Merging DNA repair with bioorthogonal conjugation enables accessible and superior asymmetric DNA catalysis

Merging DNA repair with bioorthogonal conjugation enables accessible and superior asymmetric DNA catalysis | 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 Merging DNA repair with bioorthogonal conjugation enables accessible and superior asymmetric DNA catalysis Ru-Yi Zhu, Jie Sheng, Zhaoyang Li, Kelly Kar Yun Koh, Qi Shi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3941689/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Optimizing catalysts through high-throughput screen for asymmetric catalysis is challenging due to the difficulty associated with assembling a library of catalyst analogues in a timely fashion. Here, we repurpose DNA excision repair and integrate it with bioorthogonal conjugation to construct a diverse array of DNA hybrid catalysts for highly accessible and high-throughput asymmetric DNA catalysis, enabling dramatically expedited catalyst optimization process, superior reactivity and selectivity, as well as the first atroposelective DNA catalysis. The bioorthogonality of this conjugation strategy ensures exceptional tolerance towards diverse functional groups, thereby facilitating the facile construction of 42 DNA hybrid catalysts bearing various unprotected functional groups. This unique feature holds the potential to enable catalytic modalities in asymmetric DNA catalysis that were previously deemed unattainable. Physical sciences/Chemistry/Catalysis/Asymmetric catalysis Physical sciences/Chemistry/Catalysis/Biocatalysis Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Text The majority of small-molecule drugs are typically discovered through high-throughput screening (HTS) against a large number of small molecules ( 1 ). While HTS is routinely practiced in drug discovery, its utilization in catalyst optimization for asymmetric catalysis remains underexplored presumably due to the challenges associated with assembling a library of catalyst analogues in a timely manner ( 2–4 ). Small-molecule catalysts are generally synthesized individually and subjected to evaluation through a stepwise and iterative optimization process. Likewise, enzymes are engineered through protein engineering to incorporate mutations, undergoing a similar iterative optimization process ( 5–7 ). This optimization strategy often demands labor-intensive efforts and a protracted duration for improvement (Fig. 1 A). Therefore, novel methods for facile, rapid, and effective construction of chiral catalyst analogues can potentially expedite the catalyst optimization process with HTS. From the technical aspect, enzyme engineering process starts from DNA mutations followed by transcription and translation in living organisms, which are labor-intensive and entail substantial technical challenges for most organic chemists ( 8 ). This has led us to contemplate whether DNA itself could serve as a readily available chiral scaffold for facilitating asymmetric catalysis with HTS. Since the seminal proof-of-concept study by Roelfes and Feringa on DNA-mediated asymmetric catalysis ( 9 ), the practice of bridging DNA with conventional small-molecule catalysts either non-covalently or covalently has significantly expanded the catalytic repertoire of DNA ( 10–13 ). The non-covalent supramolecular approach hinges on the attachment of catalysts to DNA-binding molecules, including intercalators and grove binders ( 9 , 14–21) . However, the inherently non-specific binding characteristics of these molecules can lead to the formation of a heterogeneous mixture of catalysts, which poses challenges to gaining a deeper understanding of the mechanisms underlying DNA-based chiral induction and potential enhancements in reaction rates. In this regard, the site-specific conjugation of small-molecule catalysts to DNA emerges as an indispensable approach, offering unambiguous insights into the precise chiral environments ( 22–28) . Moreover, covalently conjugated DNA hybrid catalysts provide superior reaction outcomes than their non-covalent counterparts ( 27 , 28 ). Consequently, the covalent anchoring strategy has garnered increased attention and adoption in the field recently. Nevertheless, a significant technical obstacle has impeded widespread engagement in DNA-based asymmetric catalysis, namely, the solid-phase oligonucleotide synthesis involving air- and moisture-sensitive phosphoramidites and more importantly, this technique remains inaccessible to most synthetic chemistry laboratories ( 29 ). These challenging aspects might have deterred many research laboratories from actively pursuing or implementing DNA-based asymmetric catalysis in their studies. Furthermore, the synthesis of customized phosphoramidites often encounters functional group compatibility issues, thereby imposing substantial restrictions on potential DNA modification (Fig. 1 B). In contrast, post-synthetic modifications present a viable avenue to circumvent the constraints associated with phosphoramidite chemistry. Despite the availability of few amino group-functionalized DNA from commercial vendors for further modifications by amide coupling, the exorbitant costs, limited chemical flexibility (e.g., fixed linker lengths and modification sites) and poor functional group compatibility render this option undesirable. Owing to the synthetic challenges and subsequent laborious work-up and purification procedures, systematically investigating the interplay between DNA sequence and reaction outcomes in a high-throughput format remains a formidable task. Therefore, there exists a pressing need for the development of a versatile, widely accessible, modular, and cost-effective method for post-synthetic modifications of DNA for the construction of DNA hybrid catalysts. To this end, we propose to harness the highly efficient DNA base excision repair pathway to site-specifically generate reactive aldehyde tags for subsequent bioorthogonal oxime or hydrazone conjugation for the synthesis of diverse small-molecule catalysts, which