Development of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides: Evaluation of the triplex-forming and photo-crosslinking properties

Development of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides: Evaluation of the triplex-forming and photo-crosslinking properties | 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 Development of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides: Evaluation of the triplex-forming and photo-crosslinking properties Yu Mikame, Haruki Toyama, Chikara Dohno, Takehiko Wada, Asako Yamayoshi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5384273/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been employed for the photodynamic regulation of gene expression by the photo-crosslinking of psoralen with the target DNA. However, stable triplex formation requires a consecutive purine base sequence in one strand of the target DNA duplexes. The pyrimidine-base interruption in the consecutive purine base sequence drastically decreases the thermodynamic stability of the corresponding triplex, which hampers the TFO application. Here, we propose a design of the Ps-TFO for stable triplex formation with target DNA sequences containing pyrimidine-base interruptions under physiological conditions. This Ps-TFO, named 1’(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a synthesized nucleoside mimic 1’-psoralen-conjugated deoxyribose to increase the thermodynamic stability of the corresponding triplex by the intercalation of psoralen. The triplex-forming abilities of the OPTO were successfully demonstrated in combination with locked nucleic acid (LNA), indicating that the use of OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation. Biological sciences/Chemical biology/DNA Biological sciences/Chemical biology/Nucleic acids Biological sciences/Chemical biology/Chemical tools Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction A triplex-forming oligonucleotide (TFO) binds to its target DNA duplex, forming a triple-helix structure. This triplex formation inhibits either the binding of transcription factors to promoter regions or transcriptional elongation, resulting in gene suppression. 1 – 4 Moreover, the cell recognizes the triplex structure as unusual, which induces a double-strand break (DSB) at the triplex-forming site by an endogenous nuclease. 5 This DSB induction by a TFO has been employed for genome editing or selectively inducing apoptosis in cancer cells. 6 – 10 The interaction of a TFO with the target DNA occurs on the major groove side of the DNA via Hoogsteen (parallel triplex; Fig. 1 a) or reverse Hoogsteen (antiparallel triplex; Fig. 1 a) hydrogen bonds. The inherent challenge of the triplex technology is the requirement for Hoogsteen hydrogen bonds to form specifically between a purine base of A/T (or G/C) base pairs in the double-stranded DNA (dsDNA) and a TFO. If the target base pair in the dsDNA changes (T/A or C/G base pair), a mismatched base pair (pyrimidine-base-interrupting site) with the TFO is produced, significantly reducing the thermodynamic stability of the triplex structure (Fig. 1 b). In the parallel motif, the N 3 position of cytosine must be protonated to facilitate hydrogen bonding. In contrast, antiparallel triplex oligonucleotides can form reverse Hoogsteen hydrogen bonds under physiological pH conditions, and they are commonly used in biological applications. However, consecutive guanine bases in the antiparallel motif can occasionally produce other higher-order structures, thereby hindering the triplex formation of TFOs with the target DNA. Therefore, we focus on parallel triplexes and attempt to devise an approach to address the abovementioned challenges. Researchers have explored various modifications to the sugar, phosphate, and base moieties of TFOs to address the primary challenges of pH-dependent thermodynamic stability and the need for polypurine sequences in target genes for parallel triplexes. 11 – 27 Notably, Brown et al. incorporated a thiazole orange (TO) intercalator into the thymine nucleobase of parallel TFOs. 25 This modification significantly increased the melting temperature (Tm) of a parallel triplex containing a TFO with three TO units and a target duplex with a single pyrimidine-base-interrupting site, even under neutral conditions (pH 7.0). Such an approach can broaden the scope of target duplexes accessible under physiological conditions; thus, it warrants further exploration, alongside other gene-directed control strategies using TFOs. Among DNA intercalators, we focus on psoralen, a well-known DNA photo-crosslinking agent. Psoralen derivatives react with pyrimidine bases through a [2 + 2] photo-cycloaddition reaction upon photo-irradiation, forming a cyclobutane ring to give monoadduct or diadduct products. 27 The crosslinking ability of psoralen has been employed to enhance and control the biological activities of TFOs. 6 , 28 – 37 Recently, our group demonstrated the inhibition of endogenous gene expression using a TFO equipped with a psoralen moiety at its 5’-end (5’-Ps-TFO) (Fig. 2 ). This illustrated that UV (365 nm) irradiation induces the crosslinking of a 5’-Ps-TFO to its target DNA, thereby significantly reducing the target gene expression. 6 j Therefore, employing psoralen for triplex stabilization is expected to enhance the biological efficacy of TFOs. In this study, we design a compound with psoralen positioned at the C-1 position of deoxyribose (1’-psoralen-conjugated deoxyribose, P ). We introduce it into the mismatch base-pair formation site in the TFO (Fig. 2 ). This proposed TFO, termed 1’(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a psoralen moiety to enhance its intercalation into the target DNA. We hypothesize that introducing P into the mismatch site of TFO can improve the thermodynamic stability of the triplex through intercalation, and psoralen can form interstrand crosslinking at the mismatch site. As the target sequence of OPTO, we select a partial sequence from the 5’-long terminal repeats (LTRs) of the human T-cell leukemia virus type 1 (HTLV-1) genome. The 5’-LTR contains critical elements for HTLV-1 replication, including promoter and enhancer sequences. HTLV-1 is responsible for adult T-cell leukemia, HTLV-1-associated myelopathy, and neurological disorders. 38 – 40 Upon HTLV-1 infection, the virus genome integrates into the host genome (provirus genome), leading to the persistent expression of viral genes. Considering the latent nature of the virus, eliminating this viral sequence from the host genome is crucial for achieving a cure for these diseases. However, current therapeutic approaches show limited efficacy. 41 – 42 If TFOs can form stable triplexes with the 5′-LTR of the HTLV-1 provirus genome, they can serve as potent candidates for the radical treatment of these diseases 43 through the further engineering of TFO, 44 using the TFO as a scaffold. However, the polypurine target (PPT) sequence in the 5′-LTR region of HTLV-1 45 contains at least two mismatched base pairs (Supplementary Fig. 1), posing a challenge for conventional TFOs to form stable triplexes. Therefore, these sequences are ideal targets for demonstrating the efficacy of the OPTO method. The OPTO formed more stable triplex with this target sequence than conventional TFO and crosslinked to the target DNA duplex upon UV irradiation. These results indicate that the use of the OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation. Results and Discussion Synthesis and functional evaluation of the 1’(one)-psoralen-conjugated triplex-forming oligonucleotide The synthesis of P began with the introduction of a cyano group into Hoffer’s chlorosugar ( 1 ), following a procedure described in the literature 46 (Scheme 1 ). The treatment of 1 with BF 3 ·OEt 2 and cyanotrimethylsilane in CH 2 Cl 2 afforded 2 in 58% yield. Compound 2 was prepared using a reported procedure, 47 with slight modifications. The subsequent treatment of 2 with sodium methoxide in MeOH/H 2 O led to nitrile hydrolysis and in situ esterification, consequently removing the toluoyl group and forming 3 in 68% yield. The two hydroxyl groups of ester 3 were protected with tert-butyldimethylsilyl (TBS) to produce 4 in 71% yield, followed by the hydrolysis of ester 4 to obtain carboxylic acid 5 in 98% yield. It was hypothesized that a C5 linker would be suitable for psoralen intercalation (Supplementary Fig. 2). Thus, linker 6 was introduced through amide condensation to produce 7 in 76% yield. Additionally, a C4 linker version of this compound was synthesized (Supplementary Methods). The primary alcohol of amide 7 was activated by converting it into a methanesulfonic acid ester ( 8 ) in 87% yield. The nucleophilic substitution of the Ms group in 8 with psoralen yielded amide 9 in 96% yield. The deprotection of the two TBS groups in 9 using tetrabutylammonium fluoride (TBAF) afforded 10 in 70% yield. For the solid-phase synthesis of OPTO, the primary alcohol of 10 was protected with a 4,4′-dimethoxytrityl group to produce 11 in 86% yield. Finally, the reaction of 11 with 1H-tetrazole and 2-cyanoethyl N , N , Nʹ , Nʹ -tetraisopropylphosphordiamidite in CH 2 Cl 2 afforded phosphoramidite ( 12 ) in 87% yield. Subsequently, 12 was employed in the solid-phase synthesis of the OPTO conducted at Ajinomoto Genedesign (Osaka, Japan), as detailed in the Supplementary method. Thereafter, the functional evaluations of the OPTO were performed. Based on the G content ratio and location, we chose a target duplex (606-Py/606-Pu) in the promoter region as the PPT. The sequences of the PPT duplex (606-Py/606-Pu), normal TFO, and OPTO are illustrated in Fig. 3 a. The OPTO incorporated P (Fig. 3 b) at the mismatch sites of the duplex, whereas normal TFO had a thymine base. The T m values of each triplex, (duplex/TFO) and (duplex/OPTO), were determined from UV-melting profiles at pH 5.3 (Fig. 3 c), and a biphasic melting profile with distinct first and second transitions was revealed. The first transition represented the triplex, and the T m values for the triplexes were 16°C (duplex/TFO) and 21°C (duplex/OPTO). Introducing P enhanced the thermodynamic stability of the triplex by 5°C, and the linker length (C4 or C5) of P did not significantly affect the stability of the triplex (Supplementary Fig. 3). Therefore, the C5 linker was selected for subsequent experiments. Synergistic effect of the locked nucleic acid and 1’(one)-psoralen-conjugated triplex-forming oligonucleotide on triplex stability Considering physiological conditions (37°C, pH 7.0), the previously described T m value of OPTO was insufficient. To enhance the thermodynamic stability of the triplex at the same PPT site, we explored a combinatorial approach using P along with other artificial nucleotides. In our previous work, we used locked nucleic acid (LNA) 11 , 48 – 49 with a bridged 2’-O,4’-C-methylene linkage structure (Fig. 4 a) for stable parallel triplex formation for gene suppression. 