . Counter selections were performed after rounds 3 and 6 against a mixture of two other siderophores, enterobactin (ENB) and ornibactin (ORB), to eliminate nonspecific binders (Fig. 1A). With the assumption that the oligonucleotide complex with PVD-Pf5 is stoichiometric, this selection protocol resulted in 60% of the round 9 oligonucleotide pool released by PVD-Pf5 (Fig. 1C). Analysis of the NextGen sequencing results showed that incremental rounds of selection were characterized by increases in the unique fraction (number of sequences in the pool divided by the pool size) and the enriched species (sequences present in the pool more than once), which is characteristic of a successful selection procedure (Fig. S1). The results of next generation sequencing for oligonucleotide pools from several rounds, including the final round of each selection provided the data for identifying potential aptamer sequences. Sequence clusters were identified with Aptasuite 20 and, based on the size of the cluster and the rise in enriched species through the rounds, several oligonucleotides were evaluated for binding the PVD-Pf5 chromophore by identifying increases in pyoverdine fluorescence when bound to the oligonucleotide. This assay tested for the ability of the aptamers to bind the chromophore, altering its structure and inducing fluorescence in a similar manner as observed for light-up aptamers such as the malachite green aptamer, Spinach, Broccoli and Mango 21 . From this analysis, two oligonucleotides were identified as potential aptamers for further investigation, which was performed using the sequences of the selected oligonucleotides stripped of their 5’ and 3’ PCR primer binding sites. These aptamers were named as 58PYO1A and 57PYO3A, according to their length and target with a unique number followed by A to identify the oligonucleotide as an aptamer. Their binding isotherms, monitored by the fluorescence change of PVD-Pf5, resulted in similar estimated K d s of 66 ± 42 nM and 98 ± 35 nM for 58PYO1A and 57PYO3A respectively (Fig. 1D) despite their differences in primary sequence (Fig. 1E). Specificity of the PVD-Pf5 binding aptamers To investigate features of the chromophore that are recognized by the aptamers, we synthesized the PVD-Pf5 chromophore and variations (Fig. 2A,B) and tested the abilities of the aptamers to increase their fluorescence (Fig. 2C). Compound 1, an intermediate for chromophore synthesis that lacks the third ring, did not bind either aptamer. Some limited evidence of binding was seen for compound 2, with a 3-ring chromophore structure and an amino group attached to the carbon position to which the peptide is attached in PVD-Pf5. The best evidence of 2’FY-RNA aptamer binding to the isolated chromophore was with compound 3, which has a carboxyl at the position that is linked by an acyl group to the peptide in PVD-Pf5. In compound 4 the acyl group is extended by two carbons, a structure that does not match that in PVD-Pf5, and fluorescence assay showed that aptamer does not bind this compound. These results suggest that the aptamers bind the chromophore in a way that is highly specific for the nature of the chemical features linked to the 1H-pyrimido[1,2-a] quinoline ring at position 5 (Chem Abstracts numbering) but that more than the chromophore is necessary for tight binding of PVD-Pf5. We tested the abilities of the aptamers to bind the two siderophores, enterobactin and ornibactin, that were used for counterselection during SELEX (Fig. 1A). The results showed that neither enterobactin nor ornibactin competed for PVD-Pf5 binding to the aptamer (Fig. 3A,B). As each fluorescent pseudomonas species synthesizes a pyoverdine with the same chromophore but different peptide moieties, we also explored the specificity of the two aptamers across a panel of five pyoverdines with the same chromophore from three different species (Fig. 3C,D). The two aptamers displayed different specificities across this panel, which suggested structural differences between the aptamers. Structural features of PVD-Pf5 aptamers To assess possible structural features of the aptamers that might be responsible for their similar binding affinities and different specificities, a motif search was performed on the clusters from which the aptamers were chosen using the MEME suite (https://meme-suite.org/meme/tools/meme) 22 . From this we identified a G-rich structural motif (Fig. 4A). The identified motif suggested the presence of G-quartets. The PENGUINN neural network (https://ml-bioinfo-ceitec.github.io/penguinn/) scores 23 identified the possible presence of G quartets (Fig. 4B), which were also predicted by 3dRNA/DNA (http://bio-phy.hust.edu.cn/new/3dRNA/create) to be formed by both aptamers (Fig. S3). To test for the presence of G-quadruplexes, the aptamers were incubated with N-methyl mesoporphyrin IX (NMM), which selectively binds to G-quadruplexes as demonstrated by its increased fluorescence 24 . Both aptamers increased NMM fluorescence at 610 nm and 670 nm at intensities equivalent to Spinach2 and Broccoli, which are confirmed G-quadruplexes (Fig. 4C). Circular dichroism spectroscopy provided additional evidence of the presence of G-quadruplexes (Fig. 4D) and identified the aptamers as forming parallel G-quadruplexes, which are characterized by a positive peak around 264 nm and a weaker negative peak at 240 nm 25 . The spectra were not different in the presence and absence of PVD-Pf5 for either aptamer (Fig. 4D). Thus, both aptamers appear to form G-quadruplexes that are not substantially reorganized upon binding PVD-Pf5, which suggests that the structural change by which these aptamers were removed from the capture oligonucleotide might not have been large. A 1D 1H-NMR study of 57PYO3A did not reveal well-resolved peaks in the imino region where the G-G Hoogsteen base pairing typical of G-quartets can be identified (Fig. 4E). However, the presence of peaks in this chemical shift range is consistent with the CD spectra and NMM binding results and with a G-quadruplex structure among others. Increasing the pressure to 2 kbar had little effect on the NMR spectrum in this region (Fig. 4E) and the spectrum taken after dropping the pressure to 1 bar (1 bar end in Fig. 4E) was identical to the starting spectrum (1 bar start) before raising the pressure to 2 kbar. This suggests that the aptamer structure is very stable, which is another feature of G-quadruplexes. To address the question of how these aptamers could form G-quadruplexes when they lack the canonical G tracts seen in many G quadruplexes and do not have sufficient appropriately distributed Gs to create 3 G-quartets, we considered the possibility that, like Oxy-1.5 from the Oxytricha nova, 58PYO1A and 57PYO3A form dimeric G-quadruplexes 26 . This question was tested by non-denaturing gel electrophoresis by which we observed evidence of dimeric forms of the aptamers (Fig. 4F). Similar evidence of dimerization was observed with the RNA equivalents of these aptamers (Fig. 4F). An additional feature of multimeric G quadruplexes is that they bind HPTA-1, increasing its fluorescence. Both aptamers increased the fluorescence of HPTA-1 whereas several other parallel and antiparallel G-quadruplex aptamers not known to form multimeric structures did not bind HPTA-1 (Fig. 4G). The increased fluorescence of HPTA-1 was manifested differently by the two aptamers with 58PYO1A causing a decided blue shift and 57PYO1A causing a slight red shift in the spectrum 58PYO3A (Fig. S4). This result suggests that the multimeric structures of the two aptamers differ around the binding site of HPTA-1, which is believed to be at the interface of the monomers. Folding dynamics The aptamer selection protocol involved annealing of complements to the 3’ and 5’ PCR primer sequences and thus did not include a refolding step that is often used to promote aptamers to fold to a homogeneous tertiary structure. G-quadruplexes are also reported to unfold and refold slowly. Therefore, we tested the outcomes of several variations of the unfolding and refolding procedure with prolonged heating and annealing periods. These included heating at 95 °C for 3 h followed by slow cooling air for 18 h and heating to 95 °C followed by slow cooling at a rate of 1 °C per minute. None of these protocols resulted in aptamers capable of binding PVD-Pf5 (Fig. 5A). The CD spectra of the refolded aptamers were also different from the untreated aptamers (Fig. 5B) whether refolded in SSMB1 salts solution or in water (Fig. 5C). As the 2’FY-RNAs are usually frozen directly after synthesis then thawed and analyzed for folding, we tested for the effect of freeze-thaw on the CD spectrum and found the same spectra and apparent size in freshly prepared samples as after freeze/thaw (Fig. S5). Thus, it appears that the G-quadruplex folds co-transcriptionally and, if denatured post-synthesis, these aptamers do not refold to form functional structures. If PVD-Pf5 was present during the refolding, the refolded aptamers showed increased fluorescence with pyoverdine indicative of pyoverdine binding to the aptamer (Fig. 5D). The quantitative increase in PVD-Pf5 fluorescence with the addition of 58PYO1A was the same for both conditions (refolded or not refolded). However, the refolding procedure resulted in an increase in fluorescence of pyoverdine that could be reproduced in the absence of the aptamer. That heating changed the pyoverdine to a different structural form was confirmed by the observation that the preheated pyoverdine (PVD-Pf5*) does not increase in fluorescence in the presence of aptamers that have not been refolded (Fig. 5D). This result can be interpreted as PVD-Pf5* does not bind the aptamers or that binding occurs but is not associated with a further increase in the fluorescence of PVD-Pf5*. To further assess if refolding in the presence of pyoverdine resulted in the formation of a G-quadruplex-like structure, we evaluated the ability of 58PYO1A to bind NMM (Fig. 5E). The control aptamer that had not experienced a refolding protocol of heating and cooling bound NMM (Fig. 5E, red bars). Fluorescence from NMM was decreased in the presence of PVD-Pf5 for the control condition suggesting competition between the two for binding (Fig. 5E, red hatched bars). NMM fluorescence was also much lower in the presence of the aptamer refolded in the presence of PVD-Pf5 and there was no change in NMM fluorescence with the addition of PVD-Pf5 (Fig. 5E, green bars). The CD spectrum was more informative with respect to probable structure as the aptamers refolded in the presence of PVD-Pf5 appeared to be well-formed G-quadruplexes (Fig. 5F). Thus, it appears that, on refolding, the aptamers become trapped in a folding energy minimum that is overcome if folded in the presence of PVD-Pf5. Salt requirements for ligand binding and aptamer structure G-quadruplex structures are often stabilized by the presence of cations, which take a central position in the structure, coordinating between G-quartets. To determine the cation requirement of the 2’FY aptamers for ligand binding, we tested the change in fluorescence of pyoverdine in the presence and absence of the SSMB1 salts. The increase in fluorescence was observed in the presence but not absence of the salt mix (Fig. 6A). Attempts to identify the required salt(s) for binding demonstrated that a single salt component of SSMB1 could not replace the salt mix in promoting PVD-Pf5 binding and none of the combinations tested promoted the same increase in binding as SSMB1 (Fig. S6). To investigate the role of salts in formation of the aptamer structures, we compared the NMM fluorescence of each