ensures high efficiency and excellent functional group compatibility (Fig. 1 C). The DNA apurinic/apyrimidinic site (AP site) is a common DNA lesion resulted from DNA glycosylase-catalyzed removal of damaged DNA bases ( 30 ). For instance, uracil-DNA glycosylase (UDG) specifically recognizes and removes deoxyuridine (dU) in DNA, which arises from the hydrolytic deamination of deoxycytidine ( 31 ). The enzymatic action results in the generation of a DNA AP site which equilibrates between a furanose ring and an open-chain free aldehyde ( 32 ). Given the characteristic bioorthogonal reactivity of aldehyde, some oxyamine, hydrazine, or hydrazide based chemical probes have been documented for biological and biomedical applications (32 –35 ). Notably, both dU-containing DNA and E. coli UDG are cheaply and widely available, potentially allowing systematic investigations into the DNA sequence effect on reaction outcomes in a high-throughput format as well as permitting site-specific modification of long and complex DNA scaffolds which are otherwise challenging using phosphoramidite chemistry. Last but not the least, the bioorthogonal conjugation involving a DNA AP site with oxyamine, hydrazine, or hydrazide decorated small-molecule catalysts would allow the presence of a wide range of unprotected functional groups in the catalysts (Fig. 1 D and 1 E). Despite the immense promise in this approach, to the best of our knowledge, this robust, cost-effective, and widely accessible bioconjugation technique has hitherto remained unexplored in the context of crafting DNA hybrid catalysts for asymmetric catalysis. Herein, by merging DNA base excision repair with bioorthogonal oxime or hydrazone formation, we demonstrate the facile synthesis of diverse DNA hybrid catalysts bearing distinct unprotected functional groups incompatible with phosphoramidite chemistry in high efficiency with simple precipitation purification. The robustness and simplicity of this strategy are further showcased by the effortless installation of various small-molecule catalysts on a complex DNA aptamer compared to a complicated post-synthetic two-step conjugation with expensive modified DNA ( 33 ). To demonstrate the feasibility of this chemoenzymatic conjugation strategy for DNA-based asymmetric catalysis, we opted to employ the well-established 2,2’-bipyridine-Cu 2+ (bpy-Cu 2+ ) system as a proof-of-concept study, in the context of the benchmark Friedel-Crafts alkylation reaction between α,β-unsaturated acylimidazoles and indoles ( 16 , 28 ). A thermostable 17-mer GC-rich hairpin DNA (hpDNA) with a GAA loop was designed because DNA duplex formation and GC-rich sequences are reported to be beneficial for achieving high stereoselectivity ( 21 , 36 ). We introduced one dU in the middle of the seven base pairs, opposites cytosine (C). We proceeded to design and synthesize four bpys bearing oxyamine (L1), hydrazide (L2 and L3), and hydrazine (L4) for subsequent bioconjugation with a DNA AP site. In brief, we denatured and reannealed 200 nmol of the dU-containing hpDNA (100 µM) in 50 mM Tris HCl pH 7.5, followed by the addition of UDG (50 U/mL). This mixture was incubated at 37°C for 1 hour. Subsequently, L1-4 (20 equivalents) were individually added to the reaction mixture and allowed to react at room temperature overnight. The resulting AP-DNA-L conjugates were purified through ethanol precipitation and verified using mass spectrometry. To evaluate the performance of these conjugates, we subjected a reaction mixture containing α,β-unsaturated acylimidazole 1a (1 mM), indole 2a (5 mM), Cu(NO 3 ) 2 (5 mol%) and AP-DNA-L (7.5 mol%) to MOPS buffer (20 mM, pH 6.5). The reaction was incubated at 5°C for 36 hours, achieving full conversions for all four AP-DNA-L conjugates tested (Fig. 2 A and Table S1 ). Encouragingly, the oxyamine-bearing bpy exhibited an 80% enantiomeric excess (ee) value. The hydrazide-based AP-DNA-L2 yielded slightly lower enantioselectivity, while AP-DNA-L3 and AP-DNA-L4, featuring shorter linkers, displayed diminished stereocontrol. This observation suggests that the flexibility or electronic properties of the bpy ligands play a pivotal role. As the bpy ligand likely occupies the empty space of the DNA AP site and closely interacts with opposite and adjacent bases, we embarked on a systematic exploration of all possible base combinations to further optimize the reaction. We first investigated three alternative opposite bases, adenine (A), guanine (G), and thymine (T), and observed that smaller pyrimidines yielded higher ee values than purines (Fig. 2 B and Table S2 ). Next, we examined all 16 possible neighboring base pair combinations, uncovering a wide range of ee values (ranging from 14–92%) (Fig. 2 C and Table S3 ). Intriguingly, the bpy-Cu 2+ catalyst sandwiched by two Gs outperformed other arrangements and the presence of a 3’ flanking G proved pivotal in maintaining high enantioselectivities. Notably, systematic investigation of neighboring sequences for asymmetric DNA catalysis has not been realized before this study. Having identified the optimal DNA sequence, we probed other reaction parameters. Variations in pH within the range of 6.5 to 9.5 exhibited minimal impact on conversion or enantioselectivity ( Table S4 ). Remarkably, reducing catalyst loadings to as low as 0.04 mol% still resulted in full conversion and 93% ee (i.e., 2500 turn-over number or TON), outperforming any previously reported DNA-based asymmetric reactions in terms of efficiency (Fig. 2 D and Table S5 ). To elucidate the origins of reactivity and enantioselectivity, we conducted a series of control experiments ( Table S6 ). No reaction occurred in the absence of Cu 2+ , and DNA was the exclusive source of chirality. Moreover, supramolecular interactions between 6,6'-dimethyl-2,2'-bipyridine (dmbpy) and the DNA AP site, intact DNA (i.e., identical sequence without an AP site), or salmon testes DNA (st-DNA) led to substantially reduced reaction rates and enantiocontrols, indicating that the DNA AP site serves as