37 The bridged structure of the LNA leads to N-type conformation, which induces a preorganized conformation of TFO similar to that of a triplex, resulting in increased binding stability. We incorporated LNA into the OPTO sequence and evaluated the T m values of the LNA-incorporated OPTO (L-OPTO1–4 = L1–4). The consecutive incorporation of LNA into a TFO can decrease the thermodynamic stability of the resulting triplex. 48 Thus, we employed an LNA mixmer as the TFO. Additionally, we used 5-methylcytosine (5mC), which has more basic N 3 than cytosine, to enhance the pH-dependent thermodynamic stability of the parallel TFO 50 (Fig. 4 b). After incorporating LNAs and 5mCs into the TFO (L-TFO), the T m value of the triplex containing L-TFO became 36°C at pH 7.0, whereas the unmodified TFO did not form a triplex at pH 7.0 (Fig. 4 c). Next, we investigated the effect of P at the mismatch site of L-TFO. Introducing one P at the mismatch site positioned between LNA (L-OPTO1) significantly enhanced the stability of the corresponding triplex ( T m = 45°C). Further, introducing one P at another mismatch site between natural nucleotides (L-OPTO2) increased the stability of the corresponding triplex ( T m = 38°C). However, the stabilizing effect of P was less pronounced compared with that in L-OPTO1. The corresponding triplex was further stabilized when P was introduced into both mismatch sites (L-OPTO3) ( T m = 50°C). Additionally, we introduced P at both the 3’ and 5’ ends of the sequence (L-OPTO4) and measured the T m . The normalized UV-melting curves indicated a single transition (Fig. 4 c, L4), suggesting the simultaneous dissociation of L-OPTO4 and the target duplex. To confirm this, we conducted the nondenaturing polyacrylamide gel electrophoresis (Native PAGE) of triplexes (duplex/L-TFO) and (duplex/(L-OPTO1–4 = L1–4)) (Fig. 4 d). The DNA samples were stained with SYBR® Gold stain (Thermo Fisher Scientific, USA) and detected as bands. The band position shifted upward proportionally to the stability of the triplex. Specifically, the triplex band of L-OPTO4 remained detectable at 37°C, whereas that of L-OPTO3 was smear indicating the triplex dissociation under experimental condition. The greater stability of the L-OPTO4 triplex compared with that of L-OPTO3 was confirmed and the estimated T m value of the L-OPTO4 triplex exceeded 60°C. Next, we investigated the impact of the positional difference of the mismatch site in the target duplex on the stability of the triplex structure to demonstrate the versatility of this approach (Fig. 5 ). We assessed the triplex formation using four PPT sequences, each with varying numbers of bases between two mismatch sites (Fig. 5 a). The triplex formed with L-OPTO5 was significantly more stable than the corresponding LNA-incorporated TFO (L-TFO2) (Fig. 5 b). For L-TFO3–5, a weak melting transition of the triplex was observed, making it challenging to accurately determine the T m values. In contrast, the UV-melting curves of each corresponding L-OPTO6–8 displayed almost a single transition like sigmoid curve, indicating stable triplex formation. This was further confirmed by Native PAGE analysis (Fig. 5 c). We also explored the effect of the position of the LNA (Supplementary Fig. 4–5: L-OPTO9-14), revealing that the overall conformation was crucial in achieving synergistic effects (it is not necessary to introduce P between the LNA). Notably, L-OPTO13, which contained four P in its sequence, also formed a significantly stable triplex with the target duplex (Supplementary Fig. 4). We assessed the specificity of this method by substituting mismatched pyrimidine base C with purine bases A or G. The thermodynamic stabilities of the corresponding triplexes were nearly identical (Supplementary Fig. 6), indicating that this stabilization effect did not rely on specific interactions between the bases and psoralen. Next, we exchanged the bases in the target duplex at adjacent or distal positions of the mismatch site and evaluated the thermodynamic stability of the corresponding triplex (Supplementary Fig. 7). Resultantly, the T m values of all of the corresponding triplexes drastically decreased, indicating that the replacement of the mismatched pyrimidine base with P did not compromise the specificity of TFO. Photo-crosslinking properties of the locked nucleic acid-incorporated 1’(one)-psoralen-conjugated triplex-forming oligonucleotide Crosslinking formation is an important factor for obtaining significant biological outcomes using TFO. We successfully demonstrated that introducing the proposed nucleotide analog ( P ) into TFO was effective in the stable triplex formation, which is a prerequisite factor for the photo-crosslinking of psoralen with the target duplex. Therefore, finally, we investigated the crosslinking profiles of OPTO using denaturing PAGE. As shown in Fig. 4 – 5 , our design allows us to select sequences with multiple pyrimidine bases as the target of TFO. Therefore, we examined whether or not we could increase the photo-adduct product in proportion to the number of psoralens that was introduced into the TFO sequence (Fig. 6 ). Each triplex sample was irradiated using blue LED light (365 nm), and the samples were collected at each irradiation time (0, 1, 5, 10, 15, 20, 25, and 30 s). The 3’ end of each L-OPTO was labeled with tetramethylrhodamine (TAMRA). Resultantly, two clusters of the bands of the crosslinked products were observed. In case of L-OPTO1 and L-OPTO2, we can simply define the top band clusters as monoadduct (OPTO crosslinked to either 606-Py or 606-Pu) and second band clusters as diadduct (OPTO crosslinked to both 606-Py and 606-Pu) by the mobility of the band because they have only one target site for photo-crosslinking. On the other hand, there is a possibility that crosslink formation at more than two sites restrain the extension of DNA complex under denaturing condition, and the band mobility will not be the same as the migration of DNA through a polyacrylamide gel depends on end-to-end distance of the DNA. Therefore, we conducted extensive photo-crosslinking study to analyze the composition of the photo-crosslinked product using either TAMRA-labeled 606-Py or 606-Pu; indicating that the second band clusters of photo-crosslinked product of L-OPTO3 and L-OPTO4 contained three strand complex (Supplementary Fig. 8). As we expected, the total crosslinked product formation rate increased in proportion to the number of psoralens in the L-OPTO sequence. 18% of L-OPTO1, 31% of L-OPTO3, and 47% of L-OPTO4 were consumed to form crosslinked product after 1 s of photo-irradiation. This might be attributed to both the number of psoralens in the sequence and the thermodynamic stability of the corresponding triplex. We also tested the crosslinking efficiency with other base combinations, it was observed that the efficient diadduct formation at single site required a 5ʹ-TA-3ʹ sequence in both target DNA strands (Supplementary Fig. 9–10). Generally, thymine base is more reactive than cytosine. This was experimentally examined in our previous study. 51 – 53 Presumably, the 5-methyl group of thymine is important for the stabilization of the radical intermediate of the [2 + 2] photo-cycloaddition reaction, and/or the hydrophobic interaction between the 5-methyl group of thymine and psoralen may contribute to the relatively high crosslinking efficiency. The photo-induced electron transfer process from the adjacent guanine base also decreases the propensity of the photo-addition of psoralen to DNA. 54 In any event, this crosslink formation is expected to enhance the biological activities of TFO upon photo-irradiation. Thus, ongoing efforts will focus on photo-dynamic gene regulation using the L-OPTO. Conclusion In summary, we developed a new approach enabling stable parallel triplex formation with pyrimidine-base-interrupting sequences under physiological conditions, leveraging the synergistic effects of P and LNA. OPTO formed stable triplexes with the 5′-LTR partial sequence of HTLV-1 and other target sequences, demonstrating the versatility of the OPTO method and its potential for expanding the range of target sequences for parallel TFO applications. Furthermore, we conducted detailed studies on the photo-crosslinking formation of OPTO and demonstrated that the internal pyrimidine base can be a target for the photo-crosslinking of psoralen. Ongoing efforts will focus on further optimizing this method and exploring photo-dynamic gene regulation using L-OPTO, with results expected in upcoming reports. Methods Chemical synthesis Detailed protocols for the synthesis of all compounds and oligonucleotides can be found in the Supporting Information of this article. Triplex formation analysis using non-denaturing polyacrylamide gel electrophoresis (Native PAGE) Sample solutions containing the ds-DNAs and TFOs (1.0 µM each, 10 mM sodium phosphate (pH 7.0), 200 mM NaCl, 0.1 mM EDTA) were denatured by heating to 95°C and cooling to 4°C at 0.5°C/min. The annealed sample was diluted with 40 wt% sucrose aq (sample: 40 wt% sucrose aq = 1:4 (v/v)). The samples were analyzed with 20% native polyacrylamide gel (PAGE) containing 5 mM Mg2 + in TBM (20–23°C, 37°C, 120 V, 90 min). The DNA bands were stained by SYBR® Gold and the gels were transferred to imaging plates. The gel images were analyzed and quantitated using ChemDoc Touch MP (BioRad, CA, U.S.A.). Evaluation of photo-crosslinking efficiency of L-OPTOs by denaturing polyacrylamide gel electrophoresis (PAGE) Sample solutions containing the ds-DNAs and TAMRA-labeled L-OPTOs (1.0 µM each, 10 mM sodium phosphate (pH 7.0), 200 mM NaCl, 0.1 mM EDTA) were denatured by heating to 95°C and cooling to 4°C at 0.5°C/min. The annealed solution was applied to a 368-well plate and irradiated by a UV spotlight (ZUV-C30H (365 nm); Omron Corp., Kyoto, Japan) at 37°C. The UV irradiated samples was diluted with formamide (sample: formamide = 1:4 (v/v)). The samples were analyzed by 15% denaturing polyacrylamide gel (PAGE) containing 7 M urea and formamide (200 V, 45 min). The gels were transferred to imaging plates, and the resulting gel images were analyzed and quantitated using by ChemDoc Touch MP (BioRad, CA, U.S.A.). Declarations Data availability The authors declare that all data supporting the findings of this study are available within the article and the Supplementary Information. Competing interests The authors declare no competing interest. Additional information The authors declare no competing interest. Author contributions YM and AY conceptualized and supervised the project with the assistance of CD and TW. YM synthesized all compounds. The functional evaluation experiments of OPTO were performed by YM and HT. The manuscript was written by the contribution of all authors. Acknowledgements This study was financially supported by the Grant-in-Aid for Transformative Research Areas (A) “Material Symbiosis” (Grant Number:20H05874 awarded to A.Y.) from MEXT, Japan. This study was also supported by JSPS KAKENHI (Grant Numbers 22H00593 and 22K14839 to A.Y. and Y.M., respectively), Japan, and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices” (Grant Numbers 20224030 and 20228004 to A.Y. and Y.M., respectively). References Li, C., Zhou, Z., Ren, C., Deng, Y., Peng, F., Wang, Q., Zhang, H., Jiang, Y. Triplex-Forming Oligonucleotides as An Anti-Gene Technique for Cancer Therapy. Front. Pharmacol. 