aptamer in water and SSMB1. Whereas there was no difference in NMM binding to either aptamer in the presence or absence of SSMB1 (Fig. 6B), NMM fluorescence with the Spinach2, the thrombin aptamer, and less so with the Broccoli aptamer, were increased in the presence of SSMB1 (Fig. 6B). Analysis of the CD spectra of 58PYO1A or 57PYO3A also showed no difference in the presence of water or SSMB1 (Fig. 6C). We examined the effects of selected cations present in SSMB1 that have been identified as stabilizing other G-quadruplex structure and their combinations (Fig. 6D) and again observed no significant effects of the cations K + , Na + and Mg 2+ or their combinations on G-quadruplex structure as assessed by NMM fluorescence. These results show that 58PYO1A and 57PYO3A form structures with features of G-quadruplexes in the absence of monovalent or divalent cations but that a complex combination of cations is required for binding the PVD-Pf5. DISCUSSION From a single SELEX experiment to isolate 2’FY-RNA aptamers that are specific for the pyoverdine, PVD-Pf5, we identified two aptamers that we have analyzed for structure and function. Although the protocol for isolating the aptamers was designed to isolate structure-switching aptamers, this property has been elusive. Otherwise, the properties of the two aptamers that we chose based on evidence of PVD-Pf5 binding, are similar in their PVD-Pf5 binding isotherms, specificity for PVD-Pf5 over ENB, ORB and PSB, and features consistent with G-quadruplex structures. The only difference identified was the red vs. blue shift in HPTA-1 fluorescence spectrum when bound to the aptamers. Such similarity of features is unexpected for two aptamers with different sequences (only 19% identical in the randomized regions) and are proposed to fold into different three-dimensional structures. Specificity and high affinities are important characteristics of aptamers intended for applications in biotechnology or medicine. To bias the selection towards aptamers that bind pyoverdines and no other common siderophores, two other siderophores, ENB and ORB, were used as counter selection targets. ENB is released by Enterobacter during iron stress whereas ORB is released by soil dwelling Burkholderia species 27 , 28 . We deliberately did not use a related pyoverdine as a counter-selection target because we were interested in the possibility that we might obtain an aptamer that binds all pyoverdines and could thus sense the presence of any Pseudomonad . Each Pseudomonad species produces a structurally unique siderophore that allows them to selectively remove iron from the environment and avoid competition from other organisms. Many of these siderophores contain the same chromophore. Although similar in many aspects including that they bind the common chromophore for many pyoverdines, these aptamers display different specificity profiles when compared against a panel of different siderophores. This observation and the relatively poor binding to the isolated chromophore support the hypothesis that these aptamers recognize the combination of chromophore and side chain. The SELEX experiment design included the use of oligonucleotides complementary to the primer regions of the library to exclude these regions from involvement in forming single-stranded structural motifs. This design would focus selection pressure on the randomized region of the library, streamline the informatics search by minimizing the length of sequence to be analyzed, and provide an opportunity to track many aptamers in screening experiments by using a single labeled complementary oligonucleotide that could be hybridized with the primer landing site of all aptamers. These outcomes were realized in the selected pool. Although predicted to form G-quartets by two computational tools, neither aptamer displays a canonical sequence expected of a series of G-quartets that could make up a G-quadruplex although each has enough Gs to contribute to two (PYO1A) or 3 (PYO3A) G-quartets. Several DNA G-quadruplexes consist of only 3 G-quartets such as the c-MYC and c-KIT promoter quadruplexes (PDB IDs: 1XAV and 2O3M, respectively) 29 , 30 , whereas the 15-mer thrombin binding aptamer (TBA, PDB1HAI) consists of only two G-quartets 31 . Also, TERRA, an RNA G-quadruplex, consists of three G-quartets 32 , 33 . Smaller nucleic acids also form multimeric G-quadruplexes 34 , 26 , 35 and our data suggests that 58PYO1A and 57PYO3A are in this category of G-quadruplex. In addition the observation that NMM competes with PVD-Pf5 suggests that, like NMM that sits on the outer G-quartet of the quadruplex 24 , the pyoverdine chromophore may also occupy this space on the aptamers. A complex combination of cations seems to be required for ligand binding, but we found no cation requirement for structure as evaluated by CD spectra and NMM binding. As an alternative to cations, long-lived water molecules were found embedded in the bimolecular d(G 3CT4G3C)2 G-quadruplex 36 . Structured water has also been found in some G-quadruplexes (PDB id 3CE5, 3NZ7, and 3UYH) 37 – 39 and demonstrated to be important for G-quadruplex-ligand interaction 37 , 36 . With embedded water and no cation, the central spaces of these aptamers might be dense. To test the density of these aptamers and their structural stability we evaluated the pressure required for structural collapse using the NMR spectrum as the readout. DNA quadruplexes usually compress in the range 1-1.5 kbar, which varies depending on the oligonucleotide sequence and the stabilizing monovalent cation 40 – 44 . RNA G-quadruplexes are more dense 45 , but still compress over the range 1–2 kbar 46 . Although the NMR spectrum of the 57PYO3A aptamer was not sufficiently resolved for assigning positions, a salient feature was that the spectra at 1 bar and 2 kbar were very similar in the imino region and showed no decrease in intensity with increased pressure. These results suggest that the aptamer structure is more stable than the DNA and RNA G-quadruplexes so far evaluated in this manner. The broad peaks in the imino-region spectrum of 57PYO3A also suggest that it may exist as an ensemble of related structures that could be in slow equilibrium. Like the multimeric quadruplexes formed by the TG 4 oligonucleotide derived from the Oxytricha nova telomeric sequence 47 and the pG 5 oligonucleotide 48 , 49 , 58PYO1A and 57PYO3A are thermally metastable reverting to alternate structures on cooling. Both aptamers can be guided by the presence of PVD-Pf5 to refold after heating to a G-quadruplex structure that binds PVD-Pf5. Although we have not yet determined the alternative structures, they might be the hairpins predicted for both sequences by RNAfold (Fig. S3). HMGB1-targeting G-quadruplex aptamers are metastable G-quadruplexes that can revert to hairpins 50 . A transition between hairpin and G-quadruplex was also reported to influence the rate of E. coli EutE mRNA translation 51 . Metastable multimeric G-quadruplexes may play important roles in nature. For example metastable G-quadruplexes have recently been proposed as features of ALS/FTD-associated nucleotide expansions 52 . Most natural RNA G4 sequences are reported not to fold as stable monomeric G-quadruplexes but required dimerization 53 . Multimeric G-quadruplexes are also proposed to play a role in mRNA aggregation during stress 53 . Cations and folding kinetics can also influence the ultimate G-quadruplex structure as shown for d(G4T4G3) quadruplexes for which conformational transitions are kinetically governed and influenced by K + concentrations 54,55 . These observations open possibilities of interventions that could influence G-quadruplex folding and multimerization for therapeutics 56 In summary, we have evaluated the features of two aptamers that were selected to bind PVD-Pf5 by a structure-switching protocol. Despite their different primary sequences and predicted tertiary structures, these aptamers were found to have similar structural features but vary in their target specificity profiles. They form multimeric metastable G-quadruplexes for which one was demonstrated stable to high pressure. Metastability and multimerization are well-documented properties of several telomeric DNAs and RNA G-quadruplexes and may be the predominant forms of RNA quadruplexes in vivo 34 , 26 , 35 . The inclusion of pyoverdine aptamers in this category broadens the repertoire of sequences capable of forming metastable multimeric G-quadruplexes. Given the emerging significance of G-quadruplex structures in mRNAs and long noncoding RNAs 57 , these aptamers, may reveal important structural features for regulating G-quadruplex structure and function that might also be employed in regulating RNA-based cellular processes. MATERIALS AND METHODS Reagents All chemicals were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Hampton, NH, USA) unless otherwise stated. N-methyl mesoporphyin IX (NMM, cat# sc-396879) was purchased from Santa Cruz Biotechnology. PVD-Pf5 (cat# PVD-Pf5), hydrazinyl pyrrolidine triazine amine (HPTA-1, cat# 23848) was from Glixx laboratories (Hopkinton, MA, USA). Enterobactin (cat# FE-ENB), and ornibactin (cat# ORNIB) were purchased from EMC microcollections (Tübingen, Germany). Pseudobactin (cat# 8374) isolated from Pseudomonas fluorescens was purchased from Sigma Aldrich. All the siderophore preparations were iron-loaded. Buffers used for this work were: 1) Buffer A: 40 mM HEPES, 125 mM KCl, 5 mM MgCl 2 , pH 7.4, 2) PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 5 mM MgCl 2 , pH 7.4, 3) SSMA2: 366 µM CaCl 2 , 3 µM CuSO 4 .5H 2 0, 5 µM MnSO 4 .H 2 0, 27 µM KI, 83 µM KH 2 PO 4 , 35 µM KOH, 1 µM ZnSO 4 .7H 2 0, 103 µM Fe (III)Na-EDTA, 68 µM MgSO 4 , 23 µM Mg (NO 3 ) 2 .6H 2 0, 94 µM MgCl 2 .6H 2 0, 64 µM NaCl, 75 µM NH 4 OAc, pH 5.9, and 4) SSMB1: SSMA2, 10 mM KCl, 1 µM H 3 BO 3, pH 5.9. Synthesis of pyoverdine chromophores Compound 1 was synthesized starting from nitro lactam A by reaction with thionyl chloride in DMF at 98°C. The resulting chloroquinoline was treated with the azetidine ester and excess triethylamine in DMF at 92°C to generate 1 . Compound 2 was prepared by treatment of 1 with HI. Compound 3 was synthesized by treatment of 2 with excess acetic anhydride. Compound 4 was synthesized starting from nitro lactam A by reaction with ethyl bromoacetate, DMF, and sodium hydride. Although a mixture of N-alkylated and O-alkylated material was produced, the desired N-alkylated product was easily separated. The nitro group was reduced using excess iron and aqueous HCl in ethanol. The resulting amine was acylated with succinic anhydride to provide amide ester acid 4 . More details regarding the synthesis (Supplemental section 1), absorbance spectra (Fig. S1 ) are part of the Supplemental materials. Nucleic acids and SELEX library design All DNAs used in this study were synthesized by Integrated DNA Technology (IDT, Coralville, IA, USA). 