a superior covalent anchoring point compared to the supramolecular approach ( Fig. S1 ). Intriguingly, the DNA AP site hosted Cu 2+ -dmbpy in a non-covalent fashion more productively in comparison to intact DNA or st-DNA, suggesting distinct binding modes. The rate acceleration observed in the presence of DNA is likely because hydrophobic and flat indoles are preferentially enriched around DNA through hydrophobic interactions under aqueous conditions, thereby increasing the local indole concentrations near the catalytic center for rate enhancements ( 20 ). Notably, the reaction rate was found to be sequence-independent with the AP-DNA-L1 system and had no correlation with observed enantioselectivities ( Table S7 ). To investigate the general applicability of this methodology, we surveyed the substrate scope of α,β-unsaturated acylimidazoles 1 and indoles 2 using 2.5 mol% of Cu(NO 3 ) 2 and 3.75 mol% DNA-L1 hybrid catalyst with significantly lower catalyst loadings compared to previously reported DNA catalysis systems (Fig. 3 ). We found that indoles bearing electron-donating substituents, such as methyl or methoxy groups at the 5-position, exhibited complete conversions and delivered excellent ee values ( 3b and 3c ). The introduction of slightly electron-withdrawing halogens at the same 5-position did not impact either the conversions or the enantioselectivity ( 3d - g ). However, the presence of a strongly electron-withdrawing cyano group at this position led to a negative effect on both conversion and enantioselectivity ( 3h ). For 6-substituted indoles, we observed that various substituents at this position successfully participated in the reaction, affording the desired products with excellent conversions and ees even with an electron-withdrawing ester group ( 3i - m ). Substitutions at the 4-position of the indole scaffold led to a modest reduction in enantioselectivity ( 3n and 3o ). Next, we explored 7-substituted indoles, revealing that they smoothly reacted with 1a , yielding full conversions and displaying excellent ees ( 3p and 3q ). While the steric hindrance imposed by 2-methylindole had a negligible effect on reactivity, it may have contributed to a slight reduction in enantioselectivity ( 3r ). 1-Methylindole demonstrated remarkable reactivity, readily undergoing the desired transformation with 1a to furnish the product in quantitative conversion and with high enantioselectivity ( 3s ). Subsequently, we examined the scope of α,β-unsaturated acylimidazoles 1 . Gratifyingly, replacing the methyl group with ethyl or propyl group, had no discernible impact on the conversions or ee values ( 3t and 3u ). With a larger cyclohexyl group, lower reactivity but higher enantioselectivity were observed ( 3v ). Furthermore, we successfully employed β-aryl substituted α,β-unsaturated acylimidazoles, although some aryl substitutions led to varying degrees of conversions and consistently yielded good to excellent ees ( 3w - aa ). Finally, several functional groups in α,β-unsaturated acylimidazoles, such as methoxy and alkenyl groups, were well tolerated, giving excellent conversions and enantioselectivities ( 3ab , 3ac ). In an attempt to expand the substrate scope to pyrrole derivatives, the optimal conditions and DNA sequence proved ineffective. A scrutiny of various Lewis acidic metal ions was undertaken, revealing that the scandium ion exhibited the highest reactivity. Employing the GG sequence, 65% ee was achieved with complete conversion, marking the first example of utilizing scandium in asymmetric DNA catalysis (Fig. 4 ). Subsequently, a systematic assessment of neighboring bases was carried out using 2-methylpyrrole as the substrate. Gratifyingly, TT sequence emerged as the optimal scaffold, resulting in an increase of ee to 82% ( 4a ). Full conversions and good enantioselectivities were obtained for non-substituted pyrrole and N -methylpyrrole ( 4b and 4c ). The reaction of 4,5,6,7-tetrahydroindole underwent smoothly to yield the desired product with 86% ee ( 4d ). Furthermore, 2,5-disubstituted pyrroles reacted at the 3-position with excellent conversions and moderate enantioselectivities ( 4e and 4f ). It is worth highlighting that only non-substituted pyrrole had been reported in asymmetric DNA catalysis prior to our study. Subsequently, our investigation delved into the more challenging Friedel-Crafts conjugate addition/enantioselective protonation reaction. A systematic exploration of the DNA sequences immediate adjacent to the catalyst was conducted, revealing that GC instead of GG or TT yielded a superior 80% ee and complete conversion (Fig. 5 ). This outcome outperformed both the recent DNA/RNA hybrid and the benchmark st-DNA ( 6a ) ( 27 , 28 ). Intriguingly, stereoinduction was observed to be reversed with certain neighboring base pairs. 5-Substituted indoles bearing methyl, methoxy, fluoro, and morpholino groups reacted smoothly with full conversions and good to excellent ee values ( 6b - e ). Substituents at other positions were also well-tolerated, affording the desired products with quantitative conversions and high ee values ( 6f - i ). Importantly, the newly devised DNA catalyst demonstrated significantly enhanced efficacy compared to the recent DNA/RNA hybrid or st-DNA given that considerably higher catalyst loadings are required in the latter cases. Taken together, we have successfully demonstrated that the new chemoenzymatic conjugation strategy yielded superior DNA catalysts for benchmark enantioselective Friedel-Crafts alkylation and protonation reactions in terms of both stereoselectivity and reactivity. Following the successful demonstration of establishing point chirality through the newly developed conjugation strategy, we questioned whether this strategy could be extended to controlling axial chirality—an aspect yet unrealized in DNA and elusive in enzyme catalysis ( 38 ). To address this, we embarked on an investigation into atroposelective Friedel-Crafts alkylation between α,β-unsaturated acylimidazole 7 and N -aryl pyrrole 8a with