13, 1007723 (2022). Hewett, P. W., Daft, E. L., Laughton, C. A., Ahmad, S., Ahmed, A., Murray, J. C. Selective Inhibition of the Human Tie-1 Promoter with Triplex-Forming Oligonucleotides Targeted to Ets Binding Sites. Mol. Med. 12, 8–16 (2006). Karympalis, V., Kalopita, K., Zarros, A., Carageorgiou, H. Regulation of Gene Expression via Triple Helical Formations. Biochemistry 69, 855–860 (2004). Young, S. L., Krawczyk, S. H., Matteucci, M. D., Toole, J. J. Triple Helix Formation Inhibits Transcription Elongation in Vitro. Proc. Natl. Acad. Sci. USA 88, 10023–10026 (1991). Moser, H. E., Dervan, P. B. Sequence-Specific Cleavage of Double Helical DNA by Triple Helix Formation. Science 238, 645–650 (1987). Faruqi, A. F., Seidman, M. M., Segal, D. J., Carroll, D., Glazer, P. M. Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells. Mol. Cell. Biol. 16, 6820–6828 (1996). Kaushik Tiwari, M., Rogers, F. A. XPD-dependent activation of apoptosis in response to triplex-induced DNA damage. Nucleic Acids Res. 41, 8979–8994 (2013). Kaushik Tiwari, M., Colon-Rios, D. A., Rao Tumu, H. C., Liu, Y., Quijano, E., Krysztofiak, A., Chan, C., Song, E., Braddock, D. T., Suh, H.-W.; et al. Direct Targeting of Amplified Gene Loci for Proapoptotic Anticancer Therapy. Nat. Biotechnol. 40, 325–334 (2022). Kaushik Tiwari, M., Adaku, N., Peart, N., Rogers, F. A. Triplex Structures Induce DNA Double Strand Breaks via Replication Fork Collapse in NER Deficient Cells. Nucleic Acids Res 44, 7742–7754 (2016). Nagatsugi, F., Sasaki, S., Miller, P. S., Seidman, M. M. Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog. Nucleic Acids Res. 31, e31 (2003). Torigoe, H., Hari, Y., Sekiguchi, M., Obika, S., Imanishi, T. 2'- O ,4'- C -methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies. J. Biol. Chem. 276, 2354–2360 (2001). Hartono, Y. D., Pabon-Martinez, Y. V., Uyar, A., Wengel, J. K., Lundin, K. E., Zain, R., Smith, C. I. E., Nilsson, L., Villa, A. Role of pseudoisocytosine tautomerization in triplex-forming oligonucleotides: In silico and in vitro studies. ACS Omega 2, 2165–2177 (2017). Rusling, D. A. Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH. Nucleic Acids Res 49, 7256–7266 (2021). Verma, S.; Miller, P. S. Interactions of Cytosine Derivatives with T·A Interruptions in Pyrimidine·Purine·Pyrimidine DNA Triplex. Bioconjugate Chem 7, 600–605 (1996). Guianvarc’h, D., Benhida, R., Fourrey, J. L., Maurisse, R., Sun, J. S. Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine·pyrimidine interruption. Chem. Commun. 18, 1814–1815 (2001). Ohkubo, A., Ohnishi, T., Nishizawa, S., Nishimura, Y., Hisamatsu, S. The ability of a triplex-forming oligonucleotide to recog-nize T-A and C-G base pairs in a DNA duplex is enhanced by incorporating N-acetyl-2,7-diaminoquinoline. Bioorg. Med. Chem. 28, 115350 (2020). Gerrard, S. R., Edrees, M. M., Bouamaied, I., Fox, K. R., Brown, T. CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides. Org. Biomol. Chem. 8, 5087–5096 (2010). Hari, Y., Akabane, M., Obika, S. 2’-4’-BNA bearing a chiral guanidinopyrrolidine-containing nucleobase with potent ability to recognize the C:G base pair in a parallel-motif DNA triplex. Chem. Commun. 49, 7421–7423 (2013). Obika, S., Uneda, T., Sugimoto, T., Nanbu, D., Minami, T., Doi, T., Imanishi, T. 2’-O,4’-C-methylene bridge nucleic acid (2’,4’-BNA): Synthesis and triplex-forming properties. Bioorg. Med. Chem. 9, 1001–1011 (2001). Sasaki, S., Taniguchi, Y., Takahashi, R., Senko, Y., Kodama, K., Nagatsugi, F., Maeda, M. Selective Formation of Stable Triplexes Including a TA or a CG Interrupting Site with New Bicyclic Nucleoside Analogue (WNA). J. Am. Chem. Soc. 126, 516–528 (2004). Inde, T., Nishizawa, S., Hattori, Y., Kanamori, T., Yuasa, H., Seio, K., Sekine, M., Ohkubo, A. Synthesis of and triplex formation in oligonucleotides containing 2’-deoxy-6-thioxanthosine. Bioorg. Med. Chem. 26, 3785–3790 (2018). Wang, E., Koshlap, K.M., Gillespie, P., Dervan, P.B., Feigon, J. Solution Structure of a Pyrimidine·Purine·Pyrimidine Triplex Con-taining the Sequence-specific Intercalating Non-natural Base D3. J. Mol. Biol. 257, 1052–1069 (1996). Carbone, G. M., McGuffie, E., Napoli, S., Flanagan, C. E., Dembech, C., Negri, U., Arcamone, F., Capobianco, M. L., Catapano, C. V. DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene. Nucleic Acids Res 32, 2396–2410 (2004). Paramasivam, M., Cogoi, S., Filichev, V. V., Bomholt, N., Pedersen, E. B., Xodo, L. E. Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation. Nucleic Acids Res 36, 3494–3507 (2008). Walsh, S., El-Sagheer, A. H., Brown, T. Fluorogenic thiazole orange TOTFO probes stabilize parallel DNA triplexes at pH 7 and above. Chem. Sci. 9, 7681–7687 (2018). Kumar, V., Kesavan, V., Gothelf, K. V. Highly stable triple helix formation by homopyrimidine (L)-acyclic threoninol nucleic acids with single stranded DNA and RNA. Org. Biomol. Chem. 13, 2366–2374 (2015). Buhimschi, D. A., Gooden, M. D., Jing, H., Fels, R. D., Hansen, S. K., Beyer, F. W., Dewhirst, W. M., Walder, H., Gasparro, P. F. Psoralen Derivatives with Enhanced Potency. Photochem. Photobiol. 96, 1014–1031 (2020). Duval-Valentin, G., Thuong, N. T., Hélène, C. Specific inhibition of transcription by triple helix-forming oligonucleotides. Proc. Natl. Acad. Sci. 89, 504–508 (1992). Grigoriev, M., Praseuth, D., Guieysse, A. L., Robin, P., Thuong, N. T., Hélène, C., Harel-Bellan, A. Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites. Proc. Natl. Acad. Sci . 90, 3501–3505 (1993). Havre, P. A., Gunther, E., Gasparro, F., Glazer, P. M. Targeted Mutagenesis of DNA Using Triple Helix-Forming Oligonucleotides Linked to Psoralen. Proc. Natl. Acad. Sci. USA 90, 7879–7883 (1993). Raha, M., Lacroix, L., Glazer, P. M. Mutagenesis Mediated by Triple Helix-Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context. Photochem. Photobiol. 67, 289–294 (1998). Faria, M., Wood, C. D., Perrouault, L., Nelson, J. S., Winter, A., White, M. R. H., Hélène, C., Giovannangeli, C. Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides. Proc. Natl. Acad. Sci . 97, 3862–3867 (2000). Diviacco, S., Rapozzi, V., Xodo, L., Hélène, C., Quadrifoglio, F., Giovannangeli, C. Site-directed inhibition of DNA replication by triple helix formation. FASEB J. 15, 2660–2668 (2001). Majumdar, A., Muniandy, P. A., Liu, J., Liu, J.-I., Liu, S.-T., Cuenoud, B., Seidman, M. M. Targeted Gene Knock-In and Sequence Modulation Mediated by a Psoralen-Linked Triplex-Forming Oligonucleotide. J. Biol. Chem. 283, 11244–11252 (2008). Liu, J., Majumdar, A., Liu, J., Thompson, L. H., Seidman, M. M. Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells. J. Biol. Chem. 285, 23198–23207 (2010). Semenyuk, A., Darian, E., Liu, J., Majumdar, A., Cuenoud, B., Miller, P. S., MacKerell, A. D., Seidman, Jr, M. M. Biochemistry 49, 7867–7878 (2010). Mikame, Y., Eshima, H., Toyama, H., Nakao, J., Matsuo, M., Yama-moto, T., Hari, Y., Komano, J. A., Yamayoshi, A. Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA. ChemMedChem 18, e202300348 (2023). Nagai, M., Osame, M. Human T-cell lymphotropic virus type I and neurological diseases. J Neurovirol 9 , 228–235 (2003). Watanabe, T. HTLV-1-associated diseases. Int J Hematol 66 , 257–278 (1997). Yasunaga, J., Matsuoka, M. Molecular mechanisms of HTLV-1 infection and pathogenesis. Int J Hematol 94, 435–442 (2011). Faris, M. Potential for molecular targeted therapy for adult T-cell leukemia/lymphoma. Int Rev Immunol 27, 71–78 (2008). Taylor, G. P., Matsuoka, M. Natural history of adult T-cell leukemia/lymphoma and approaches to therapy. Oncogene 24, 6047–6057 (2005). Tanaka, A., Takeda, S., Kariya, R., Matsuda, K., Urano, S., Okada, S., Komano, J. A novel therapeutic molecule against HTLV-1 infection taregeting provirus. Leukemia 27, 1621–1627 (2013). Mikame, Y., Yamayoshi, A. Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids. Pharmaceutics 15 2515 (2023). Inoue, J., Watanabe, T., Sato, M., Oda, A., Toyoshima, K., Yoshida, M., Seiki, M. Nucleotide sequence of the protease-coding region in an infectious DNA of simian retrovirus (STLV) of the HTLV-I family. Virology 150, 187–195 (1986). Jazouli, M., Guianvarc’h, D., Soufiaoui, M., Bougrin, K., Vierling, P., Benhida, R. A Short and efficient synthesis of 2ʹ-deoxybenzo and pyridoimidazole C-nucleosides. Tetrahedron Letters 44, 5807–5810 (2003). Martín-Nieves, V., Fàbrega, C., Guasch, M., Fernández, S., Sanghvi, Y. S., Ferrero, M., Eritja, R. Oligonucleotides Containing 1-Aminomethyl or 1-Mercaptomethyl-2-deoxy-D-ribofuranoses: Synthesis, Purification, Characterization, and Conjugation with Fluorophores and Lipids. Bioconjugate Chem 32, 350–366 (2021). Obika, S., Uneda, T., Sugimoto, T., Nanbu, D., Minami, T., Doi, T., Imanishi, T. 2'-O,4'-C-Methylene bridged nucleic acid (2',4'-BNA): synthesis and triplex-forming properties. Bioorg. Med. Chem. 9, 1001–1011 (2001). Brunet, E., Alberti, P., Perrouault, L., Babu, R., Wengel, J., Giovannangeli, C. Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis. J. Biol. Chem. 280, 2007–20086 (2005). Lee, J. S., Woodsworth, M. L., Latimer, L. J., Morgan, A. R. Poly(pyrimidine)·poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. Nucleic Acids Res 12, 6603–6614 (1984). Yamayoshi, A., Matsuyama, Y., Kushida M., Kobori, A., Murakami A. Novel Photodynamic Effect of a Psoralen-Conjugated Oligonucleotide for the Discrimination of the Methylation of Cytosine in DNA. Photochem. Photobiol. 90, 716–722 (2014). Kojima, A., Nakao, J., Shimada, N., Yoshida, N., Abe, Y., Mikame, Y., Yamamoto, T., Wada, T., Maruyama, A., Yamayoshi, A. Selective Photo-Crosslinking Detection of Methylated Cytosine in DNA Duplex Aided by a Cationic Comb-Type Copolymer. ACS Biomater. Sci. Eng. 8, 1799–1805 (2022). Nakao, J., Mikame, Y., Eshima, H., Yamamoto, T., Dohno, C., Wada, T., Yamayoshi, A. Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N -Hydroxysuccinimide Esters. ChemBioChem e202200789 (2023). Bertling, J., Thom, K. A., Geenen, S., Jeuken, H., Presser, L., Müller, T. J. J., Gilch, P. Synthesis and Photophysics of Water-Soluble Psoralens with Red-Shifted Absorption. Photochem. Photobiol. 97, 1534–1547 (2021). Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files CommunicationChemistrySuppXXXXXXXXXXXXXXX.pdf scheme1.png Scheme 1. Synthesis of 1’-psoralen-conjugated deoxyribose phosphoramidite (12). Compound 12 was prepared by 10 steps from Hoffer’s chlorosugar (1) as a starting material. Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5384273","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":377237925,"identity":"bd3ea513-db7b-4164-a380-24c3a8459670","order_by":0,"name":"Yu Mikame","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYLCChANAgr0BwpFghooyNhDSwnOAFC0MINUSCVAthNxkcCOBdcODM3bR/JJvD378UlMnL9nOwCbBUGPHwDwbuzVALWw3Em4k586cnZcsLXPssOFsZpCWY8kMjHMO4NHygTl3w+0cA2kJtgOM85j5v0kwsB1gYJyRgE9Lfe7+m2eMf0v8q7OfB7blHyEtNw7nbpDgMZP82MacCHYYYxtuLZJnHgC1nDmeO+NMjpk1Y9/h5JnNDMwWiX3JPLj8wnc8ge3mj2PVuf3tZ4xv/vhWZzvj/AHGGx++2ckZ4ggxhQP8H+AcZh4YC+gkHsMZWHUwyCObxPgDRYpgpI6CUTAKRsEIAQDTamLz55hfQQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5239-533X","institution":"Nagasaki University","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Mikame","suffix":""},{"id":377237926,"identity":"c9a282fa-74f8-4ce1-9beb-cfc447900734","order_by":1,"name":"Haruki Toyama","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haruki","middleName":"","lastName":"Toyama","suffix":""},{"id":377237927,"identity":"e59dab2b-ab71-4c2d-83b4-42ab66e03240","order_by":2,"name":"Chikara Dohno","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chikara","middleName":"","lastName":"Dohno","suffix":""},{"id":377237928,"identity":"696980c2-ad63-4894-a666-1cb4d834cac0","order_by":3,"name":"Takehiko Wada","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Takehiko","middleName":"","lastName":"Wada","suffix":""},{"id":377237929,"identity":"41640b88-9fd0-4ca2-89d1-335f910dd5e8","order_by":4,"name":"Asako Yamayoshi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Asako","middleName":"","lastName":"Yamayoshi","suffix":""}],"badges":[],"createdAt":"2024-11-04 02:45:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5384273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5384273/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-025-01416-2","type":"published","date":"2025-01-22T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70569003,"identity":"4ac77371-36c8-4a9c-9fdf-1b6d0ea532b1","added_by":"auto","created_at":"2024-12-04 13:16:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":352613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural features of the triplex-forming oligonucleotides.\u003c/strong\u003ea) The parallel triplex forms via a T·A:T triad and C+·G:C triad. The antiparallel triplex forms via a (A or T)·A:T triad and G·G:C triad. b) Natural bases do not strongly recognize the T/A or C/G base pairs, making stable triplex formation with these pyrimidine-base-interrupting sequences challenging.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/2dd40deb31238908b559bbb6.png"},{"id":70569952,"identity":"6f9edea5-81ef-4b6a-8393-296793727ad4","added_by":"auto","created_at":"2024-12-04 13:32:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":322084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe working hypothesis of this research.\u003c/strong\u003e The established structure of a psoralen-conjugated triplex-forming oligonucleotide (TFO) with the proposed design (1’(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO)) is shown. The optimized orientation of psoralen in OPTO is better for intercalating psoralen into the target DNA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/8c8dce7698224941a7bb81ce.png"},{"id":70569007,"identity":"8d2e0fe5-4fe4-4434-9002-a3b1672c617f","added_by":"auto","created_at":"2024-12-04 13:16:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe UV-melting profile of the triplex of OPTO.\u003c/strong\u003e a) sequences of the polypurine target (PPT) duplex (606-Py/606-Pu), TFO, and OPTO. b) Structure of the 1’-psoralen-conjugated deoxyribose (P). c) Normalized UV-melting curves of triplexes (duplex/TFO) and (duplex/OPTO) at pH 5.3. The final concentration of duplex and TFO were 3 μM each. The melting temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) values are displayed as the mean ± standard deviation (s.d.) for n = 3 replicates.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/6bd32058b0a644fa5dcf9749.png"},{"id":70569009,"identity":"db8faabd-8543-4784-8b7d-218140fa40ef","added_by":"auto","created_at":"2024-12-04 13:16:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1098918,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe UV-melting profiles of\u003c/strong\u003e \u003cstrong\u003ethe triplex of L-OPTO.\u003c/strong\u003e a) Structures of locked nucleic acid (LNA) and the conformational differences of the ribose moiety. b) Sequences of the PPT duplex (606-Py/606-Pu), LNA-incorporated TFO (L-TFO), and LNA-incorporated OPTO (L-OPTO1–4). c) Normalized UV-melting curves of triplexes (duplex/L-TFO) and (duplex/(L-OPTO1–4 = L1–4)) at pH 7.0 and the corresponding \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of the triplexes. The final concentration of duplex and TFO were 3 μM each. The \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values are displayed as the mean ± s.d. for n = 3 replicates. d) Nondenaturing polyacrylamide gel electrophoresis (Native PAGE) of triplexes (duplex/L-TFO) and (duplex/(L-OPTO1–4 = L1–4)) at pH 7.0 and 31°C or 37°C. The final concentration of duplex and TFO were 1 μM each. The bands were detected using SYBR\u003csup\u003e®\u003c/sup\u003e Gold stain (Thermo Fisher Scientific, USA).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/93b2e962d16e56a4e2f78c34.png"},{"id":70569004,"identity":"0d48bb4d-20f4-442c-9d42-3d184ff8e74c","added_by":"auto","created_at":"2024-12-04 13:16:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":602060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe UV-melting profiles of\u003c/strong\u003e \u003cstrong\u003ethe triplex of L-OPTO\u003c/strong\u003e \u003cstrong\u003ewith varied sequences.\u003c/strong\u003e a) Sequences of four PPT duplexes (Py-1/Pu-1, Py-2/Pu-2, Py-3/Pu-3, and Py-4/Pu-4), LNA-incorporated TFO (L-TFO2–5), and LNA-incorporated OPTO (L-OPTO5–8). b) Normalized UV-melting curves of each triplex at pH 7.0 and their respective \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values. The \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of L6-8 were determined by differential method from the region between the dotted line. The final concentration of duplex and TFO were 3 μM each. The \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values are displayed as the mean ± s.d. for n = 3 replicates. c) Native PAGE of the triplexes containing L-OPTO6–8 (L6–8) at pH 7.0 and 37°C. The final concentration of duplex and TFO were 1 μM each. The bands were visualized using SYBR® Gold stain.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/349501b781cd630ef656f462.png"},{"id":70569777,"identity":"70a9bb65-0654-4293-a817-78422002e862","added_by":"auto","created_at":"2024-12-04 13:24:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":267616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe photo-crosslinking properties of L-OPTO.\u003c/strong\u003e a) Photo-crosslinking of psoralen with the pyrimidine base and the sequence of the PPT duplexes. The correlation between the crosslinking efficiency and the number of psoralen molecules that were introduced into the TFO sequence (L-OPTO2-4) was examined. b) The photo-irradiation (365 nm) was conducted at pH 7.0 and 37°C. The final concentration of duplex and TFO were 1 μM each. The samples were collected at each irradiation time (0, 1, 5, 10, 15, 20, 25, and 30 s), and the product formation was analyzed by denaturing \u003cstrong\u003eP\u003c/strong\u003e. The 3’ end of the L-OPTOs was labeled with tetramethylrhodamine (TAMRA). The graphs show the quantification results of the photo-crosslinking efficiency of each sequence. The data are displayed as the mean ± s.d. for n = 3 replicates\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/dfea380a8909cd642dfb8147.png"},{"id":74532899,"identity":"1af35a42-e280-4588-81a5-95537f47d0c1","added_by":"auto","created_at":"2025-01-23 08:06:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3616830,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/f0d5988e-0ddf-4bf0-b085-5dd919953488.pdf"},{"id":70569008,"identity":"0eb76bcd-4bf3-43f1-aca4-743115efee3c","added_by":"auto","created_at":"2024-12-04 13:16:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6808212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"CommunicationChemistrySuppXXXXXXXXXXXXXXX.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/6d79485d43bf01633e4a074b.pdf"},{"id":70569778,"identity":"338fd357-d7d0-4dc3-82c7-404621a6bdcc","added_by":"auto","created_at":"2024-12-04 13:24:58","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":78291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Synthesis of 1’-psoralen-conjugated deoxyribose phosphoramidite (12).\u003c/strong\u003e Compound \u003cstrong\u003e12\u003c/strong\u003e was prepared by 10 steps from Hoffer’s chlorosugar (\u003cstrong\u003e1\u003c/strong\u003e) as a starting material.\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-5384273/v1/9bd05d079f0aba0ac45ca633.