2’FY-RNAs were synthesized as described under 2’ FY-RNA synthesis and purification. Table S1 lists all nucleic acids, and their sequences described in this work. All chemically modified oligonucleotides were purified using HPLC whereas unmodified oligonucleotide sequences were purified by standard desalting unless otherwise stated. A single stranded DNA library was used (IDT, Coralville, IA) with each oligonucleotide having a length of 100 bases, which includes three constant regions and two random regions: 5'-GCCTGTTGTGAGCCTCCTGTCGAA (10N) GGAGCGAATG (35N) GAGCGTTTATTCTTGTCTCCC-3’. In this library, N represents an equimolar combination of A, T, C and G. The 5’ and 3’ ends of the constant regions were primer binding sites for PCR amplification and the 10 base long constant region between 10N and 35N was used to capture the 2’ FY-RNA library components during selection. Nucleic Acid synthesis Conversion of ssDNA SELEX library to dsDNA The ssDNA pool was converted to the dsDNA template for in vitro transcription (IVT) by primer extension from oligonucleotide 5627. Each reaction contained 1.7 µM ssDNA pool, 3.3 µM oligonucleotide 5627 (primer), 0.2 mM dNTP mix, 0.05 U/µL DNA Taq Polymerase (GenScript, Piscataway NJ, USA, cat# E00007), 50 mM KCl, 10 mM Tris-HCl (pH 8.55), 1.5 mM MgCl 2 , 0.1% TritonX-100. The mixture was incubated at 94°C for 5 min, 65°C, for 15 min, then 72°C for 99 min using a thermocycler (Quant Studio 3, Thermofisher, Waltham, MA, USA). The generated dsDNA was resolved through a 2% agarose gel and purified using a PCR cleanup column from Syd labs (Hopkinton, MA, USA). The dsDNA was quantified based on its uv/vis absorption spectrum (Nanodrop, Thermofisher). 2’ FY-RNA and RNA synthesis and purification Reverse transcription was performed using Superscript IV reverse transcriptase (Thermofisher, cat# 18090050) and PCR to generate the template for in vitro transcription was done using Taq DNA polymerase (GenScript, Piscataway NJ, USA, cat# E00007) 2’ FY-RNA was prepared by in vitro transcription using a Durascribe T7 transcription kit from (Epicentre, Madison WI, USA, cat# DS010925) or with recombinant His-tagged T7(Y639F/H784A) RNA polymerase purified from transformed XL-1 Blue E. coli by Ni-NTA affinity resin (eluted with 250 mM imidazole). Around 1.6 nmoles dsDNA from the extended pool was incubated with 5 mM ATP, 5 mM GTP, 5 mM 2’FY-UTP, 5 mM 2’FY-CTP, 5 mM dithiothreitol, 5% DMSO, 0.8 µg/µL T7(Y639F/H784A) RNA polymerase, 40 mM Tris-HCl, 25 mM MgCl 2 , 2.5mM spermidine, 0.01% Triton X-100, pH 7.8, at 37 or 42°C for 12 h then digested with 1 MBU DNAse I for 15 min. RNA was prepared from templates created by PCR amplification or by annealing complementary oligonucleotides using the Hyperscribe T7 RNA synthesis kit (Apexbio, Houston, Texas, cat# K1047). To separate the residual NTPs and abortive transcripts from the desired transcript, the 2’ FY-RNA or RNA was resolved through an 8% (19:1 acrylamide:bisacrylamide) gel containing 7 M urea with TBE (100 mM Tris, 100 mM Borate, 1 mM EDTA, pH 9) as running buffer. The 2’FY-RNA or RNA was eluted by crushing the section of gel containing the RNA and incubating with TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 18 h at 37°C followed by removing the gel by centrifugation. SELEX: In vitro selection of aptamers for PVD-Pf5 SELEX was performed with PVD-Pf5 as target to select 2’FY-RNA aptamers. For the first round of selection a library of 10 15 molecules of 2’ FY-RNA was used. The biotinylated capture oligonucleotides, which are complementary to the short constant region in the library sequence, were attached to streptavidin coated magnetic beads. The 2’FY-RNA libraries were first hybridized with oligonucleotides 5625 and 5617 (complementary to the PCR primer regions), then immobilized on the beads by hybridization with the capture oligonucleotide. The length of the capture oligonucleotide varied with the SELEX round as did the hybridization temperature and the oligonucleotide:PVD-Pf5 molar ratio (Table S1 ). The bound oligonucleotides were captured using a magnet and purified by ethanol precipitation in the presence of 0.13 µg/mL linear polyacrylamide (LPA, Sigma-Aldrich). Purified 2’FY-RNA was reverse transcribed and amplified by low fidelity Taq polymerase (cat#: E00007; Genscript, Piscataway, NJ, USA). The ratio of 2’FY-RNA to PVD-Pf5 was reduced and the length of capture oligonucleotide increased in successive SELEX rounds to increase selection pressure (Table S2). Nine rounds of selection were performed, and two sequential rounds of counter selection were done after rounds 3 and 6. The targets for counter selection were a combination of enterobactin and ornibactin each at a 1:1 molar ratio with the oligonucleotide library. All targets used in selection and counter-selection were the Fe 3+ -bound forms. The buffer used during selection was SSMA2. PCR-amplified libraries were prepared for NGS using Nextera Illumina sequencing primers and the appropriate bar coding. Next generation sequencing was performed by the Novogene Facility (Sacramento, California, USA) using HiSeq PE150 platform. Bioinformatics analysis and data preprocessing of SELEX results The raw NGS data was demultiplexed and assigned to respective rounds by open-source software AptaSuite 20 . Barcodes were used to assign DNA pools from different rounds of selection. A zero-nucleotide mismatch between the reference and matched barcodes, and two mismatches between the reference and matched primers were criteria for demultiplexing. Further analysis was done with the sequences that met this standard. Clustering was done with 10 iterations of locality sensitive hashing, sampling 55% of the indices in the randomized regions and allowing no more than 10 nucleotide mismatches between the seed sequence of a cluster to each remaining member. Clusters were extracted for further analysis and aptamer screening. MEME was run with version 5.4.1 using default parameters whereas secondary structures were predicted using Mfold version 3.6 with standard parameters in the RNA mode 22 . The probability of G quadruplex formation was analyzed by the neural network PENGUINN 23 and QGRS mapper 58 . Tertiary structures of RNAs were predicted by 3dRNA/RNA 59 . Fluorescence spectroscopy All assays were performed at 23–24°C in SSMB1 unless otherwise stated. Pyoverdine binding assay Fluorescence emissions by the pyoverdine chromophore were measured by a Cary eclipse fluorimeter (Varian, Palo Alto, CA, USA) and a Synergy 2 (Biotek, Winooski, VT, USA) plate reader. Ten nM PVD-Pf5 was incubated with various concentrations of 2’ FY-RNA up to 1 µM at 23°C for 30 minutes in SSMB1. Fluorescence intensities were measured with excitation at λ ex = 400 nm and λ em = 460 nm with 5 nm slit widths. The dissociation constants (K d ) of the aptamer-ligand complexes were determined from the fluorescence output as a function of aptamer concentration. Nucleic acid binding to N methyl mesoporphyrin IX Two µM each of aptamer and N methyl mesoporphyrin IX (NMM) from Santa Cruz Biotechnology Inc. (Dallas, TX, USA, cat# 396879) in SSMB1 were incubated for 10 minutes at 23°C. Fluorescence was measured at λ ex = 399 nm, λ em = 410–700 nm, slit = 5 nm PMT + 800V using a Cary Eclipse fluorimeter (Agilent, Santa Clara, CA, USA). Pyoverdine binding and fluorescence competition assay PVD-Pf5 was incubated with aptamers in SSMB1 for 30 minutes at 23°C. For competition assays, a 100-fold to 1000-fold excess of ENB or ferric ORB was included, and the samples were incubated at 23°C for another 30 minutes. Fluorescence was measured at λ ex = 400 nm, λ em = 411–600 nm, slit = 5nm PMT + 800V using a Cary eclipse fluorimeter. Circular dichroism spectroscopy Two µM each of aptamer and PVD-Pf5 were incubated for 20 min in SSMB1 then evaluated for circular dichroism using an MOS-500 monochromator, an ALX-250 light source (Bio-Logic Science Instruments, Seyssinet-Pariset, France), and a quartz cell (pathlength 1 mm). Each spectrum was determined from the average of three accumulated scans from 220 to 320 nm from which the baseline (buffer alone) was subtracted. Two or more independently obtained spectra were averaged for many of the spectra shown in figures. NMR NMR spectra were collected on Bruker Avance III HD 800 MHz Spectrometer and pressure applied using Daedalus Xtreme-60 High Pressure NMR accessory at Iowa State University, Ames, USA. Two hundred and fifty-six scans were performed for each spectrum and mixing time was 200 ms. The internal standard for the chemical shift was DSS (2,2-dimethyl-2-silapentane-5-sulfonate). NMR spectra were collected in H 2 O/D 2 O (95%/5%) and the aptamer concentration was 0.5 mM. The NMR data was processed using Topspin. Analysis of binding isotherms, spectra and statistical evaluations The binding data was fit to Hill’s equation Hill n: \(\:B={B}_{min}+\frac{{B}_{max}{L}^{n}}{{L}^{n}+{K}_{d}^{n}}\) with the Hill n set to 1 and Sigmaplot was used to obtain statistical parameters for binding isotherms. Reported values for Kd passed the Normality (Shapiro-Wilk) and the Constant Variance (Spearman Rank Correlation) tests. Except for the standard deviations of the fit calculated by Sigmaplot, the standard errors of the mean (SEM) were calculated for all data due to variations in the numbers of samples contributing to each average value. When a background was subtracted, these were calculated to include the errors in the average values in the blanks by the formula: \(\:SEM=\:\sqrt{{Se}^{2}+{Be}^{2}}\) where Se = SEM of the sample and Be = SEM of the subtracted blank. Spectra (fluorescent and CD) were smoothed by simple exponential smoothing. Raw and smoothed spectra for all data shown in the figures are found in the Supplemental materials (Fig. S7-S12). Declarations Data availability The datasets generated and analyzed for this study are available from the authors on reasonable request. Next gen sequencing data are available through the following links: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE224685 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM7029482 Competing Interests Statement MNH owns Aptalogic Inc. MNH is a member of the editorial board of Scientific Reports. Author Contributions Author contributions were as follows: MNH and SA participated in conceptualization, data analysis and writing the original draft and revisions. SA designed all experiments with aptamers and collected and performed preliminary analyses. JA and GAK developed the chemical protocols, synthesized and validated the chromophore derivatives, and prepared the figure showing the protocols and chemical structures. MNH prepared the remaining figures, secured funding, and administered the project. All authors have read and agree to the published version of the manuscript. Funding This research was supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) through the Ames Laboratory. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. DE-AC02-07CH11358. Partial support of Dr. Anisuzzaman and supplies to complete the project were provided by Aptalogic Inc. Acknowledgments We gratefully acknowledge the contributions of Lee Bendickson for help with NextGen sequence analysis, Soma Banerjee for identifying the effect on aptamer function of the folding protocol, and Jeffrey Purslow for assistance in obtaining and analyzing the NMR spectra. References Adrian, M., Heddi, B., and Phan, A.T. NMR spectroscopy of G-quadruplexes. Methods 57, 11-24 10.1016/j.ymeth.2012.05.003 (2012). 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15:12:15","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164882,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/8139fd833417887f2c916bcb.html"},{"id":95127153,"identity":"375469d3-bcb3-4f70-9b6c-ec43ea30f24d","added_by":"auto","created_at":"2025-11-04 15:12:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":522480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSELEX design and selected aptamers. (a) \u003c/strong\u003eFlow diagram for SELEX protocol for PYO-Pf5, \u003cstrong\u003e(b)\u003c/strong\u003eDesign of the SELEX library (N10 and N35 are random regions, additional 10nt constant region binds to capture oligo (CO), Oligo P1 and P2 are primer blocking oligonucleotides, \u003cstrong\u003e(c) \u003c/strong\u003eThe fraction maximum release with the assumption of a 1:1 stoichiometry. Full release (1.0) is the release of oligonucleotides at a 1:1 molar ratio to the pyoverdine presented to the captured oligos, \u003cstrong\u003e(d)\u003c/strong\u003e Binding isotherms for the two chosen aptamers. The fitted lines were calculated by the Hill’s equation with the restriction of n=1. The data shown for 58PYO1A and 57PYO3A are the averages of 4 and 3 independently performed experiments respectively, \u003cstrong\u003e(e)\u003c/strong\u003e Sequences of 58PYO1A and 57PYO3A. The bolded letters are in identical positions in both aptamers. The red sequence is the complement to the capture oligonucleotide (CO). The green dots identify the positions of the Gs in each sequence.