the aim of constructing axial chiral C‒N bonds ( Fig. 4 ) . Systematic sequence evaluation yielded promising results, achieving a 76% ee with the TT sequence when utilizing 1 mol% of Cu(OAc) 2 at room temperature in a single screen ( Table S8 ). Subsequent enhancement of the enantioselectivity to 92% was achieved by increasing the catalyst loading to 5 mol% and conducting the reaction at 5°C. Notably, the stereoinduction exhibited a pronounced dependency on neighboring sequences, with several sequences yielding opposite enantioselectivities. Under the optimized conditions, an exploration of the substrate scope for N -aryl pyrroles was conducted. Ortho iso-propyl, methyl, propyl, or trifluoromethyl substituted N -aryl pyrrole smoothly underwent Friedel-Crafts alkylation, yielding good to excellent conversions and excellent ee values ( 9a - d ). Halogen-substituted substrates were also effectively employed, yielding the desired products in high conversion and displaying high ee values ( 9e - g ). Furthermore, N -naphthyl 2-methylpyrrole readily reacted with 7 , resulting in the product with 90% ee ( 9h ). Phenyl groups fused with 6- or 5-membered alkyl rings proved sufficiently hindered to induce axial chirality, furnishing the product in 87% ee or 84% ee, respectively ( 9i , 9j ). A series of functional groups, such as vinyl, cyano, and nitro groups, were well tolerated, affording the desired products with good stereocontrols. It is worth noting that this study marks the first example of atroposelective DNA catalysis. The structural diversity of small molecular catalysts covalently attached to DNA has been largely constrained by phosphoramidite chemistry-based solid-phase synthesis primarily due to its limited accessibility and poor functional group tolerance. Consequently, a vast majority of catalytic modalities has remained incompatible with DNA catalysis. In order to expand the scope of asymmetric DNA catalysis to include more previously challenging catalytic modalities, we embarked on the synthesis of a library of DNA-small molecule hybrid catalysts bearing a wide array of unprotected functional groups using the newly established conjugation strategy. (Fig. 5 A). Small molecules decorated with amino, hydroxyl, or carboxyl groups could be readily transformed into corresponding oxyamine or hydrazide for subsequent conjugation (Fig. 5 B). In addition to the introduction of four bidentate bipyridine ligands, we successfully attached tridentate terpyridine ligands through a hydroxyl or a carboxyl group to DNA in good yields (Fig. 5 C, L1 and L2). Monodentate phosphine ligands, including a carboxyl-modified RuPhos, were effectively introduced to DNA for a broad spectrum of transition-metal-catalyzed transformations (L7-9). A secondary amine, a valuable organo-catalyst for enamine catalysis, was seamlessly incorporated into DNA without the need for protecting groups (L10). Subsequently, we incorporated a versatile organo-catalyst TEMPO to DNA without compromising the integrity of the oxygen radical (L11). A carboxyl-functionalized 4-dimethylaminopyridine (DMAP) was conjugated to DNA with moderate yield (L12) to potentially facilitate DNA-catalyzed acyl transfer reactions. Pyridoxamine, one form of vitamin B6 utilized as a biomimetic catalyst for asymmetric catalysis, was successfully installed, bearing free hydroxyl and amino groups (L13). Another organo-catalyst, thiourea, was introduced onto DNA with good yield (L14). The inclusion of free sulfonic acid aimed to enable potential Brønsted acid DNA catalysis was realized in decent yield (L15). Lastly, a redox-active nicotinamide adenine dinucleotide hydrogen (NADH) derivative was introduced into DNA for potential DNA photocatalysis (L16). All these small-molecule catalysts were incorporated into a 17-mer hpDNA. To demonstrate the robustness of this conjugation strategy, a 44-mer DNA aptamer capable of adopting a three-dimensional structure was selected ( 37 ). A panel of small-molecule catalysts, each possessing distinctive structures, were successfully incorporated at predetermined positions with satisfactory yields. Notably, this methodology allows for the programmable placement of small-molecule catalysts at virtually any position within DNA without compromising their efficacy (Fig. 5 D). Taken together, this chemoenzymatic conjugation strategy exhibits the capacity to effectively couple virtually any DNA sequence with small molecules harboring diverse functional groups and features simple non-chromatographic purification. Through the integration of UDG-mediated DNA AP site generation with bioorthogonal conjugation, we have showcased the development of a myriad of DNA catalysts for asymmetric catalysis in a high-throughput manner, thereby overcoming challenges related to accessibility, functional group tolerance, and low-throughput issues. The utilization of DNA AP site-based conjugation has revealed a significantly broader scope with superior efficiency and selectivity across all investigated reactions. Additionally, the first DNA-based atroposelective catalysis has been realized with excellent stereocontrol. For all reactions, the catalyst optimization process has been dramatically expedited with HTS, yielding satisfactory results in a matter of a few days. Last but not the least, a diverse array of catalysts has been effectively immobilized on DNA, thereby affording prospects for a multitude of catalytic modalities for asymmetric catalysis previously unattainable. Declarations Acknowledgments: We thank Prof. Anh Tuân Phan at Nanyang Technological University for the assistance of MALDI-TOF analysis. We thank Prof. Ye Zhu at National University of Singapore for providing the precursor of L9. Funding: National University of Singapore start-up grant A-0008363-00-00 (R.-Y.Z.) National University of Singapore white space funding A-0008363-01-00 (R.-Y.Z.) Academic Research Fund Tier 1 A-8000476-00-00 (R.-Y.Z.) Author contributions: R.-Y.Z. directed the research. R.