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Development of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides: Evaluation of the triplex-forming and photo-crosslinking properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA triplex-forming oligonucleotide (TFO) binds to its target DNA duplex, forming a triple-helix structure. This triplex formation inhibits either the binding of transcription factors to promoter regions or transcriptional elongation, resulting in gene suppression.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Moreover, the cell recognizes the triplex structure as unusual, which induces a double-strand break (DSB) at the triplex-forming site by an endogenous nuclease.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This DSB induction by a TFO has been employed for genome editing or selectively inducing apoptosis in cancer cells.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e The interaction of a TFO with the target DNA occurs on the major groove side of the DNA via Hoogsteen (parallel triplex; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) or reverse Hoogsteen (antiparallel triplex; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) hydrogen bonds. The inherent challenge of the triplex technology is the requirement for Hoogsteen hydrogen bonds to form specifically between a purine base of A/T (or G/C) base pairs in the double-stranded DNA (dsDNA) and a TFO. If the target base pair in the dsDNA changes (T/A or C/G base pair), a mismatched base pair (pyrimidine-base-interrupting site) with the TFO is produced, significantly reducing the thermodynamic stability of the triplex structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the parallel motif, the \u003cem\u003eN\u003c/em\u003e3 position of cytosine must be protonated to facilitate hydrogen bonding. In contrast, antiparallel triplex oligonucleotides can form reverse Hoogsteen hydrogen bonds under physiological pH conditions, and they are commonly used in biological applications. However, consecutive guanine bases in the antiparallel motif can occasionally produce other higher-order structures, thereby hindering the triplex formation of TFOs with the target DNA. Therefore, we focus on parallel triplexes and attempt to devise an approach to address the abovementioned challenges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResearchers have explored various modifications to the sugar, phosphate, and base moieties of TFOs to address the primary challenges of pH-dependent thermodynamic stability and the need for polypurine sequences in target genes for parallel triplexes.\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Notably, Brown et al. incorporated a thiazole orange (TO) intercalator into the thymine nucleobase of parallel TFOs.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e This modification significantly increased the melting temperature (Tm) of a parallel triplex containing a TFO with three TO units and a target duplex with a single pyrimidine-base-interrupting site, even under neutral conditions (pH 7.0). Such an approach can broaden the scope of target duplexes accessible under physiological conditions; thus, it warrants further exploration, alongside other gene-directed control strategies using TFOs. Among DNA intercalators, we focus on psoralen, a well-known DNA photo-crosslinking agent. Psoralen derivatives react with pyrimidine bases through a [2\u0026thinsp;+\u0026thinsp;2] photo-cycloaddition reaction upon photo-irradiation, forming a cyclobutane ring to give monoadduct or diadduct products.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The crosslinking ability of psoralen has been employed to enhance and control the biological activities of TFOs.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Recently, our group demonstrated the inhibition of endogenous gene expression using a TFO equipped with a psoralen moiety at its 5\u0026rsquo;-end (5\u0026rsquo;-Ps-TFO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This illustrated that UV (365 nm) irradiation induces the crosslinking of a 5\u0026rsquo;-Ps-TFO to its target DNA, thereby significantly reducing the target gene expression.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003ej\u003c/sup\u003e Therefore, employing psoralen for triplex stabilization is expected to enhance the biological efficacy of TFOs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we design a compound with psoralen positioned at the C-1 position of deoxyribose (1\u0026rsquo;-psoralen-conjugated deoxyribose, \u003cb\u003eP\u003c/b\u003e). We introduce it into the mismatch base-pair formation site in the TFO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This proposed TFO, termed 1\u0026rsquo;(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a psoralen moiety to enhance its intercalation into the target DNA. We hypothesize that introducing \u003cb\u003eP\u003c/b\u003e into the mismatch site of TFO can improve the thermodynamic stability of the triplex through intercalation, and psoralen can form interstrand crosslinking at the mismatch site. As the target sequence of OPTO, we select a partial sequence from the 5\u0026rsquo;-long terminal repeats (LTRs) of the human T-cell leukemia virus type 1 (HTLV-1) genome. The 5\u0026rsquo;-LTR contains critical elements for HTLV-1 replication, including promoter and enhancer sequences. HTLV-1 is responsible for adult T-cell leukemia, HTLV-1-associated myelopathy, and neurological disorders.\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Upon HTLV-1 infection, the virus genome integrates into the host genome (provirus genome), leading to the persistent expression of viral genes. Considering the latent nature of the virus, eliminating this viral sequence from the host genome is crucial for achieving a cure for these diseases. However, current therapeutic approaches show limited efficacy.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e If TFOs can form stable triplexes with the 5\u0026prime;-LTR of the HTLV-1 provirus genome, they can serve as potent candidates for the radical treatment of these diseases\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e through the further engineering of TFO,\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e using the TFO as a scaffold. However, the polypurine target (PPT) sequence in the 5\u0026prime;-LTR region of HTLV-1\u003csup\u003e45\u003c/sup\u003e contains at least two mismatched base pairs (Supplementary Fig.\u0026nbsp;1), posing a challenge for conventional TFOs to form stable triplexes. Therefore, these sequences are ideal targets for demonstrating the efficacy of the OPTO method. The OPTO formed more stable triplex with this target sequence than conventional TFO and crosslinked to the target DNA duplex upon UV irradiation. These results indicate that the use of the OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and functional evaluation of the 1\u0026rsquo;(one)-psoralen-conjugated triplex-forming oligonucleotide\u003c/h2\u003e \u003cp\u003eThe synthesis of \u003cb\u003eP\u003c/b\u003e began with the introduction of a cyano group into Hoffer\u0026rsquo;s chlorosugar (\u003cb\u003e1\u003c/b\u003e), following a procedure described in the literature\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The treatment of \u003cb\u003e1\u003c/b\u003e with BF\u003csub\u003e3\u003c/sub\u003e\u0026middot;OEt\u003csub\u003e2\u003c/sub\u003e and cyanotrimethylsilane in CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e afforded \u003cb\u003e2\u003c/b\u003e in 58% yield. Compound \u003cb\u003e2\u003c/b\u003e was prepared using a reported procedure,\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e with slight modifications. The subsequent treatment of \u003cb\u003e2\u003c/b\u003e with sodium methoxide in MeOH/H\u003csub\u003e2\u003c/sub\u003eO led to nitrile hydrolysis and in situ esterification, consequently removing the toluoyl group and forming \u003cb\u003e3\u003c/b\u003e in 68% yield. The two hydroxyl groups of ester \u003cb\u003e3\u003c/b\u003e were protected with tert-butyldimethylsilyl (TBS) to produce \u003cb\u003e4\u003c/b\u003e in 71% yield, followed by the hydrolysis of ester \u003cb\u003e4\u003c/b\u003e to obtain carboxylic acid \u003cb\u003e5\u003c/b\u003e in 98% yield. It was hypothesized that a C5 linker would be suitable for psoralen intercalation (Supplementary Fig.\u0026nbsp;2). Thus, linker \u003cb\u003e6\u003c/b\u003e was introduced through amide condensation to produce \u003cb\u003e7\u003c/b\u003e in 76% yield. Additionally, a C4 linker version of this compound was synthesized (Supplementary Methods). The primary alcohol of amide \u003cb\u003e7\u003c/b\u003e was activated by converting it into a methanesulfonic acid ester (\u003cb\u003e8\u003c/b\u003e) in 87% yield. The nucleophilic substitution of the Ms group in \u003cb\u003e8\u003c/b\u003e with psoralen yielded amide \u003cb\u003e9\u003c/b\u003e in 96% yield. The deprotection of the two TBS groups in \u003cb\u003e9\u003c/b\u003e using tetrabutylammonium fluoride (TBAF) afforded \u003cb\u003e10\u003c/b\u003e in 70% yield. For the solid-phase synthesis of OPTO, the primary alcohol of \u003cb\u003e10\u003c/b\u003e was protected with a 4,4\u0026prime;-dimethoxytrityl group to produce \u003cb\u003e11\u003c/b\u003e in 86% yield. Finally, the reaction of \u003cb\u003e11\u003c/b\u003e with 1H-tetrazole and 2-cyanoethyl \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eNʹ\u003c/em\u003e,\u003cem\u003eNʹ\u003c/em\u003e-tetraisopropylphosphordiamidite in CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e afforded phosphoramidite (\u003cb\u003e12\u003c/b\u003e) in 87% yield. Subsequently, \u003cb\u003e12\u003c/b\u003e was employed in the solid-phase synthesis of the OPTO conducted at Ajinomoto Genedesign (Osaka, Japan), as detailed in the Supplementary method. Thereafter, the functional evaluations of the OPTO were performed. Based on the G content ratio and location, we chose a target duplex (606-Py/606-Pu) in the promoter region as the PPT. The sequences of the PPT duplex (606-Py/606-Pu), normal TFO, and OPTO are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The OPTO incorporated \u003cb\u003eP\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) at the mismatch sites of the duplex, whereas normal TFO had a thymine base. The \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of each triplex, (duplex/TFO) and (duplex/OPTO), were determined from UV-melting profiles at pH 5.3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and a biphasic melting profile with distinct first and second transitions was revealed. The first transition represented the triplex, and the \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values for the triplexes were 16\u0026deg;C (duplex/TFO) and 21\u0026deg;C (duplex/OPTO). Introducing \u003cb\u003eP\u003c/b\u003e enhanced the thermodynamic stability of the triplex by 5\u0026deg;C, and the linker length (C4 or C5) of \u003cb\u003eP\u003c/b\u003e did not significantly affect the stability of the triplex (Supplementary Fig.