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/d536cbc822c9897609fc3230.png"},{"id":95226151,"identity":"4c0b483b-043a-4d22-a14e-8b4cc515720d","added_by":"auto","created_at":"2025-11-05 16:26:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":319988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe PVD chromophore as an aptamer target. (a) \u003c/strong\u003eSynthesis of pyoverdine chromophore and related compounds \u003cstrong\u003e(b) \u003c/strong\u003eStructure of PVD-Pf5, \u003cstrong\u003e(c) \u003c/strong\u003eChange in fluorescence (λ\u003csup\u003eex\u003c/sup\u003e = 310 or 360nm,\u0026nbsp; λ\u003csup\u003eem \u003c/sup\u003e= 321 or 371nm) due to aptamer presence. λ\u003csup\u003eex\u003c/sup\u003e and\u0026nbsp; λ\u003csup\u003eem \u003c/sup\u003ewere chosen according to the absorption spectrum for each chromophore shown in Fig. S2. 58PYO1A was tested in both the 2’FY-RNA and RNA forms and 57PYO3A was tested in the RNA form. The number of independent estimates contributing to each value is shown above the relevant bar.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/4bfb4aad3bd9eb60af926d30.png"},{"id":95127155,"identity":"6db1d02f-c294-4e8a-be5e-8602c2f0a617","added_by":"auto","created_at":"2025-11-04 15:12:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":350640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAptamer specificity. (a) \u003c/strong\u003eTo determine if ENB or ORB compete with the aptamers for binding PVD-Pf5, 100\u003cstrong\u003e \u003c/strong\u003enM 57PYO3A and 7 µM PVD-Pf5 were incubated in SSMB1 with competitors, ENB and ORB, at 140:1 (competitor:PVD-Pf5) and a fluorescence spectrum obtained. This data is representative of the results of two independently performed experiments, \u003cstrong\u003e(b) \u003c/strong\u003ePeak fluorescence when 58PYO1A (blue) or 57PYO3A (red) bind to PVD-Pf5 (empty bars) and when they bind PVD-Pf5 in the presence of ENB (right angle hatched) or ORB (left angle hatched bars). Each spectrum is the average o independently collected spectra.\u003cstrong\u003e (c,d) \u003c/strong\u003eFluorescence changes when pyoverdines from \u003cem\u003eP. Protegens \u003c/em\u003ePf5 (pf5), \u003cem\u003eP. aeruginosa \u003c/em\u003eATCC 27853 (Pa1), \u003cem\u003eP. aeruginosa \u003c/em\u003eATCC 15692(Pa2), \u003cem\u003eP. fluorescens (\u003c/em\u003ePSB), \u003cem\u003eP. aeruginosa \u003c/em\u003ePYO-Pa6 (Pa6) incubated with 58PYO1A and 57PYO3A). Asterisks represent the results of standard Ttests comparing the PVD-Pf5 fluorescence in the presence of the identified pyoverdine with the increase due to the presence of pseudobactin (PSB). ***, p value \u0026lt; 10\u003csup\u003e-3\u003c/sup\u003e.\u003csup\u003e \u003c/sup\u003eAll incubations were performed in SSMB1 at 23-24 ⁰C. The numbers above each bar identify the number of independent estimates averaged to obtain the values and errors shown. The error bars show the SEM.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/9185845bd526f01d7427d8ef.png"},{"id":95127164,"identity":"6652c1e8-d887-4d1e-9ac8-e29576820b77","added_by":"auto","created_at":"2025-11-04 15:12:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":654876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAptamer structure. (a) \u003c/strong\u003eMotif analysis of the top 100 clusters of selected oligonucleotides with MEME suite identifies a G motif in aptamer, \u003cstrong\u003e(b) \u003c/strong\u003eOutput from PENGUINN with the bases in each aptamer predicted to form a G-quadruplex, \u003cstrong\u003e(c) \u003c/strong\u003eIncubation with NMM indicates the presence of G quadruplex in 58PYO1A (1A) and 57PYO3A (3A\u003cem\u003e)\u003c/em\u003e. Representative spectra of five spectra for each aptamer, three spectra for BRC and the control DNA (oligo 5637) and two spectra for SPN with all spectra determined with separately prepared samples. \u003cstrong\u003e(d)\u003c/strong\u003e CD Spectra of 58PYO1A and 57PYO3A in the presence or absence of PVD-Pf5. Representative spectra of two independent experiments for each aptamer.\u0026nbsp; \u003cstrong\u003e(e) \u003c/strong\u003eThe 1D-H’ NMR spectrum of 57PYO3A starting at 1 bar (blue) then after shifting to 2K bar (red) and finally after shifting back to 1 bar (purple), and \u003cstrong\u003e(f) \u003c/strong\u003eNon-denaturing acrylamide gel electrophoresis of 57PYO3A in the form of 2’FY-RNA (2’FY) and RNA (2’OH) run beside a control DNA (C) of the same size, \u003cstrong\u003e(g) \u003c/strong\u003eThe fluorescence of HPTA-1 in combination with various oligonucleotides is plotted as the ratio of the peak fluorescence (420-426 nm) in the presence of oligonucleotide to that in its presence with λ\u003csup\u003eex \u003c/sup\u003e= 304 nm and a scan for λ\u003csup\u003eem \u003c/sup\u003efrom 340-590 nm. Due to a small blue shift in the peak position when the dye binds a multimeric G-quadruplex, the peak position was determined independently for each spectrum.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/8757372a33cbf64c6ef147dd.png"},{"id":95224814,"identity":"b769ba84-755d-480f-8835-3642918a75ab","added_by":"auto","created_at":"2025-11-05 16:24:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":523606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of the folding protocol on aptamer structure and function. (a) \u003c/strong\u003e200 nM 57PYO3A was either refolded or not refolded then incubated with 7 μM PVD-Pf5 for 30 min, followed by fluorescence spectroscopy to measure PYO-Pf5 fluorescence\u003cstrong\u003e (b) \u003c/strong\u003e2 μM 58PYO1A or 57PYO3A were refolded in presence of SSMB1 and compared with non-refolded aptamers by CD spectroscopy. Shown are the average, smoothed spectra of 2 (58PYO1A folded), 6 (57PYO3A folded), 3 (58PYO1A not folded), and 7 (57PYO3A not folded) independently collected spectra. \u003cstrong\u003e(c) \u003c/strong\u003e2 μM 58PYO1A or 57PYO3A were refolded in water and compared with non-refolded aptamers by CD spectroscopy. Shown are the average, smoothed spectra of 2 independently collected spectra, \u003cstrong\u003e(d) \u003c/strong\u003e400 nM 57PYO3A was refolded in the presence or absence of 7 μM PV-Pf5 then examined by fluorescence spectroscopy for PVD-Pf5 fluorescence. \u003cstrong\u003e(e) \u003c/strong\u003e2 μM NMM was incubated for 10 min alone (Blank) or with 1 μM 58PYO1A that was either not refolded (Control) or refolded (Refolded) followed by fluorescence spectroscopy to measure NMM fluorescence.\u003cstrong\u003e (f) \u003c/strong\u003e2 μM 58PYO1A was refolded with 2 μM PYO-Pf5 then examined by CD spectroscopy. Shown is the smoothed average of two independently collected CD spectra. All incubations were performed at 23-24 ⁰C.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/f59f7131a60026b89bb82a6c.png"},{"id":95225143,"identity":"0c1aab95-3da6-46bf-a2b9-533800b1e43b","added_by":"auto","created_at":"2025-11-05 16:24:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":525727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of salts on aptamer structure and function. (a) \u003c/strong\u003e400 nM 58PYO1A and 57PYO3A were incubated in water or SSMB1 with 1 μM PVD-Pf5 for 30 min, followed by fluorescence spectroscopy to measure pyoverdine fluorescence , \u003cstrong\u003e(b) \u003c/strong\u003e2 μM\u0026nbsp; of either 58PYO1A, 57PYO3A, Broccoli (BRC), Spinach2 (SPN), or thrombin aptamer (THR) were incubated in water or SSMB1 with 2 μM NMM for 10 min, followed by fluorescence spectroscopy to measure NMM fluorescence, \u003cstrong\u003e(c)\u003c/strong\u003e 2 μM 58PYO1A and 57PYO3A\u0026nbsp; were incubated in SSMB1 or water and examined by CD spectroscopy \u003cstrong\u003e(d)\u003c/strong\u003e The effects of K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e and Mg\u003csup\u003e2+ \u003c/sup\u003eat 10 mM each and with chloride as the anion on binding of NMM by 58PYO1A (PYO1A) and 57PYO3A (PYO3A). Ttests are in comparison with the water control. The absence of an asterisk denotes no significant difference from the water control. All incubations were performed at 23-24 ⁰C.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/6eb9bc3156cd2bbb97dc8ad3.png"},{"id":95230654,"identity":"e4867839-aec1-4f5b-8c60-72a627be6461","added_by":"auto","created_at":"2025-11-05 16:38:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3463240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/c9100947-8a88-428a-9ef2-308b5197649d.pdf"},{"id":95127166,"identity":"0803d6ac-111d-4640-90be-b69ec6af7817","added_by":"auto","created_at":"2025-11-04 15:12:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4810067,"visible":true,"origin":"","legend":"","description":"","filename":"Anisuzzamanpyoverdine2FYaptamersGquadSupplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7885693/v1/a3ceb8c015f46b5793b4f0e3.pdf"}],"financialInterests":"Competing interest reported. MNH owns Aptalogic Inc. MNH is a member of the editorial board of Scientific Reports.","formattedTitle":"2’FY-RNA aptamers form metastable multimeric G-quadruplexes that selectively bind pyoverdines","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAptamers can exquisitely selective for their molecular target and are therefore being developed as possible therapeutic agents\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, receptors for tissue and cell-specific drug targeting\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, targeted protein degradation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and recognition elements on sensors\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The improving technology for introducing RNAs into cells is opening avenues for applying aptamers as drugs to inhibit intracellular functions\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and for binding metabolites to alter metabolic flow\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and drug intake\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. As short but structured oligonucleotides, aptamers can also be informative for understanding nucleic acid structure and interactions between structural units. Folding patterns and binding strategies of aptamers can reveal principles that will help to decode the architecture and function of natural RNA and DNA systems, including riboswitches, ribozymes, and regulatory complexes.