-Y.Z. conceived the work and designed the experiments. J.S. and Z.L. conducted most experiments on DNA catalysts syntheses, optimization and substrate scope. K.K.Y.K., Q.S., A.F., P.M.L.T., T.-K.K., X.W. and L.F. helped J.S. and Z.L. with above mentioned experiments. R.-Y.Z. wrote the manuscript with the input from J.S. and others. Competing interests: The authors declare no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. References and Notes Blay, V., Tolani, B., Ho, S. P., Arkin, M. R. High-throughput screening: today's biochemical and cell-based approaches. Drug. Discov. Today. 25 , 1807–1821 (2020). Jäkel, C., Paciello, R. High-throughput and parallel screening methods in asymmetric hydrogenation. Chem. Rev. 106 , 2912–2942 (2006). Wagen, C. C., McMinn, S. E., Kwan, E. E., Jacobsen, E. N. Screening for generality in asymmetric catalysis. Nature 610 , 680–686 (2022). Rein, J. et al . Generality-oriented optimization of enantioselective aminoxyl radical catalysis. Science 380 , 706–712 (2023). Yang, Y., Arnold, F. H. Navigating the unnatural reaction space: directed evolution of heme proteins for selective carbene and nitrene transfer. Acc. Chem. Res. 54 , 1209–1225 (2021). Drienovská, I., Roelfes, G. Expanding the enzyme universe with genetically encoded unnatural amino acids. Nat. Catal. 3 , 193–202 (2020). Davis, H. J., Ward, T. R. Artificial metalloenzymes: Challenges and opportunities. ACS Cent. Sci. 5 , 1120–1136 (2019). Wang, Y. et al . Directed evolution: Methodologies and applications. Chem. Rev. 121 , 12384–12444 (2021). Roelfes, G., Feringa, B. L. DNA-based asymmetric catalysis. Angew. Chem. Int. Ed. 44 , 3230–3232 (2005). Silverman, S. K. DNA as a versatile chemical component for catalysis, encoding, and stereocontrol. Angew. Chem. Int. Ed. 49 , 7180–7201 (2010). Park, S., Sugiyama, H. DNA-based hybrid catalysts for asymmetric organic synthesis. Angew. Chem. Int. Ed. 49 , 3870–3878 (2010). Rioz-Martínez, A., Roelfes, G. DNA-based hybrid catalysis. Curr. Opin. Chem. Biol. 25 , 80–87 (2015). Aubert, S., Duchemin, N., Zhang, J.-L., Smietana, M., Arseniyadis, S. DNA-based asymmetric catalysis: Past, present and future. Topics in Enantioselective Catalysis , pp. 1–30 (2022). Roelfes, G., Boersma, A. J., Feringa, B. L. Highly enantioselective DNA-based catalysis. Chem. Commun. 635–637 (2006). Coquière, D., Feringa, B. L., Roelfes, G. DNA-based catalytic enantioselective Michael reactions in water. Angew. Chem. Int. Ed. 46 , 9308–9311 (2007). Boersma, A. J., Feringa, B. L., Roelfes, G. Enantioselective Friedel-Crafts reactions in water using a DNA-based catalyst. Angew. Chem. Int. Ed. 48 , 3346–3348 (2009). Boersma, A. J. et al . Nat. Chem. 2 , 991–995 (2010). Oelerich, J., Roelfes, G. Chem. Sci. 4 , 2013–2017 (2013). Amirbekyan, K. et al. Design, synthesis, and binding affinity evaluation of Hoechst 33258 derivatives for the development of sequence-specific DNA-based asymmetric catalysts. ACS Catal. 6 , 3096–3105 (2016). García-Fernández, A., Megens, R. P., Villarino, L., Roelfes, G. DNA-accelerated copper catalysis of Friedel-Crafts conjugate addition/enantioselective protonation reactions in water. J. Am. Chem. Soc. 138 , 16308–16314 (2016). Guo, J. et al. A localized enantioselective catalytic site on short DNA sequences and their amphiphiles. JACS Au 2 , 483–491 (2022). Ropartz, L., Meeuwenoord, N. J., van der Marel, G. A., van Leeuwen, P. W. N. M., Slawin, A. M. Z., Kamer, P. C. J. Chem. Commun. 1556–1558 (2007). Oltra, N. S., Roelfes, G. Modular assembly of novel DNA-based catalysts. Chem. Commun. 6039–6041 (2008). Fournier, P., Fiammengo, R., Jäschke, A. Allylic amination by a DNA–diene–iridium(I) hybrid catalyst. Angew. Chem. Int. Ed. 48 , 4426–4429 (2009). Gjonaj, L., Roelfes, G. Novel catalyst design by using Cisplatin to covalently anchor catalytically active copper complexes to DNA. ChemCatChem. 5 , 1718–1721 (2013). Park, S. et al. Development of DNA-based hybrid catalysts through direct ligand incorporation: Toward understanding of DNA-based asymmetric catalysis. ACS Catal. 4 , 4070–4073 (2014). Mansot, J. et al. A rational quest for selectivity through precise ligand-positioning in tandem DNA-catalysed Friedel–Crafts alkylation/asymmetric protonation. Chem. Sci. 10 , 2875–2881 (2019). Duchemin, N. et al . New benchmark in DNA-based asymmetric catalysis: prevalence of modified DNA/RNA hybrid systems. JACS Au. 2 , 1910–1917 (2022). Matteucci, M. D., Caruthers, M. H. Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 103 , 3185–3191 (1981). Kim, Y. J., Wilson, D. M. III. Overview of base excision repair biochemistry. Curr. Mol. Pharmacol. 5 , 3–13 (2012). Schormann, N., Ricciardi, R., Chattopadhyay, D. Uracil-DNA glycosylases-structural and functional perspectives on an essential family of DNA repair enzymes. Protein Sci. 23 , 1667–1685 (2014). Kölmel, D. K., Kool, E. T. Oximes and hydrazones in bioconjugation: Mechanism and catalysis. Chem. Rev. 117 , 10358–10376 (2017). Atamna, H., Cheung, T., Ames, B. N. A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. Proc. Natl. Acad. Sci. U S A. 97 , 686–691 (2000). Kojima, N., Takebayashi, T., Mikami, A., Ohtsuka, E., Komatsu, Y. Construction of highly reactive probes for abasic site detection by introduction of an aromatic and a guanidine residue into an aminooxy group. J. Am. Chem. Soc. 131 , 13208–13209 (2009). Wilson, D. L., Kool, E. T. Ultrafast oxime formation enables efficient fluorescence light-up measurement of DNA base excision. J. Am. Chem. Soc. 141 , 19379–19388 (2019). Yoshizawa, S., Kawai, G., Watanabe, K., Miura, K. Hirao, I. GNA trinucleotide loop sequences producing extraordinarily stable DNA minihairpins. Biochemistry. 