\u0026nbsp;3). Therefore, the C5 linker was selected for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynergistic effect of the locked nucleic acid and 1’(one)-psoralen-conjugated triplex-forming oligonucleotide on triplex stability\u003c/h3\u003e\n\u003cp\u003eConsidering physiological conditions (37\u0026deg;C, pH 7.0), the previously described \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of OPTO was insufficient. To enhance the thermodynamic stability of the triplex at the same PPT site, we explored a combinatorial approach using \u003cb\u003eP\u003c/b\u003e along with other artificial nucleotides. In our previous work, we used locked nucleic acid (LNA)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e with a bridged 2\u0026rsquo;-O,4\u0026rsquo;-C-methylene linkage structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) for stable parallel triplex formation for gene suppression.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e The bridged structure of the LNA leads to N-type conformation, which induces a preorganized conformation of TFO similar to that of a triplex, resulting in increased binding stability. We incorporated LNA into the OPTO sequence and evaluated the \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of the LNA-incorporated OPTO (L-OPTO1\u0026ndash;4\u0026thinsp;=\u0026thinsp;L1\u0026ndash;4). The consecutive incorporation of LNA into a TFO can decrease the thermodynamic stability of the resulting triplex.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e Thus, we employed an LNA mixmer as the TFO. Additionally, we used 5-methylcytosine (5mC), which has more basic \u003cem\u003eN\u003c/em\u003e3 than cytosine, to enhance the pH-dependent thermodynamic stability of the parallel TFO\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After incorporating LNAs and 5mCs into the TFO (L-TFO), the \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of the triplex containing L-TFO became 36\u0026deg;C at pH 7.0, whereas the unmodified TFO did not form a triplex at pH 7.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Next, we investigated the effect of \u003cb\u003eP\u003c/b\u003e at the mismatch site of L-TFO. Introducing one \u003cb\u003eP\u003c/b\u003e at the mismatch site positioned between LNA (L-OPTO1) significantly enhanced the stability of the corresponding triplex (\u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 45\u0026deg;C). Further, introducing one \u003cb\u003eP\u003c/b\u003e at another mismatch site between natural nucleotides (L-OPTO2) increased the stability of the corresponding triplex (\u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 38\u0026deg;C). However, the stabilizing effect of \u003cb\u003eP\u003c/b\u003e was less pronounced compared with that in L-OPTO1. The corresponding triplex was further stabilized when \u003cb\u003eP\u003c/b\u003e was introduced into both mismatch sites (L-OPTO3) (\u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 50\u0026deg;C). Additionally, we introduced \u003cb\u003eP\u003c/b\u003e at both the 3\u0026rsquo; and 5\u0026rsquo; ends of the sequence (L-OPTO4) and measured the \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e. The normalized UV-melting curves indicated a single transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, L4), suggesting the simultaneous dissociation of L-OPTO4 and the target duplex. To confirm this, we conducted the nondenaturing polyacrylamide gel electrophoresis (Native PAGE) of triplexes (duplex/L-TFO) and (duplex/(L-OPTO1\u0026ndash;4\u0026thinsp;=\u0026thinsp;L1\u0026ndash;4)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The DNA samples were stained with SYBR\u0026reg; Gold stain (Thermo Fisher Scientific, USA) and detected as bands. The band position shifted upward proportionally to the stability of the triplex. Specifically, the triplex band of L-OPTO4 remained detectable at 37\u0026deg;C, whereas that of L-OPTO3 was smear indicating the triplex dissociation under experimental condition. The greater stability of the L-OPTO4 triplex compared with that of L-OPTO3 was confirmed and the estimated \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value of the L-OPTO4 triplex exceeded 60\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we investigated the impact of the positional difference of the mismatch site in the target duplex on the stability of the triplex structure to demonstrate the versatility of this approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We assessed the triplex formation using four PPT sequences, each with varying numbers of bases between two mismatch sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The triplex formed with L-OPTO5 was significantly more stable than the corresponding LNA-incorporated TFO (L-TFO2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). For L-TFO3\u0026ndash;5, a weak melting transition of the triplex was observed, making it challenging to accurately determine the \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values. In contrast, the UV-melting curves of each corresponding L-OPTO6\u0026ndash;8 displayed almost a single transition like sigmoid curve, indicating stable triplex formation. This was further confirmed by Native PAGE analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). We also explored the effect of the position of the LNA (Supplementary Fig.\u0026nbsp;4\u0026ndash;5: L-OPTO9-14), revealing that the overall conformation was crucial in achieving synergistic effects (it is not necessary to introduce \u003cb\u003eP\u003c/b\u003e between the LNA). Notably, L-OPTO13, which contained four \u003cb\u003eP\u003c/b\u003e in its sequence, also formed a significantly stable triplex with the target duplex (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe assessed the specificity of this method by substituting mismatched pyrimidine base C with purine bases A or G. The thermodynamic stabilities of the corresponding triplexes were nearly identical (Supplementary Fig.\u0026nbsp;6), indicating that this stabilization effect did not rely on specific interactions between the bases and psoralen. Next, we exchanged the bases in the target duplex at adjacent or distal positions of the mismatch site and evaluated the thermodynamic stability of the corresponding triplex (Supplementary Fig.\u0026nbsp;7). Resultantly, the \u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of all of the corresponding triplexes drastically decreased, indicating that the replacement of the mismatched pyrimidine base with \u003cb\u003eP\u003c/b\u003e did not compromise the specificity of TFO.\u003c/p\u003e\n\u003ch3\u003ePhoto-crosslinking properties of the locked nucleic acid-incorporated 1’(one)-psoralen-conjugated triplex-forming oligonucleotide\u003c/h3\u003e\n\u003cp\u003eCrosslinking formation is an important factor for obtaining significant biological outcomes using TFO. We successfully demonstrated that introducing the proposed nucleotide analog (\u003cb\u003eP\u003c/b\u003e) into TFO was effective in the stable triplex formation, which is a prerequisite factor for the photo-crosslinking of psoralen with the target duplex. Therefore, finally, we investigated the crosslinking profiles of OPTO using denaturing PAGE. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, our design allows us to select sequences with multiple pyrimidine bases as the target of TFO. Therefore, we examined whether or not we could increase the photo-adduct product in proportion to the number of psoralens that was introduced into the TFO sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Each triplex sample was irradiated using blue LED light (365 nm), and the samples were collected at each irradiation time (0, 1, 5, 10, 15, 20, 25, and 30 s). The 3\u0026rsquo; end of each L-OPTO was labeled with tetramethylrhodamine (TAMRA). Resultantly, two clusters of the bands of the crosslinked products were observed. In case of L-OPTO1 and L-OPTO2, we can simply define the top band clusters as monoadduct (OPTO crosslinked to either 606-Py or 606-Pu) and second band clusters as diadduct (OPTO crosslinked to both 606-Py and 606-Pu) by the mobility of the band because they have only one target site for photo-crosslinking. On the other hand, there is a possibility that crosslink formation at more than two sites restrain the extension of DNA complex under denaturing condition, and the band mobility will not be the same as the migration of DNA through a polyacrylamide gel depends on end-to-end distance of the DNA. Therefore, we conducted extensive photo-crosslinking study to analyze the composition of the photo-crosslinked product using either TAMRA-labeled 606-Py or 606-Pu; indicating that the second band clusters of photo-crosslinked product of L-OPTO3 and L-OPTO4 contained three strand complex (Supplementary Fig.\u0026nbsp;8). As we expected, the total crosslinked product formation rate increased in proportion to the number of psoralens in the L-OPTO sequence. 18% of L-OPTO1, 31% of L-OPTO3, and 47% of L-OPTO4 were consumed to form crosslinked product after 1 s of photo-irradiation. This might be attributed to both the number of psoralens in the sequence and the thermodynamic stability of the corresponding triplex. We also tested the crosslinking efficiency with other base combinations, it was observed that the efficient diadduct formation at single site required a 5ʹ-TA-3ʹ sequence in both target DNA strands (Supplementary Fig.\u0026nbsp;9\u0026ndash;10). Generally, thymine base is more reactive than cytosine. This was experimentally examined in our previous study.\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e Presumably, the 5-methyl group of thymine is important for the stabilization of the radical intermediate of the [2\u0026thinsp;+\u0026thinsp;2] photo-cycloaddition reaction, and/or the hydrophobic interaction between the 5-methyl group of thymine and psoralen may contribute to the relatively high crosslinking efficiency. The photo-induced electron transfer process from the adjacent guanine base also decreases the propensity of the photo-addition of psoralen to DNA.