\u003c/p\u003e\u003cp\u003eModification of the ribose sugar with fluorine, as in 2\u0026rsquo;FY-RNA in which the pyrimidine-linked sugars are 2\u0026rsquo;F, confers the resulting molecules with more helical stability and nuclease resistance than their RNA equivalents\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These features are important for applications of RNAs in therapeutics or on sensors\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePseudomonads are an important genus of bacteria with species that range from beneficial to deleterious to animal and plant health and that populate many habitats\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Fluorescent pseudomonads, produce a characteristic fluorescent pyoverdine, which is a siderophore that usually contains a 2,3-diamino-6,7-dihydroxyquinoline fluorophore to which is attached a polypeptide produced by a non-ribosomal synthetic pathway\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The polypeptides bind iron with affinities around 10\u003csup\u003e32\u003c/sup\u003e M\u003csup\u003e16\u003c/sup\u003e and enable these pseudomonad species to survive in hospitals where they are a serious threat to already compromised individuals and in the soil, where the released siderophores can protect plants from pathogens by mechanisms that include iron sequestration\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Each species produces unique siderophores with strain-specific peptide chains\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere we describe the selection and structural analysis of 2\u0026rsquo;FY-RNA aptamers that recognize pyoverdine-Pf5 (PVD-Pf5), a unique product of \u003cem\u003ePseudomonas protegens\u003c/em\u003e, that signals the presence of this microbe. These aptamers bind the pyoverdine chromophore and structural elements of the peptides with different patterns of specificity for a range of pyoverdines and display properties consistent with high structural stability. Although their structural signatures appear independent of cations, pyoverdine binding has a complex dependence on cations. Both aptamers are thermally metastable and have spectral and other characteristics that are consistent with their forming multimeric G-quadruplexes.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003ch2\u003eAptamer selection\u003c/h2\u003e\n\u003cp\u003eAptamer selection was performed as described in Materials and Methods (Fig. 1A, B). This protocol was designed to isolate aptamers with the characteristics of high affinities and specificities for the ligand and for switching structure on binding the ligand. In addition, the presence of complementary oligonucleotides to the PCR primer sequences during selection limited structure selection to the single-stranded central randomized region. Aptamer selection started with a high molar ratio of pool to PVD-Pf5 (1:1) and a harmonic increase of pool to target PVD-Pf5 in later cycles to increase the selection pressure\u003csup\u003e19\u003c/sup\u003e. Counter selections were performed after rounds 3 and 6 against a mixture of two other siderophores, enterobactin (ENB) and ornibactin (ORB), to eliminate nonspecific binders (Fig. 1A). With the assumption that the oligonucleotide complex with PVD-Pf5 is stoichiometric, this selection protocol resulted in 60% of the round 9 oligonucleotide pool released by PVD-Pf5 (Fig. 1C). Analysis of the NextGen sequencing results showed that incremental rounds of selection were characterized by increases in the unique fraction (number of sequences in the pool divided by the pool size) and the enriched species (sequences present in the pool more than once), which is characteristic of a successful selection procedure (Fig. S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results of next generation sequencing for oligonucleotide pools from several rounds, including the final round of each selection provided the data for identifying potential aptamer sequences. Sequence clusters were identified with Aptasuite\u003csup\u003e20\u003c/sup\u003e and, based on the size of the cluster and the rise in enriched species through the rounds, several oligonucleotides were evaluated for binding the PVD-Pf5 chromophore by identifying increases in pyoverdine fluorescence when bound to the oligonucleotide. This assay tested for the ability of the aptamers to bind the chromophore, altering its structure and inducing fluorescence in a similar manner as observed for light-up aptamers such as the malachite green aptamer, Spinach, Broccoli and Mango\u003csup\u003e21\u003c/sup\u003e. From this analysis, two oligonucleotides were identified as potential aptamers for further investigation, which was performed using the sequences of the selected oligonucleotides stripped of their 5\u0026rsquo; and 3\u0026rsquo; PCR primer binding sites.\u0026nbsp;These aptamers were named as 58PYO1A and 57PYO3A, according to their length and target with a unique number followed by A to identify the oligonucleotide as an aptamer.\u0026nbsp;Their binding isotherms, monitored by the fluorescence change of PVD-Pf5, resulted in similar estimated K\u003csub\u003ed\u003c/sub\u003es of 66 \u0026plusmn; 42 nM and 98 \u0026plusmn; 35 nM for 58PYO1A and 57PYO3A respectively (Fig. 1D) despite their differences in primary sequence (Fig. 1E).\u003c/p\u003e\n\u003ch2\u003eSpecificity of the PVD-Pf5 binding aptamers\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo investigate features of the chromophore that are recognized by the aptamers, we synthesized the PVD-Pf5 chromophore and variations (Fig. 2A,B) and tested the abilities of the aptamers to increase their fluorescence (Fig. 2C). Compound 1, an intermediate for chromophore synthesis that lacks the third ring, did not bind either aptamer. Some limited evidence of binding was seen for compound 2, with a 3-ring chromophore structure and an amino group attached to the carbon position to which the peptide is attached in PVD-Pf5. \u0026nbsp;The best evidence of 2\u0026rsquo;FY-RNA aptamer binding to the isolated chromophore was with compound 3, which has a carboxyl at the position that is linked by an acyl group to the peptide in PVD-Pf5. In compound 4 the acyl group is extended by two carbons, a structure that does not match that in PVD-Pf5, and fluorescence assay showed that aptamer does not bind this compound. These results suggest that the aptamers bind the chromophore in a way that is highly specific for the nature of the chemical features linked to the 1H-pyrimido[1,2-a] quinoline ring at position 5 (Chem Abstracts numbering) but that more than the chromophore is necessary for tight binding of PVD-Pf5.\u003c/p\u003e\n\u003cp\u003eWe tested the abilities of the aptamers to bind the two siderophores, enterobactin and ornibactin, that were used for counterselection during SELEX (Fig. 1A). The results showed that neither enterobactin nor ornibactin competed for PVD-Pf5 binding to the aptamer (Fig. 3A,B).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;As each fluorescent pseudomonas species synthesizes a pyoverdine with the same chromophore but different peptide moieties, we also explored the specificity of the two aptamers across a panel of five pyoverdines with the same chromophore from three different species (Fig. 3C,D). The two aptamers displayed different specificities across this panel, which suggested structural differences between the aptamers.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStructural features of PVD-Pf5 aptamers\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo assess possible structural features of the aptamers that might be responsible for their similar binding affinities and different specificities, a motif search was performed on the clusters from which the aptamers were chosen using the MEME suite (https://meme-suite.org/meme/tools/meme)\u003csup\u003e22\u003c/sup\u003e. From this we identified a G-rich structural motif (Fig. 4A). The identified motif suggested the presence of G-quartets. The PENGUINN neural network (https://ml-bioinfo-ceitec.github.io/penguinn/) scores\u003csup\u003e23\u003c/sup\u003e identified the possible presence of G quartets (Fig. 4B), which were also predicted by 3dRNA/DNA (http://bio-phy.hust.edu.cn/new/3dRNA/create) to be formed by both aptamers (Fig. S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test for the presence of G-quadruplexes, the aptamers were incubated with N-methyl mesoporphyrin IX (NMM), which selectively binds to G-quadruplexes as demonstrated by its increased fluorescence\u003csup\u003e24\u003c/sup\u003e. Both aptamers increased NMM fluorescence at 610 nm and 670 nm at intensities equivalent to Spinach2 and Broccoli, which are confirmed G-quadruplexes (Fig. 4C). Circular dichroism spectroscopy provided additional evidence of the presence of G-quadruplexes (Fig. 4D) and identified the aptamers as forming parallel G-quadruplexes, which are characterized by a positive peak around 264 nm and a weaker negative peak at 240 nm\u003csup\u003e25\u003c/sup\u003e. The spectra were not different in the presence and absence of PVD-Pf5 for either aptamer (Fig. 4D). Thus, both aptamers appear to form G-quadruplexes that are not substantially reorganized upon binding PVD-Pf5, which suggests that the structural change by which these aptamers were removed from the capture oligonucleotide might not have been large.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA 1D 1H-NMR study of 57PYO3A did not reveal well-resolved peaks in the imino region where the G-G Hoogsteen base pairing typical of G-quartets can be identified (Fig. 4E). However, the presence of peaks in this chemical shift range is consistent with the CD spectra and NMM binding results and with a G-quadruplex structure among others. Increasing the pressure to 2 kbar had little effect on the NMR spectrum in this region (Fig. 4E) and the spectrum taken after dropping the pressure to 1 bar (1 bar end in Fig. 4E) was identical to the starting spectrum (1 bar start) before \u0026nbsp;raising the pressure to 2 kbar. This suggests that the aptamer structure is very stable, which is another feature of G-quadruplexes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo address the question of how these aptamers could form G-quadruplexes when they lack the canonical G tracts seen in many G quadruplexes and do not have sufficient appropriately distributed Gs to create 3 G-quartets, we considered the possibility that, like Oxy-1.5 from the \u003cem\u003eOxytricha nova,\u0026nbsp;\u003c/em\u003e58PYO1A and 57PYO3A form dimeric G-quadruplexes\u003csup\u003e26\u003c/sup\u003e. This question was tested by non-denaturing gel electrophoresis by which we observed evidence of dimeric forms of the aptamers (Fig. 4F). Similar evidence of dimerization was observed with the RNA equivalents of these aptamers (Fig. 4F). An additional feature of multimeric G quadruplexes is that they bind HPTA-1, increasing its fluorescence. Both aptamers increased the fluorescence of HPTA-1 whereas several other parallel and antiparallel G-quadruplex aptamers not known to form multimeric structures did not bind HPTA-1 (Fig. 4G). The increased fluorescence of HPTA-1 was manifested differently by the two aptamers with 58PYO1A causing a decided blue shift and 57PYO1A causing a slight red shift in the spectrum 58PYO3A (Fig. S4). This result suggests that the multimeric structures of the two aptamers differ around the binding site of HPTA-1, which is believed to be at the interface of the monomers.