36 , 4761–4767 (1997). Nakatsuka, N. et al. Aptamer–field-effect transistors overcome Debye length imitations for small-molecule sensing. Science 362 , 319–324 (2018). Watts, O. F. B., Berreur, J., Collins, B. S. L., Clayden, J. Biocatalytic enantioselective synthesis of atropisomers. Acc. Chem. Res. 55 , 3362–3375 (2022). Additional Declarations There is NO Competing Interest. Supplementary Files SIZhu.pdf Supporting Information Cite Share Download PDF Status: Posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3941689","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":276221568,"identity":"e15674b2-6ca8-414a-8636-e5539a72fda2","order_by":0,"name":"Ru-Yi Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYFACHgaGDxCWAfFaGGeQrIWZhyQt5v1nj0nb7jhsz8DevE2C4U8tYS0yB86lSeeeOZzYwHOsTIKx7ThhLRKMPWbSuW2HExgkcswkGBuOEaGFmcdM2rIN6DD5N2ZAhxGjhQ2ohbHtMGODBA9QC1sNEVp4eIwte9vSE9t40ootEtsOEKGF/4zhjZ9t1vb87Ic33vjwp46wFjhgAxEJDIdJ0AIFpNgyCkbBKBgFIwUAAEgoLxsZRpSJAAAAAElFTkSuQmCC","orcid":"","institution":"National University of Singapore","correspondingAuthor":true,"prefix":"","firstName":"Ru-Yi","middleName":"","lastName":"Zhu","suffix":""},{"id":276221569,"identity":"1250a2c1-0929-4066-b193-be4f99e6c6bc","order_by":1,"name":"Jie Sheng","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Sheng","suffix":""},{"id":276221570,"identity":"c4547af4-6151-4279-9347-a07fc8ad32a6","order_by":2,"name":"Zhaoyang Li","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Zhaoyang","middleName":"","lastName":"Li","suffix":""},{"id":276221571,"identity":"c87f19eb-9e2f-4e27-ae5e-ecc8dd284493","order_by":3,"name":"Kelly Kar Yun Koh","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"Kar Yun","lastName":"Koh","suffix":""},{"id":276221572,"identity":"f0c86e43-298a-4036-9827-c9d39c8a142c","order_by":4,"name":"Qi Shi","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Shi","suffix":""},{"id":276221573,"identity":"9cfb8178-3c77-419b-a875-0e0347db819f","order_by":5,"name":"Angel Foo","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Angel","middleName":"","lastName":"Foo","suffix":""},{"id":276221574,"identity":"dc01af16-bd44-4dae-8dcd-41ba8fe6ecd6","order_by":6,"name":"Philip Mark Leetiong Tan","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"Mark Leetiong","lastName":"Tan","suffix":""},{"id":276221575,"identity":"a7043874-8f79-4743-b579-e254995f7d4b","order_by":7,"name":"Tuan-Khoa Kha","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Tuan-Khoa","middleName":"","lastName":"Kha","suffix":""},{"id":276221576,"identity":"2fad9b86-b810-4d3e-8dda-86cab3ce4772","order_by":8,"name":"Xujie Wang","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Xujie","middleName":"","lastName":"Wang","suffix":""},{"id":276221577,"identity":"0015aa44-f023-448f-aeb8-d79cdcb28cb6","order_by":9,"name":"Leonard Fang","email":"","orcid":"","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Leonard","middleName":"","lastName":"Fang","suffix":""}],"badges":[],"createdAt":"2024-02-09 02:40:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3941689/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3941689/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52004612,"identity":"c3a94a04-447f-4f68-8f9f-d0053af13aae","added_by":"auto","created_at":"2024-03-05 08:45:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMerging DNA repair with bioorthogonal conjugation for asymmetric DNA catalysis.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Expediting the catalyst optimization process with HTS. (\u003cstrong\u003eb\u003c/strong\u003e) Phosphoramidite-based synthesis of DNA hybrid catalysts faces three major challenges. (\u003cstrong\u003ec\u003c/strong\u003e) Merging DNA base excision repair with bioorthogonal conjugation ensures accessible and efficient construction of diverse DNA catalysts with excellent functional group compatibility. (\u003cstrong\u003ed\u003c/strong\u003e) Selected functional groups incorporated to DNA without any protection. (\u003cstrong\u003ee\u003c/strong\u003e) Programmable small-molecule catalyst installation on complex DNA structure.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/fe9935a738b28b3f06b14635.jpg"},{"id":52004613,"identity":"8a4dafe9-c8c1-468c-b3ad-43a40e6ca582","added_by":"auto","created_at":"2024-03-05 08:45:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReaction optimization.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Evaluation of linkers between bipyridine and DNA. (\u003cstrong\u003eb\u003c/strong\u003e) Evaluation of opposite bases. (\u003cstrong\u003ec\u003c/strong\u003e) Evaluation of all 16 neighboring base pairs. (\u003cstrong\u003ed\u003c/strong\u003e) Turnover numbers at different catalyst loadings.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/132210599782d91423e1ee9a.jpg"},{"id":52004615,"identity":"3165c4a7-e982-4ef9-8d1a-aef6dbbd52f9","added_by":"auto","created_at":"2024-03-05 08:45:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":935832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate scope.\u003c/strong\u003e \u003csup\u003ea\u003c/sup\u003e-5 °C, 60 hours. \u003csup\u003eb\u003c/sup\u003eGG sequence. \u003csup\u003ec\u003c/sup\u003eConditions for DNA/RNA hybrid: Cu(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (13 mol%), DNA/RNA (16.5 mol%), MES (pH 5.0), 4 °C, 72 hours. \u003csup\u003ed\u003c/sup\u003eConditions for st-DNA: Cu(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (30 mol%), st-DNA (0.58 mM), MES (pH 5.0), 4 °C, 72 hours. \u003csup\u003ee\u003c/sup\u003e-5 °C, 60 hours.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/e186fb369b7851ae95bddf4d.jpg"},{"id":52004616,"identity":"f53765a3-dceb-4646-9fc9-48503c406672","added_by":"auto","created_at":"2024-03-05 08:45:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":391630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate scope of atroposelective DNA catalysis.\u003c/strong\u003e Conditions for sequence screening: \u003cstrong\u003e7\u003c/strong\u003e (1 mM), \u003cstrong\u003e8a \u003c/strong\u003e(10 mM), Cu(OAc)\u003csub\u003e2\u003c/sub\u003e (1 mol%), AP-DNA-L1 (1.5 mol%), PBS (20 mM, pH 7), room temperature, 18 hours.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/7d6d1f1f2935b443fc15a516.jpg"},{"id":52004617,"identity":"7f8357e1-e27a-4a21-87fa-b7d4972cc021","added_by":"auto","created_at":"2024-03-05 08:45:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":553802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of diverse DNA hybrid catalysts.