\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e In any event, this crosslink formation is expected to enhance the biological activities of TFO upon photo-irradiation. Thus, ongoing efforts will focus on photo-dynamic gene regulation using the L-OPTO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we developed a new approach enabling stable parallel triplex formation with pyrimidine-base-interrupting sequences under physiological conditions, leveraging the synergistic effects of \u003cb\u003eP\u003c/b\u003e and LNA. OPTO formed stable triplexes with the 5\u0026prime;-LTR partial sequence of HTLV-1 and other target sequences, demonstrating the versatility of the OPTO method and its potential for expanding the range of target sequences for parallel TFO applications. Furthermore, we conducted detailed studies on the photo-crosslinking formation of OPTO and demonstrated that the internal pyrimidine base can be a target for the photo-crosslinking of psoralen. Ongoing efforts will focus on further optimizing this method and exploring photo-dynamic gene regulation using L-OPTO, with results expected in upcoming reports.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChemical synthesis\u003c/h2\u003e \u003cp\u003eDetailed protocols for the synthesis of all compounds and oligonucleotides can be found in the Supporting Information of this article.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTriplex formation analysis using non-denaturing polyacrylamide gel electrophoresis (Native PAGE)\u003c/h3\u003e\n\u003cp\u003eSample solutions containing the ds-DNAs and TFOs (1.0 \u0026micro;M each, 10 mM sodium phosphate (pH 7.0), 200 mM NaCl, 0.1 mM EDTA) were denatured by heating to 95\u0026deg;C and cooling to 4\u0026deg;C at 0.5\u0026deg;C/min. The annealed sample was diluted with 40 wt% sucrose aq (sample: 40 wt% sucrose aq\u0026thinsp;=\u0026thinsp;1:4 (v/v)). The samples were analyzed with 20% native polyacrylamide gel (PAGE) containing 5 mM Mg2\u0026thinsp;+\u0026thinsp;in TBM (20\u0026ndash;23\u0026deg;C, 37\u0026deg;C, 120 V, 90 min). The DNA bands were stained by SYBR\u0026reg; Gold and the gels were transferred to imaging plates. The gel images were analyzed and quantitated using ChemDoc Touch MP (BioRad, CA, U.S.A.).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of photo-crosslinking efficiency of L-OPTOs by denaturing polyacrylamide gel electrophoresis (PAGE)\u003c/h2\u003e \u003cp\u003eSample solutions containing the ds-DNAs and TAMRA-labeled L-OPTOs (1.0 \u0026micro;M each, 10 mM sodium phosphate (pH 7.0), 200 mM NaCl, 0.1 mM EDTA) were denatured by heating to 95\u0026deg;C and cooling to 4\u0026deg;C at 0.5\u0026deg;C/min. The annealed solution was applied to a 368-well plate and irradiated by a UV spotlight (ZUV-C30H (365 nm); Omron Corp., Kyoto, Japan) at 37\u0026deg;C. The UV irradiated samples was diluted with formamide (sample: formamide\u0026thinsp;=\u0026thinsp;1:4 (v/v)). The samples were analyzed by 15% denaturing polyacrylamide gel (PAGE) containing 7 M urea and formamide (200 V, 45 min). The gels were transferred to imaging plates, and the resulting gel images were analyzed and quantitated using by ChemDoc Touch MP (BioRad, CA, U.S.A.).\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors declare that all data supporting the findings of this study are available within the article and the Supplementary Information.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAdditional information\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eYM and AY conceptualized and supervised the project with the assistance of CD and TW. YM synthesized all compounds. The functional evaluation experiments of OPTO were performed by YM and HT. The manuscript was written by the contribution of all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was financially supported by the Grant-in-Aid for Transformative Research Areas (A) \u0026ldquo;Material Symbiosis\u0026rdquo; (Grant Number:20H05874 awarded to A.Y.) from MEXT, Japan. This study was also supported by JSPS KAKENHI (Grant Numbers 22H00593 and 22K14839 to A.Y. and Y.M., respectively), Japan, and the Cooperative Research Program of \u0026ldquo;Network Joint Research Center for Materials and Devices\u0026rdquo; (Grant Numbers 20224030 and 20228004 to A.Y. and Y.M., respectively).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, C., Zhou, Z., Ren, C., Deng, Y., Peng, F., Wang, Q., Zhang, H., Jiang, Y. Triplex-Forming Oligonucleotides as An Anti-Gene Technique for Cancer Therapy. Front. Pharmacol. 13, 1007723 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHewett, P. W., Daft, E. L., Laughton, C. A., Ahmad, S., Ahmed, A., Murray, J. C. Selective Inhibition of the Human Tie-1 Promoter with Triplex-Forming Oligonucleotides Targeted to Ets Binding Sites. Mol. Med. 12, 8\u0026ndash;16 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarympalis, V., Kalopita, K., Zarros, A., Carageorgiou, H. Regulation of Gene Expression via Triple Helical Formations. Biochemistry 69, 855\u0026ndash;860 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung, S. L., Krawczyk, S. H., Matteucci, M. D., Toole, J. J. Triple Helix Formation Inhibits Transcription Elongation in Vitro. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e 88, 10023\u0026ndash;10026 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoser, H. E., Dervan, P. B. Sequence-Specific Cleavage of Double Helical DNA by Triple Helix Formation. Science 238, 645\u0026ndash;650 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaruqi, A. F., Seidman, M. M., Segal, D. J., Carroll, D., Glazer, P. M. Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells. Mol. Cell. Biol. 16, 6820\u0026ndash;6828 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaushik Tiwari, M., Rogers, F. A. XPD-dependent activation of apoptosis in response to triplex-induced DNA damage. Nucleic Acids Res. 41, 8979\u0026ndash;8994 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaushik Tiwari, M., Colon-Rios, D. A., Rao Tumu, H. C., Liu, Y., Quijano, E., Krysztofiak, A., Chan, C., Song, E., Braddock, D. T., Suh, H.-W.; et al. Direct Targeting of Amplified Gene Loci for Proapoptotic Anticancer Therapy. Nat. Biotechnol. 40, 325\u0026ndash;334 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaushik Tiwari, M., Adaku, N., Peart, N., Rogers, F. A. Triplex Structures Induce DNA Double Strand Breaks via Replication Fork Collapse in NER Deficient Cells. Nucleic Acids Res 44, 7742\u0026ndash;7754 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagatsugi, F., Sasaki, S., Miller, P. S., Seidman, M. M. Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog. Nucleic Acids Res. 31, e31 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorigoe, H., Hari, Y., Sekiguchi, M., Obika, S., Imanishi, T. 2'-\u003cem\u003eO\u003c/em\u003e,4'-\u003cem\u003eC\u003c/em\u003e-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies. J. Biol. Chem. 276, 2354\u0026ndash;2360 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartono, Y. D., Pabon-Martinez, Y. V., Uyar, A., Wengel, J. K., Lundin, K. E., Zain, R., Smith, C. I. E., Nilsson, L., Villa, A. Role of pseudoisocytosine tautomerization in triplex-forming oligonucleotides: In silico and in vitro studies. ACS Omega 2, 2165\u0026ndash;2177 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRusling, D. A. Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH. Nucleic Acids Res 49, 7256\u0026ndash;7266 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma, S.; Miller, P. S. Interactions of Cytosine Derivatives with T\u0026middot;A Interruptions in Pyrimidine\u0026middot;Purine\u0026middot;Pyrimidine DNA Triplex. Bioconjugate Chem 7, 600\u0026ndash;605 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuianvarc\u0026rsquo;h, D., Benhida, R., Fourrey, J. L., Maurisse, R., Sun, J. S. Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine\u0026middot;pyrimidine interruption. Chem. Commun. 18, 1814\u0026ndash;1815 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhkubo, A., Ohnishi, T., Nishizawa, S., Nishimura, Y., Hisamatsu, S. The ability of a triplex-forming oligonucleotide to recog-nize T-A and C-G base pairs in a DNA duplex is enhanced by incorporating N-acetyl-2,7-diaminoquinoline. Bioorg. Med. Chem. 28, 115350 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerrard, S. R., Edrees, M. M., Bouamaied, I., Fox, K. R., Brown, T. CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides. Org. Biomol. Chem. 8, 5087\u0026ndash;5096 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHari, Y., Akabane, M., Obika, S. 2\u0026rsquo;-4\u0026rsquo;-BNA bearing a chiral guanidinopyrrolidine-containing nucleobase with potent ability to recognize the C:G base pair in a parallel-motif DNA triplex. Chem. Commun. 49, 7421\u0026ndash;7423 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObika, S., Uneda, T., Sugimoto, T., Nanbu, D., Minami, T., Doi, T., Imanishi, T. 2\u0026rsquo;-O,4\u0026rsquo;-C-methylene bridge nucleic acid (2\u0026rsquo;,4\u0026rsquo;-BNA): Synthesis and triplex-forming properties. Bioorg. Med. Chem. 9, 1001\u0026ndash;1011 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasaki, S., Taniguchi, Y., Takahashi, R., Senko, Y., Kodama, K., Nagatsugi, F., Maeda, M. Selective Formation of Stable Triplexes Including a TA or a CG Interrupting Site with New Bicyclic Nucleoside Analogue (WNA). J. Am. Chem. Soc. 126, 516\u0026ndash;528 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInde, T., Nishizawa, S., Hattori, Y., Kanamori, T., Yuasa, H., Seio, K., Sekine, M., Ohkubo, A. Synthesis of and triplex formation in oligonucleotides containing 2\u0026rsquo;-deoxy-6-thioxanthosine. Bioorg. Med. Chem. 26, 3785\u0026ndash;3790 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, E., Koshlap, K.M., Gillespie, P., Dervan, P.B., Feigon, J. Solution Structure of a Pyrimidine\u0026middot;Purine\u0026middot;Pyrimidine Triplex Con-taining the Sequence-specific Intercalating Non-natural Base D3. J. Mol. Biol. 257, 1052\u0026ndash;1069 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarbone, G. M., McGuffie, E., Napoli, S., Flanagan, C. E., Dembech, C., Negri, U., Arcamone, F., Capobianco, M. L., Catapano, C. V. DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene. Nucleic Acids Res 32, 2396\u0026ndash;2410 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParamasivam, M., Cogoi, S., Filichev, V. V., Bomholt, N., Pedersen, E. B., Xodo, L. E. Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation. Nucleic Acids Res 36, 3494\u0026ndash;3507 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalsh, S., El-Sagheer, A. H., Brown, T. Fluorogenic thiazole orange TOTFO probes stabilize parallel DNA triplexes at pH 7 and above. Chem. Sci. 9, 7681\u0026ndash;7687 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, V., Kesavan, V., Gothelf, K. V. Highly stable triple helix formation by homopyrimidine (L)-acyclic threoninol nucleic acids with single stranded DNA and RNA. Org. Biomol. Chem. 13, 2366\u0026ndash;2374 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuhimschi, D. A., Gooden, M. D., Jing, H., Fels, R. D., Hansen, S. K., Beyer, F. W., Dewhirst, W. M., Walder, H., Gasparro, P. F. Psoralen Derivatives with Enhanced Potency. Photochem. Photobiol. 96, 1014\u0026ndash;1031 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuval-Valentin, G., Thuong, N. T., H\u0026eacute;l\u0026egrave;ne, C. Specific inhibition of transcription by triple helix-forming oligonucleotides. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 89, 504\u0026ndash;508 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrigoriev, M., Praseuth, D., Guieysse, A. L., Robin, P., Thuong, N. T., H\u0026eacute;l\u0026egrave;ne, C., Harel-Bellan, A. Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites. \u003cem\u003eProc. Natl. Acad. Sci\u003c/em\u003e. 90, 3501\u0026ndash;3505 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHavre, P. A., Gunther, E., Gasparro, F., Glazer, P. M. Targeted Mutagenesis of DNA Using Triple Helix-Forming Oligonucleotides Linked to Psoralen. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e 90, 7879\u0026ndash;7883 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaha, M., Lacroix, L., Glazer, P. M. Mutagenesis Mediated by Triple Helix-Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context. Photochem. Photobiol. 67, 289\u0026ndash;294 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaria, M., Wood, C. D., Perrouault, L., Nelson, J. S., Winter, A., White, M. R. H., H\u0026eacute;l\u0026egrave;ne, C., Giovannangeli, C. Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides. \u003cem\u003eProc. Natl. Acad. Sci\u003c/em\u003e. 97, 3862\u0026ndash;3867 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiviacco, S., Rapozzi, V., Xodo, L., H\u0026eacute;l\u0026egrave;ne, C., Quadrifoglio, F., Giovannangeli, C. Site-directed inhibition of DNA replication by triple helix formation. FASEB J. 15, 2660\u0026ndash;2668 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajumdar, A., Muniandy, P. A., Liu, J., Liu, J.-I., Liu, S.-T., Cuenoud, B., Seidman, M. M. Targeted Gene Knock-In and Sequence Modulation Mediated by a Psoralen-Linked Triplex-Forming Oligonucleotide. J. Biol. Chem. 283, 11244\u0026ndash;11252 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, J., Majumdar, A., Liu, J., Thompson, L. H., Seidman, M. M. Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells. J. Biol. Chem. 285, 23198\u0026ndash;23207 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemenyuk, A., Darian, E., Liu, J., Majumdar, A., Cuenoud, B., Miller, P. S., MacKerell, A. D., Seidman, Jr, M. M. Biochemistry 49, 7867\u0026ndash;7878 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikame, Y., Eshima, H., Toyama, H., Nakao, J., Matsuo, M., Yama-moto, T., Hari, Y., Komano, J. A., Yamayoshi, A. Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA. \u003cem\u003eChemMedChem\u003c/em\u003e 18, e202300348 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagai, M., Osame, M. Human T-cell lymphotropic virus type I and neurological diseases. J Neurovirol \u003cem\u003e9\u003c/em\u003e, 228\u0026ndash;235 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe, T. HTLV-1-associated diseases. Int J Hematol \u003cem\u003e66\u003c/em\u003e, 257\u0026ndash;278 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasunaga, J., Matsuoka, M. Molecular mechanisms of HTLV-1 infection and pathogenesis. Int J Hematol 94, 435\u0026ndash;442 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaris, M. Potential for molecular targeted therapy for adult T-cell leukemia/lymphoma. Int Rev Immunol 27, 71\u0026ndash;78 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor, G. P., Matsuoka, M. Natural history of adult T-cell leukemia/lymphoma and approaches to therapy. Oncogene 24, 6047\u0026ndash;6057 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, A., Takeda, S., Kariya, R., Matsuda, K., Urano, S., Okada, S., Komano, J. A novel therapeutic molecule against HTLV-1 infection taregeting provirus. Leukemia 27, 1621\u0026ndash;1627 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikame, Y., Yamayoshi, A. Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids. \u003cem\u003ePharmaceutics\u003c/em\u003e 15 2515 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue, J., Watanabe, T., Sato, M., Oda, A., Toyoshima, K., Yoshida, M., Seiki, M. Nucleotide sequence of the protease-coding region in an infectious DNA of simian retrovirus (STLV) of the HTLV-I family. Virology 150, 187\u0026ndash;195 (1986).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJazouli, M., Guianvarc\u0026rsquo;h, D., Soufiaoui, M., Bougrin, K., Vierling, P., Benhida, R. A Short and efficient synthesis of 2ʹ-deoxybenzo and pyridoimidazole C-nucleosides. Tetrahedron Letters 44, 5807\u0026ndash;5810 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;n-Nieves, V., F\u0026agrave;brega, C., Guasch, M., Fern\u0026aacute;ndez, S., Sanghvi, Y. S., Ferrero, M., Eritja, R. Oligonucleotides Containing 1-Aminomethyl or 1-Mercaptomethyl-2-deoxy-D-ribofuranoses: Synthesis, Purification, Characterization, and Conjugation with Fluorophores and Lipids. Bioconjugate Chem 32, 350\u0026ndash;366 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObika, S., Uneda, T., Sugimoto, T., Nanbu, D., Minami, T., Doi, T., Imanishi, T. 2'-O,4'-C-Methylene bridged nucleic acid (2',4'-BNA): synthesis and triplex-forming properties. Bioorg. Med. Chem. 9, 1001\u0026ndash;1011 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunet, E., Alberti, P., Perrouault, L., Babu, R., Wengel, J., Giovannangeli, C. Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis. J. Biol. Chem. 280, 2007\u0026ndash;20086 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, J. S., Woodsworth, M. L., Latimer, L. J., Morgan, A. R. Poly(pyrimidine)\u0026middot;poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. Nucleic Acids Res 12, 6603\u0026ndash;6614 (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamayoshi, A., Matsuyama, Y., Kushida M., Kobori, A., Murakami A. Novel Photodynamic Effect of a Psoralen-Conjugated Oligonucleotide for the Discrimination of the Methylation of Cytosine in DNA. Photochem. Photobiol. 90, 716\u0026ndash;722 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKojima, A., Nakao, J., Shimada, N., Yoshida, N., Abe, Y., Mikame, Y., Yamamoto, T., Wada, T., Maruyama, A., Yamayoshi, A. Selective Photo-Crosslinking Detection of Methylated Cytosine in DNA Duplex Aided by a Cationic Comb-Type Copolymer. ACS Biomater. Sci. Eng. 8, 1799\u0026ndash;1805 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakao, J., Mikame, Y., Eshima, H., Yamamoto, T., Dohno, C., Wada, T., Yamayoshi, A. Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen \u003cem\u003eN\u003c/em\u003e-Hydroxysuccinimide Esters. \u003cem\u003eChemBioChem\u003c/em\u003e e202200789 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertling, J., Thom, K. A., Geenen, S., Jeuken, H., Presser, L., M\u0026uuml;ller, T. J. J., Gilch, P. Synthesis and Photophysics of Water-Soluble Psoralens with Red-Shifted Absorption. Photochem. Photobiol. 97, 1534\u0026ndash;1547 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5384273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5384273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePsoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been employed for the photodynamic regulation of gene expression by the photo-crosslinking of psoralen with the target DNA. However, stable triplex formation requires a consecutive purine base sequence in one strand of the target DNA duplexes. The pyrimidine-base interruption in the consecutive purine base sequence drastically decreases the thermodynamic stability of the corresponding triplex, which hampers the TFO application. Here, we propose a design of the Ps-TFO for stable triplex formation with target DNA sequences containing pyrimidine-base interruptions under physiological conditions. This Ps-TFO, named 1\u0026rsquo;(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a synthesized nucleoside mimic 1\u0026rsquo;-psoralen-conjugated deoxyribose to increase the thermodynamic stability of the corresponding triplex by the intercalation of psoralen. The triplex-forming abilities of the OPTO were successfully demonstrated in combination with locked nucleic acid (LNA), indicating that the use of OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation.\u003c/p\u003e","manuscriptTitle":"Development of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides: Evaluation of the triplex-forming and photo-crosslinking properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-04 13:16:53","doi":"10.21203/rs.3.rs-5384273/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"527f0793-592b-40d2-8cf7-73e08412de48","owner":[],"postedDate":"December 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40159031,"name":"Biological sciences/Chemical biology/DNA"},{"id":40159032,"name":"Biological sciences/Chemical biology/Nucleic acids"},{"id":40159033,"name":"Biological sciences/Chemical biology/Chemical tools"}],"tags":[],"updatedAt":"2025-01-23T08:06:49+00:00","versionOfRecord":{"articleIdentity":"rs-5384273","link":"https://doi.org/10.1038/s42004-025-01416-2","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2025-01-22 05:00:00","publishedOnDateReadable":"January 22nd, 2025"},"versionCreatedAt":"2024-12-04 13:16:53","video":"","vorDoi":"10.1038/s42004-025-01416-2","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01416-2","workflowStages":[]},"version":"v1","identity":"rs-5384273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5384273","identity":"rs-5384273","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}