\u003c/p\u003e\n\u003ch2\u003eFolding dynamics\u003c/h2\u003e\n\u003cp\u003eThe aptamer selection protocol involved annealing of complements to the 3\u0026rsquo; and 5\u0026rsquo; PCR primer sequences and thus did not include a refolding step that is often used to promote aptamers to fold to a homogeneous tertiary structure. G-quadruplexes are also reported to unfold and refold slowly. Therefore, we tested the outcomes of several variations of the unfolding and refolding procedure with prolonged heating and annealing periods. These included heating at 95 \u0026deg;C for 3 h followed by slow cooling air for 18 h and heating to 95 \u0026deg;C followed by slow cooling at a rate of 1 \u0026deg;C per minute. None of these protocols resulted in aptamers capable of binding PVD-Pf5 (Fig. 5A). The CD spectra of the refolded aptamers were also different from the untreated aptamers (Fig. 5B) whether refolded in SSMB1 salts solution or in water (Fig. 5C). As the 2\u0026rsquo;FY-RNAs are usually frozen directly after synthesis then thawed and analyzed for folding, we tested for the effect of freeze-thaw on the CD spectrum and found the same spectra and apparent size in freshly prepared samples as after freeze/thaw (Fig. S5). Thus, it appears that the G-quadruplex folds co-transcriptionally and, if denatured post-synthesis, these aptamers do not refold to form functional structures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIf PVD-Pf5 was present during the refolding, the refolded aptamers showed increased fluorescence with pyoverdine indicative of pyoverdine binding to the aptamer (Fig. 5D). The quantitative increase in PVD-Pf5 fluorescence with the addition of 58PYO1A was the same for both conditions (refolded or not refolded). However, the refolding procedure resulted in an increase in fluorescence of pyoverdine that could be reproduced in the absence of the aptamer. That heating changed the pyoverdine to a different structural form was confirmed by the observation that the preheated pyoverdine (PVD-Pf5*) does not increase in fluorescence in the presence of aptamers that have not been refolded (Fig. 5D). This result can be interpreted as PVD-Pf5* does not bind the aptamers or that binding occurs but is not associated with a further increase in the fluorescence of PVD-Pf5*.\u003c/p\u003e\n\u003cp\u003eTo further assess if refolding in the presence of pyoverdine resulted in the formation of a G-quadruplex-like structure, we evaluated the ability of 58PYO1A to bind NMM (Fig. 5E). The control aptamer that had not experienced a refolding protocol of heating and cooling bound NMM (Fig. 5E, red bars). Fluorescence from NMM was decreased in the presence of PVD-Pf5 for the control condition suggesting competition between the two for binding (Fig. 5E, red hatched bars). NMM fluorescence was also much lower in the presence of the aptamer refolded in the presence of PVD-Pf5 and there was no change in NMM fluorescence with the addition of PVD-Pf5 (Fig. 5E, green bars). The CD spectrum was more informative with respect to probable structure as the aptamers refolded in the presence of PVD-Pf5 appeared to be well-formed G-quadruplexes (Fig. 5F). Thus, it appears that, on refolding, the aptamers become trapped in a folding energy minimum that is overcome if folded in the presence of PVD-Pf5.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSalt requirements for ligand binding and aptamer structure\u003c/h2\u003e\n\u003cp\u003eG-quadruplex structures are often stabilized by the presence of cations, which take a central position in the structure, coordinating between G-quartets. To determine the cation requirement of the 2\u0026rsquo;FY aptamers for ligand binding, we tested the change in fluorescence of pyoverdine in the presence and absence of the SSMB1 salts. The increase in fluorescence was observed in the presence but not absence of the salt mix (Fig. 6A). Attempts to identify the required salt(s) for binding demonstrated that a single salt component of SSMB1 could not replace the salt mix in promoting PVD-Pf5 binding and none of the combinations tested promoted the same increase in binding as SSMB1 (Fig. S6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate the role of salts in formation of the aptamer structures, we compared the NMM fluorescence of each aptamer in water and SSMB1. Whereas there was no difference in NMM binding to either aptamer in the presence or absence of SSMB1 (Fig. 6B), NMM fluorescence with the Spinach2, the thrombin aptamer, and less so with the Broccoli aptamer, were increased in the presence of SSMB1 (Fig. 6B). Analysis of the CD spectra of 58PYO1A or 57PYO3A also showed no difference in the presence of water or SSMB1 (Fig. 6C). We examined the effects of selected cations present in SSMB1 that have been identified as stabilizing other G-quadruplex structure and their combinations (Fig. 6D) and again observed no significant effects of the cations K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e or their combinations on G-quadruplex structure as assessed by NMM fluorescence. These results show that 58PYO1A and 57PYO3A form structures with features of G-quadruplexes in the absence of monovalent or divalent cations but that a complex combination of cations is required for binding the PVD-Pf5.\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eFrom a single SELEX experiment to isolate 2\u0026rsquo;FY-RNA aptamers that are specific for the pyoverdine, PVD-Pf5, we identified two aptamers that we have analyzed for structure and function. Although the protocol for isolating the aptamers was designed to isolate structure-switching aptamers, this property has been elusive. Otherwise, the properties of the two aptamers that we chose based on evidence of PVD-Pf5 binding, are similar in their PVD-Pf5 binding isotherms, specificity for PVD-Pf5 over ENB, ORB and PSB, and features consistent with G-quadruplex structures. The only difference identified was the red vs. blue shift in HPTA-1 fluorescence spectrum when bound to the aptamers. Such similarity of features is unexpected for two aptamers with different sequences (only 19% identical in the randomized regions) and are proposed to fold into different three-dimensional structures.\u003c/p\u003e\u003cp\u003eSpecificity and high affinities are important characteristics of aptamers intended for applications in biotechnology or medicine. To bias the selection towards aptamers that bind pyoverdines and no other common siderophores, two other siderophores, ENB and ORB, were used as counter selection targets. ENB is released by \u003cem\u003eEnterobacter\u003c/em\u003e during iron stress whereas ORB is released by soil dwelling \u003cem\u003eBurkholderia\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We deliberately did not use a related pyoverdine as a counter-selection target because we were interested in the possibility that we might obtain an aptamer that binds all pyoverdines and could thus sense the presence of any \u003cem\u003ePseudomonad\u003c/em\u003e. Each \u003cem\u003ePseudomonad\u003c/em\u003e species produces a structurally unique siderophore that allows them to selectively remove iron from the environment and avoid competition from other organisms. Many of these siderophores contain the same chromophore. Although similar in many aspects including that they bind the common chromophore for many pyoverdines, these aptamers display different specificity profiles when compared against a panel of different siderophores. This observation and the relatively poor binding to the isolated chromophore support the hypothesis that these aptamers recognize the combination of chromophore and side chain.\u003c/p\u003e\u003cp\u003eThe SELEX experiment design included the use of oligonucleotides complementary to the primer regions of the library to exclude these regions from involvement in forming single-stranded structural motifs. This design would focus selection pressure on the randomized region of the library, streamline the informatics search by minimizing the length of sequence to be analyzed, and provide an opportunity to track many aptamers in screening experiments by using a single labeled complementary oligonucleotide that could be hybridized with the primer landing site of all aptamers. These outcomes were realized in the selected pool.\u003c/p\u003e\u003cp\u003eAlthough predicted to form G-quartets by two computational tools, neither aptamer displays a canonical sequence expected of a series of G-quartets that could make up a G-quadruplex although each has enough Gs to contribute to two (PYO1A) or 3 (PYO3A) G-quartets. Several DNA G-quadruplexes consist of only 3 G-quartets such as the c-MYC and c-KIT promoter quadruplexes (PDB IDs: 1XAV and 2O3M, respectively)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, whereas the 15-mer thrombin binding aptamer (TBA, PDB1HAI) consists of only two G-quartets\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Also, TERRA, an RNA G-quadruplex, consists of three G-quartets\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Smaller nucleic acids also form multimeric G-quadruplexes\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and our data suggests that 58PYO1A and 57PYO3A are in this category of G-quadruplex. In addition the observation that NMM competes with PVD-Pf5 suggests that, like NMM that sits on the outer G-quartet of the quadruplex\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, the pyoverdine chromophore may also occupy this space on the aptamers. A complex combination of cations seems to be required for ligand binding, but we found no cation requirement for structure as evaluated by CD spectra and NMM binding. As an alternative to cations, long-lived water molecules were found embedded in the bimolecular d(G 3CT4G3C)2 G-quadruplex\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Structured water has also been found in some G-quadruplexes (PDB id 3CE5, 3NZ7, and 3UYH)\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and demonstrated to be important for G-quadruplex-ligand interaction\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. With embedded water and no cation, the central spaces of these aptamers might be dense. To test the density of these aptamers and their structural stability we evaluated the pressure required for structural collapse using the NMR spectrum as the readout. DNA quadruplexes usually compress in the range 1-1.5 kbar, which varies depending on the oligonucleotide sequence and the stabilizing monovalent cation\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. RNA G-quadruplexes are more dense\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, but still compress over the range 1\u0026ndash;2 kbar\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Although the NMR spectrum of the 57PYO3A aptamer was not sufficiently resolved for assigning positions, a salient feature was that the spectra at 1 bar and 2 kbar were very similar in the imino region and showed no decrease in intensity with increased pressure. These results suggest that the aptamer structure is more stable than the DNA and RNA G-quadruplexes so far evaluated in this manner. The broad peaks in the imino-region spectrum of 57PYO3A also suggest that it may exist as an ensemble of related structures that could be in slow equilibrium.