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic illustration of the chemoenzymatic conjugation. (\u003cstrong\u003eB\u003c/strong\u003e) Different DNAs and conjugation chemistries employed in this study. (\u003cstrong\u003eC\u003c/strong\u003e) Chemical structures of small-molecule catalysts installed on the 17-mer hpDNA. (\u003cstrong\u003eD\u003c/strong\u003e) Incorporation of various small-molecule catalysts to the DNA aptamer at different sites.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/3ad075ac2e2fba83df9f1d37.jpg"},{"id":52005322,"identity":"398852e1-de8d-44f9-b312-1351b0d9955f","added_by":"auto","created_at":"2024-03-05 09:01:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":915609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/f7fe4ba5-19a5-41cb-93d6-7262c9ee8921.pdf"},{"id":52004618,"identity":"c163cbba-9b3c-43e2-9d96-c02dc68e8f7c","added_by":"auto","created_at":"2024-03-05 08:45:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17898798,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Information\u003c/p\u003e","description":"","filename":"SIZhu.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3941689/v1/8c79b02e75678db972bfff66.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Merging DNA repair with bioorthogonal conjugation enables accessible and superior asymmetric DNA catalysis","fulltext":[{"header":"Main Text","content":"\u003cp\u003eThe majority of small-molecule drugs are typically discovered through high-throughput screening (HTS) against a large number of small molecules (\u003cem\u003e1\u003c/em\u003e). While HTS is routinely practiced in drug discovery, its utilization in catalyst optimization for asymmetric catalysis remains underexplored presumably due to the challenges associated with assembling a library of catalyst analogues in a timely manner (\u003cem\u003e2\u0026ndash;4\u003c/em\u003e). Small-molecule catalysts are generally synthesized individually and subjected to evaluation through a stepwise and iterative optimization process. Likewise, enzymes are engineered through protein engineering to incorporate mutations, undergoing a similar iterative optimization process (\u003cem\u003e5\u0026ndash;7\u003c/em\u003e). This optimization strategy often demands labor-intensive efforts and a protracted duration for improvement (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Therefore, novel methods for facile, rapid, and effective construction of chiral catalyst analogues can potentially expedite the catalyst optimization process with HTS. From the technical aspect, enzyme engineering process starts from DNA mutations followed by transcription and translation in living organisms, which are labor-intensive and entail substantial technical challenges for most organic chemists (\u003cem\u003e8\u003c/em\u003e). This has led us to contemplate whether DNA itself could serve as a readily available chiral scaffold for facilitating asymmetric catalysis with HTS.\u003c/p\u003e\n\u003cp\u003eSince the seminal proof-of-concept study by Roelfes and Feringa on DNA-mediated asymmetric catalysis (\u003cem\u003e9\u003c/em\u003e), the practice of bridging DNA with conventional small-molecule catalysts either non-covalently or covalently has significantly expanded the catalytic repertoire of DNA (\u003cem\u003e10\u0026ndash;13\u003c/em\u003e). The non-covalent supramolecular approach hinges on the attachment of catalysts to DNA-binding molecules, including intercalators and grove binders (\u003cem\u003e9\u003c/em\u003e, \u003cem\u003e14\u0026ndash;21)\u003c/em\u003e. However, the inherently non-specific binding characteristics of these molecules can lead to the formation of a heterogeneous mixture of catalysts, which poses challenges to gaining a deeper understanding of the mechanisms underlying DNA-based chiral induction and potential enhancements in reaction rates. In this regard, the site-specific conjugation of small-molecule catalysts to DNA emerges as an indispensable approach, offering unambiguous insights into the precise chiral environments (\u003cem\u003e22\u0026ndash;28)\u003c/em\u003e. Moreover, covalently conjugated DNA hybrid catalysts provide superior reaction outcomes than their non-covalent counterparts (\u003cem\u003e27\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e). Consequently, the covalent anchoring strategy has garnered increased attention and adoption in the field recently.\u003c/p\u003e\n\u003cp\u003eNevertheless, a significant technical obstacle has impeded widespread engagement in DNA-based asymmetric catalysis, namely, the solid-phase oligonucleotide synthesis involving air- and moisture-sensitive phosphoramidites and more importantly, this technique remains inaccessible to most synthetic chemistry laboratories (\u003cem\u003e29\u003c/em\u003e). These challenging aspects might have deterred many research laboratories from actively pursuing or implementing DNA-based asymmetric catalysis in their studies. Furthermore, the synthesis of customized phosphoramidites often encounters functional group compatibility issues, thereby imposing substantial restrictions on potential DNA modification (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, post-synthetic modifications present a viable avenue to circumvent the constraints associated with phosphoramidite chemistry. Despite the availability of few amino group-functionalized DNA from commercial vendors for further modifications by amide coupling, the exorbitant costs, limited chemical flexibility (e.g., fixed linker lengths and modification sites) and poor functional group compatibility render this option undesirable. Owing to the synthetic challenges and subsequent laborious work-up and purification procedures, systematically investigating the interplay between DNA sequence and reaction outcomes in a high-throughput format remains a formidable task. Therefore, there exists a pressing need for the development of a versatile, widely accessible, modular, and cost-effective method for post-synthetic