\u003c/p\u003e\u003cp\u003eLike the multimeric quadruplexes formed by the TG\u003csub\u003e4\u003c/sub\u003e oligonucleotide derived from the \u003cem\u003eOxytricha nova\u003c/em\u003e telomeric sequence\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and the pG\u003csub\u003e5\u003c/sub\u003e oligonucleotide\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, 58PYO1A and 57PYO3A are thermally metastable reverting to alternate structures on cooling. Both aptamers can be guided by the presence of PVD-Pf5 to refold after heating to a G-quadruplex structure that binds PVD-Pf5. Although we have not yet determined the alternative structures, they might be the hairpins predicted for both sequences by RNAfold (Fig. S3). HMGB1-targeting G-quadruplex aptamers are metastable G-quadruplexes that can revert to hairpins\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. A transition between hairpin and G-quadruplex was also reported to influence the rate of \u003cem\u003eE. coli EutE\u003c/em\u003e mRNA translation\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMetastable multimeric G-quadruplexes may play important roles in nature. For example metastable G-quadruplexes have recently been proposed as features of ALS/FTD-associated nucleotide expansions\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Most natural RNA G4 sequences are reported not to fold as stable monomeric G-quadruplexes but required dimerization\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Multimeric G-quadruplexes are also proposed to play a role in mRNA aggregation during stress\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Cations and folding kinetics can also influence the ultimate G-quadruplex structure as shown for d(G4T4G3) quadruplexes for which conformational transitions are kinetically governed and influenced by K\u003csup\u003e+\u003c/sup\u003e concentrations\u003csup\u003e54,55\u003c/sup\u003e. These observations open possibilities of interventions that could influence G-quadruplex folding and multimerization for therapeutics\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn summary, we have evaluated the features of two aptamers that were selected to bind PVD-Pf5 by a structure-switching protocol. Despite their different primary sequences and predicted tertiary structures, these aptamers were found to have similar structural features but vary in their target specificity profiles. They form multimeric metastable G-quadruplexes for which one was demonstrated stable to high pressure. Metastability and multimerization are well-documented properties of several telomeric DNAs and RNA G-quadruplexes and may be the predominant forms of RNA quadruplexes \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The inclusion of pyoverdine aptamers in this category broadens the repertoire of sequences capable of forming metastable multimeric G-quadruplexes. Given the emerging significance of G-quadruplex structures in mRNAs and long noncoding RNAs\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, these aptamers, may reveal important structural features for regulating G-quadruplex structure and function that might also be employed in regulating RNA-based cellular processes.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eReagents\u003c/p\u003e\u003cp\u003eAll chemicals were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Hampton, NH, USA) unless otherwise stated. N-methyl mesoporphyin IX (NMM, cat# sc-396879) was purchased from Santa Cruz Biotechnology. PVD-Pf5 (cat# PVD-Pf5), hydrazinyl pyrrolidine triazine amine (HPTA-1, cat# 23848) was from Glixx laboratories (Hopkinton, MA, USA). Enterobactin (cat# FE-ENB), and ornibactin (cat# ORNIB) were purchased from EMC microcollections (T\u0026uuml;bingen, Germany). Pseudobactin (cat# 8374) isolated from \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e was purchased from Sigma Aldrich. All the siderophore preparations were iron-loaded.\u003c/p\u003e\u003cp\u003eBuffers used for this work were: 1) Buffer A: 40 mM HEPES, 125 mM KCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4, 2) PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 7.4, 3) SSMA2: 366 \u0026micro;M CaCl\u003csub\u003e2\u003c/sub\u003e, 3 \u0026micro;M CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003e0, 5 \u0026micro;M MnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003e0, 27 \u0026micro;M KI, 83 \u0026micro;M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 35 \u0026micro;M KOH, 1 \u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e .7H\u003csub\u003e2\u003c/sub\u003e0, 103 \u0026micro;M Fe (III)Na-EDTA, 68 \u0026micro;M MgSO\u003csub\u003e4\u003c/sub\u003e, 23 \u0026micro;M Mg (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003e0, 94 \u0026micro;M MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003e0, 64 \u0026micro;M NaCl, 75 \u0026micro;M NH\u003csub\u003e4\u003c/sub\u003eOAc, pH 5.9, and 4) SSMB1: SSMA2, 10 mM KCl, 1 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3,\u003c/sub\u003e pH 5.9.\u003c/p\u003e\u003cp\u003eSynthesis of pyoverdine chromophores\u003c/p\u003e\u003cp\u003eCompound \u003cb\u003e1\u003c/b\u003e was synthesized starting from nitro lactam \u003cb\u003eA\u003c/b\u003e by reaction with thionyl chloride in DMF at 98\u0026deg;C. The resulting chloroquinoline was treated with the azetidine ester and excess triethylamine in DMF at 92\u0026deg;C to generate \u003cb\u003e1\u003c/b\u003e. Compound \u003cb\u003e2\u003c/b\u003e was prepared by treatment of \u003cb\u003e1\u003c/b\u003e with HI. Compound \u003cb\u003e3\u003c/b\u003e was synthesized by treatment of \u003cb\u003e2\u003c/b\u003e with excess acetic anhydride. Compound \u003cb\u003e4\u003c/b\u003e was synthesized starting from nitro lactam \u003cb\u003eA\u003c/b\u003e by reaction with ethyl bromoacetate, DMF, and sodium hydride. Although a mixture of N-alkylated and O-alkylated material was produced, the desired N-alkylated product was easily separated. The nitro group was reduced using excess iron and aqueous HCl in ethanol. The resulting amine was acylated with succinic anhydride to provide amide ester acid \u003cb\u003e4\u003c/b\u003e. More details regarding the synthesis (Supplemental section 1), absorbance spectra (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) are part of the Supplemental materials.\u003c/p\u003e\u003cp\u003eNucleic acids and SELEX library design\u003c/p\u003e\u003cp\u003eAll DNAs used in this study were synthesized by Integrated DNA Technology (IDT, Coralville, IA, USA). 2\u0026rsquo;FY-RNAs were synthesized as described under 2\u0026rsquo; FY-RNA synthesis and purification. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e lists all nucleic acids, and their sequences described in this work. All chemically modified oligonucleotides were purified using HPLC whereas unmodified oligonucleotide sequences were purified by standard desalting unless otherwise stated.\u003c/p\u003e\u003cp\u003eA single stranded DNA library was used (IDT, Coralville, IA) with each oligonucleotide having a length of 100 bases, which includes three constant regions and two random regions: 5'-GCCTGTTGTGAGCCTCCTGTCGAA (10N) GGAGCGAATG (35N) GAGCGTTTATTCTTGTCTCCC-3\u0026rsquo;. In this library, N represents an equimolar combination of A, T, C and G. The 5\u0026rsquo; and 3\u0026rsquo; ends of the constant regions were primer binding sites for PCR amplification and the 10 base long constant region between 10N and 35N was used to capture the 2\u0026rsquo; FY-RNA library components during selection.\u003c/p\u003e\u003cp\u003eNucleic Acid synthesis\u003c/p\u003e\n\u003ch3\u003eConversion of ssDNA SELEX library to dsDNA\u003c/h3\u003e\n\u003cp\u003eThe ssDNA pool was converted to the dsDNA template for \u003cem\u003ein vitro\u003c/em\u003e transcription (IVT) by primer extension from oligonucleotide 5627. Each reaction contained 1.7 \u0026micro;M ssDNA pool, 3.3 \u0026micro;M oligonucleotide 5627 (primer), 0.2 mM dNTP mix, 0.05 U/\u0026micro;L DNA Taq Polymerase (GenScript, Piscataway NJ, USA, cat# E00007), 50 mM KCl, 10 mM Tris-HCl (pH 8.55), 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1% TritonX-100. The mixture was incubated at 94\u0026deg;C for 5 min, 65\u0026deg;C, for 15 min, then 72\u0026deg;C for 99 min using a thermocycler (Quant Studio 3, Thermofisher, Waltham, MA, USA). The generated dsDNA was resolved through a 2% agarose gel and purified using a PCR cleanup column from Syd labs (Hopkinton, MA, USA). The dsDNA was quantified based on its uv/vis absorption spectrum (Nanodrop, Thermofisher).\u003c/p\u003e\u003cp\u003e2\u0026rsquo; FY-RNA and RNA synthesis and purification\u003c/p\u003e\u003cp\u003eReverse transcription was performed using Superscript IV reverse transcriptase (Thermofisher, cat# 18090050) and PCR to generate the template for \u003cem\u003ein vitro\u003c/em\u003e transcription was done using Taq DNA polymerase (GenScript, Piscataway NJ, USA, cat# E00007)\u003c/p\u003e\u003cp\u003e2\u0026rsquo; FY-RNA was prepared by \u003cem\u003ein vitro\u003c/em\u003e transcription using a Durascribe T7 transcription kit from (Epicentre, Madison WI, USA, cat# DS010925) or with recombinant His-tagged T7(Y639F/H784A) RNA polymerase purified from transformed XL-1 Blue \u003cem\u003eE. coli\u003c/em\u003e by Ni-NTA affinity resin (eluted with 250 mM imidazole). Around 1.6 nmoles dsDNA from the extended pool was incubated with 5 mM ATP, 5 mM GTP, 5 mM 2\u0026rsquo;FY-UTP, 5 mM 2\u0026rsquo;FY-CTP, 5 mM dithiothreitol, 5% DMSO, 0.8 \u0026micro;g/\u0026micro;L T7(Y639F/H784A) RNA polymerase, 40 mM Tris-HCl, 25 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2.5mM spermidine, 0.01% Triton X-100, pH 7.8, at 37 or 42\u0026deg;C for 12 h then digested with 1 MBU DNAse I for 15 min.\u003c/p\u003e\u003cp\u003eRNA was prepared from templates created by PCR amplification or by annealing complementary oligonucleotides using the Hyperscribe T7 RNA synthesis kit (Apexbio, Houston, Texas, cat# K1047). To separate the residual NTPs and abortive transcripts from the desired transcript, the 2\u0026rsquo; FY-RNA or RNA was resolved through an 8% (19:1 acrylamide:bisacrylamide) gel containing 7 M urea with TBE (100 mM Tris, 100 mM Borate, 1 mM EDTA, pH 9) as running buffer. The 2\u0026rsquo;FY-RNA or RNA was eluted by crushing the section of gel containing the RNA and incubating with TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 18 h at 37\u0026deg;C followed by removing the gel by centrifugation.\u003c/p\u003e\u003cp\u003eSELEX: \u003cem\u003eIn vitro\u003c/em\u003e selection of aptamers for PVD-Pf5\u003c/p\u003e\u003cp\u003eSELEX was performed with PVD-Pf5 as target to select 2\u0026rsquo;FY-RNA aptamers. For the first round of selection a library of 10\u003csup\u003e15\u003c/sup\u003e molecules of 2\u0026rsquo; FY-RNA was used. The biotinylated capture oligonucleotides, which are complementary to the short constant region in the library sequence, were attached to streptavidin coated magnetic beads. The 2\u0026rsquo;FY-RNA libraries were first hybridized with oligonucleotides 5625 and 5617 (complementary to the PCR primer regions), then immobilized on the beads by hybridization with the capture oligonucleotide. The length of the capture oligonucleotide varied with the SELEX round as did the hybridization temperature and the oligonucleotide:PVD-Pf5 molar ratio (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The bound oligonucleotides were captured using a magnet and purified by ethanol precipitation in the presence of 0.13 \u0026micro;g/mL linear polyacrylamide (LPA, Sigma-Aldrich). Purified 2\u0026rsquo;FY-RNA was reverse transcribed and amplified by low fidelity Taq polymerase (cat#: E00007; Genscript, Piscataway, NJ, USA). The ratio of 2\u0026rsquo;FY-RNA to PVD-Pf5 was reduced and the length of capture oligonucleotide increased in successive SELEX rounds to increase selection pressure (Table S2). Nine rounds of selection were performed, and two sequential rounds of counter selection were done after rounds 3 and 6. The targets for counter selection were a combination of enterobactin and ornibactin each at a 1:1 molar ratio with the oligonucleotide library. All targets used in selection and counter-selection were the Fe\u003csup\u003e3+\u003c/sup\u003e-bound forms. The buffer used during selection was SSMA2. PCR-amplified libraries were prepared for NGS using Nextera Illumina sequencing primers and the appropriate bar coding. Next generation sequencing was performed by the Novogene Facility (Sacramento, California, USA) using HiSeq PE150 platform.\u003c/p\u003e\n\u003ch3\u003eBioinformatics analysis and data preprocessing of SELEX results\u003c/h3\u003e\n\u003cp\u003eThe raw NGS data was demultiplexed and assigned to respective rounds by open-source software AptaSuite\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Barcodes were used to assign DNA pools from different rounds of selection. A zero-nucleotide mismatch between the reference and matched barcodes, and two mismatches between the reference and matched primers were criteria for demultiplexing. Further analysis was done with the sequences that met this standard. Clustering was done with 10 iterations of locality sensitive hashing, sampling 55% of the indices in the randomized regions and allowing no more than 10 nucleotide mismatches between the seed sequence of a cluster to each remaining member. Clusters were extracted for further analysis and aptamer screening. MEME was run with version 5.4.1 using default parameters whereas secondary structures were predicted using Mfold version 3.6 with standard parameters in the RNA mode\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The probability of G quadruplex formation was analyzed by the neural network PENGUINN\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and QGRS mapper\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Tertiary structures of RNAs were predicted by 3dRNA/RNA\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFluorescence spectroscopy\u003c/p\u003e\u003cp\u003eAll assays were performed at 23\u0026ndash;24\u0026deg;C in SSMB1 unless otherwise stated.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePyoverdine binding assay\u003c/h2\u003e\u003cp\u003eFluorescence emissions by the pyoverdine chromophore were measured by a Cary eclipse fluorimeter (Varian, Palo Alto, CA, USA) and a Synergy 2 (Biotek, Winooski, VT, USA) plate reader. Ten nM PVD-Pf5 was incubated with various concentrations of 2\u0026rsquo; FY-RNA up to 1 \u0026micro;M at 23\u0026deg;C for 30 minutes in SSMB1. Fluorescence intensities were measured with excitation at λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;400 nm and λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;460 nm with 5 nm slit widths. The dissociation constants (K\u003csub\u003ed\u003c/sub\u003e) of the aptamer-ligand complexes were determined from the fluorescence output as a function of aptamer concentration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eNucleic acid binding to N methyl mesoporphyrin IX\u003c/h2\u003e\u003cp\u003eTwo \u0026micro;M each of aptamer and N methyl mesoporphyrin IX (NMM) from Santa Cruz Biotechnology Inc. (Dallas, TX, USA, cat# 396879) in SSMB1 were incubated for 10 minutes at 23\u0026deg;C. Fluorescence was measured at λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;399 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;410\u0026ndash;700 nm, slit\u0026thinsp;=\u0026thinsp;5 nm PMT\u0026thinsp;+\u0026thinsp;800V using a Cary Eclipse fluorimeter (Agilent, Santa Clara, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePyoverdine binding and fluorescence competition assay\u003c/h2\u003e\u003cp\u003ePVD-Pf5 was incubated with aptamers in SSMB1 for 30 minutes at 23\u0026deg;C. For competition assays, a 100-fold to 1000-fold excess of ENB or ferric ORB was included, and the samples were incubated at 23\u0026deg;C for another 30 minutes. Fluorescence was measured at λ\u003csup\u003eex\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;400 nm, λ\u003csup\u003eem\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;411\u0026ndash;600 nm, slit\u0026thinsp;=\u0026thinsp;5nm PMT\u0026thinsp;+\u0026thinsp;800V using a Cary eclipse fluorimeter.\u003c/p\u003e\u003cp\u003eCircular dichroism spectroscopy\u003c/p\u003e\u003cp\u003eTwo \u0026micro;M each of aptamer and PVD-Pf5 were incubated for 20 min in SSMB1 then evaluated for circular dichroism using an MOS-500 monochromator, an ALX-250 light source (Bio-Logic Science Instruments, Seyssinet-Pariset, France), and a quartz cell (pathlength 1 mm). Each spectrum was determined from the average of three accumulated scans from 220 to 320 nm from which the baseline (buffer alone) was subtracted. Two or more independently obtained spectra were averaged for many of the spectra shown in figures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eNMR\u003c/h2\u003e\u003cp\u003eNMR spectra were collected on Bruker Avance III HD 800 MHz Spectrometer and pressure applied using Daedalus Xtreme-60 High Pressure NMR accessory at Iowa State University, Ames, USA. Two hundred and fifty-six scans were performed for each spectrum and mixing time was 200 ms. The internal standard for the chemical shift was DSS (2,2-dimethyl-2-silapentane-5-sulfonate). NMR spectra were collected in H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO (95%/5%) and the aptamer concentration was 0.5 mM. The NMR data was processed using Topspin.\u003c/p\u003e\u003cp\u003eAnalysis of binding isotherms, spectra and statistical evaluations\u003c/p\u003e\u003cp\u003eThe binding data was fit to Hill\u0026rsquo;s equation Hill n: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:B={B}_{min}+\\frac{{B}_{max}{L}^{n}}{{L}^{n}+{K}_{d}^{n}}\\)\u003c/span\u003e\u003c/span\u003e with the Hill n set to 1 and Sigmaplot was used to obtain statistical parameters for binding isotherms. Reported values for Kd passed the Normality (Shapiro-Wilk) and the Constant Variance (Spearman Rank Correlation) tests. Except for the standard deviations of the fit calculated by Sigmaplot, the standard errors of the mean (SEM) were calculated for all data due to variations in the numbers of samples contributing to each average value. When a background was subtracted, these were calculated to include the errors in the average values in the blanks by the formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:SEM=\\:\\sqrt{{Se}^{2}+{Be}^{2}}\\)\u003c/span\u003e\u003c/span\u003e where Se\u0026thinsp;=\u0026thinsp;SEM of the sample and Be =\u0026thinsp;SEM of the subtracted blank.\u003c/p\u003e\u003cp\u003eSpectra (fluorescent and CD) were smoothed by simple exponential smoothing. Raw and smoothed spectra for all data shown in the figures are found in the Supplemental materials (Fig. S7-S12).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and analyzed for this study are available from the authors on reasonable request. Next gen sequencing data are available through the following links: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE224685 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM7029482\u003c/p\u003e\n\u003ch2\u003eCompeting Interests Statement\u003c/h2\u003e\n\u003cp\u003eMNH owns Aptalogic Inc. MNH is a member of the editorial board of Scientific Reports.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eAuthor contributions were as follows: MNH and SA participated in conceptualization, data analysis and writing the original draft and revisions. SA designed all experiments with aptamers and collected and performed preliminary analyses. JA and GAK developed the chemical protocols, synthesized and validated the chromophore derivatives, and prepared the figure showing the protocols and chemical structures. MNH prepared the remaining figures, secured funding, and administered the project. All authors have read and agree to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) through the Ames Laboratory. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. DE-AC02-07CH11358. Partial support of Dr. Anisuzzaman and supplies to complete the project were provided by Aptalogic Inc.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe gratefully acknowledge the contributions of Lee Bendickson for help with NextGen sequence analysis, Soma Banerjee for identifying the effect on aptamer function of the folding protocol, and Jeffrey Purslow for assistance in obtaining and analyzing the NMR spectra.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdrian, M., Heddi, B., and Phan, A.T. 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QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e34,\u003c/strong\u003e W676-W682 10.1093/nar/gkl253 (2006).\u003c/li\u003e\n\u003cli\u003eWang, J., Wang, J., Huang, Y., and Xiao, Y. 3dRNA v2.0: An Updated Web Server for RNA 3D Structure Prediction. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e20,\u003c/strong\u003e 10.3390/ijms20174116 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"aptamer, 2’FY-RNA2, pyoverdine5, G-quadruplex1, metastable","lastPublishedDoi":"10.21203/rs.3.rs-7885693/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7885693/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwo 2\u0026rsquo;FY-RNA aptamers with distinct sequences were selected for specific binding to pyoverdine-Pf5 (PVD-Pf5), increasing chromophore fluorescence upon binding. They also recognized the peptide portion of pyoverdines, as shown by their differential specificity for related variants. Computational analysis and experimental data (NMM binding, CD spectra) identified G-quadruplex structures that were thermally metastable but reformed in the presence of PVD-Pf5. Further structural studies mainly with one aptamer revealed imino proton peaks in 1D H-NMR and pressure stability up to 2 kbar. Electrophoretic evidence identified dimeric G-quadruplexes formed by the 2\u0026rsquo;FY-RNA aptamers and their RNA equivalents. While cations were necessary for PVD-Pf5 binding, they were not required for G-quadruplex formation. Given the established role of G-quadruplexes as protein interaction sites, multimeric G-quadruplexes offer a potential framework for structure-based regulatory mechanisms in cellular RNAs. In addition to previously characterized multimeric G-quadruplexes, these aptamers contribute novel sequences that expand the repertoire of known multimeric G-quadruplexes.\u003c/p\u003e","manuscriptTitle":"2’FY-RNA aptamers form metastable multimeric G-quadruplexes that selectively bind pyoverdines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 15:12:10","doi":"10.21203/rs.3.rs-7885693/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-30T08:49:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-26T11:34:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324112599919900803160986932337961625475","date":"2026-01-13T15:52:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90155537392836933484621452211623520709","date":"2026-01-12T20:37:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T04:54:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126831921323142113923143709576332542310","date":"2025-11-15T12:52:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-15T08:20:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252603214446314214270532042186825972467","date":"2025-10-28T00:32:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T05:35:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-23T04:16:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-18T05:12:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-18T05:10:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-17T10:22:09+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":"4411824d-f87b-4fe4-a1bc-36181ece86e3","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57284665,"name":"Biological sciences/Biochemistry"},{"id":57284666,"name":"Biological sciences/Biophysics"},{"id":57284667,"name":"Biological sciences/Chemical biology"},{"id":57284668,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2026-04-28T06:39:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-04 15:12:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7885693","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7885693","identity":"rs-7885693","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}