modifications of DNA for the construction of DNA hybrid catalysts. To this end, we propose to harness the highly efficient DNA base excision repair pathway to site-specifically generate reactive aldehyde tags for subsequent bioorthogonal oxime or hydrazone conjugation for the synthesis of diverse small-molecule catalysts, which ensures high efficiency and excellent functional group compatibility (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eThe DNA apurinic/apyrimidinic site (AP site) is a common DNA lesion resulted from DNA glycosylase-catalyzed removal of damaged DNA bases (\u003cem\u003e30\u003c/em\u003e). For instance, uracil-DNA glycosylase (UDG) specifically recognizes and removes deoxyuridine (dU) in DNA, which arises from the hydrolytic deamination of deoxycytidine (\u003cem\u003e31\u003c/em\u003e). The enzymatic action results in the generation of a DNA AP site which equilibrates between a furanose ring and an open-chain free aldehyde (\u003cem\u003e32\u003c/em\u003e). Given the characteristic bioorthogonal reactivity of aldehyde, some oxyamine, hydrazine, or hydrazide based chemical probes have been documented for biological and biomedical applications (32\u003cem\u003e\u0026ndash;35\u003c/em\u003e). Notably, both dU-containing DNA and \u003cem\u003eE. coli\u003c/em\u003e UDG are cheaply and widely available, potentially allowing systematic investigations into the DNA sequence effect on reaction outcomes in a high-throughput format as well as permitting site-specific modification of long and complex DNA scaffolds which are otherwise challenging using phosphoramidite chemistry. Last but not the least, the bioorthogonal conjugation involving a DNA AP site with oxyamine, hydrazine, or hydrazide decorated small-molecule catalysts would allow the presence of a wide range of unprotected functional groups in the catalysts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Despite the immense promise in this approach, to the best of our knowledge, this robust, cost-effective, and widely accessible bioconjugation technique has hitherto remained unexplored in the context of crafting DNA hybrid catalysts for asymmetric catalysis. Herein, by merging DNA base excision repair with bioorthogonal oxime or hydrazone formation, we demonstrate the facile synthesis of diverse DNA hybrid catalysts bearing distinct unprotected functional groups incompatible with phosphoramidite chemistry in high efficiency with simple precipitation purification. The robustness and simplicity of this strategy are further showcased by the effortless installation of various small-molecule catalysts on a complex DNA aptamer compared to a complicated post-synthetic two-step conjugation with expensive modified DNA (\u003cem\u003e33\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eTo demonstrate the feasibility of this chemoenzymatic conjugation strategy for DNA-based asymmetric catalysis, we opted to employ the well-established 2,2\u0026rsquo;-bipyridine-Cu\u003csup\u003e2+\u003c/sup\u003e (bpy-Cu\u003csup\u003e2+\u003c/sup\u003e) system as a proof-of-concept study, in the context of the benchmark Friedel-Crafts alkylation reaction between \u0026alpha;,\u0026beta;-unsaturated acylimidazoles and indoles (\u003cem\u003e16\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e). A thermostable 17-mer GC-rich hairpin DNA (hpDNA) with a GAA loop was designed because DNA duplex formation and GC-rich sequences are reported to be beneficial for achieving high stereoselectivity (\u003cem\u003e21\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e). We introduced one dU in the middle of the seven base pairs, opposites cytosine (C). We proceeded to design and synthesize four bpys bearing oxyamine (L1), hydrazide (L2 and L3), and hydrazine (L4) for subsequent bioconjugation with a DNA AP site. In brief, we denatured and reannealed 200 nmol of the dU-containing hpDNA (100 \u0026micro;M) in 50 mM Tris HCl pH 7.5, followed by the addition of UDG (50 U/mL). This mixture was incubated at 37\u0026deg;C for 1 hour. Subsequently, L1-4 (20 equivalents) were individually added to the reaction mixture and allowed to react at room temperature overnight. The resulting AP-DNA-L conjugates were purified through ethanol precipitation and verified using mass spectrometry. To evaluate the performance of these conjugates, we subjected a reaction mixture containing \u0026alpha;,\u0026beta;-unsaturated acylimidazole \u003cstrong\u003e1a\u003c/strong\u003e (1 mM), indole \u003cstrong\u003e2a\u003c/strong\u003e (5 mM), Cu(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (5 mol%) and AP-DNA-L (7.5 mol%) to MOPS buffer (20 mM, pH 6.5). The reaction was incubated at 5\u0026deg;C for 36 hours, achieving full conversions for all four AP-DNA-L conjugates tested (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). Encouragingly, the oxyamine-bearing bpy exhibited an 80% enantiomeric excess (ee) value. The hydrazide-based AP-DNA-L2 yielded slightly lower enantioselectivity, while AP-DNA-L3 and AP-DNA-L4, featuring shorter linkers, displayed diminished stereocontrol. This observation suggests that the flexibility or electronic properties of the bpy ligands play a pivotal role. As the bpy ligand likely occupies the empty space of the DNA AP site and closely interacts with opposite and adjacent bases, we embarked on a systematic exploration of all possible base combinations to further optimize the reaction. We first investigated three alternative opposite bases, adenine (A), guanine (G), and thymine (T), and observed that smaller pyrimidines yielded higher ee values than purines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cstrong\u003eTable S2\u003c/strong\u003e). Next, we examined all 16 possible neighboring base pair combinations, uncovering a wide range of ee values (ranging from 14\u0026ndash;92%) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cstrong\u003eTable S3\u003c/strong\u003e). Intriguingly, the bpy-Cu\u003csup\u003e2+\u003c/sup\u003e catalyst sandwiched by two Gs outperformed other arrangements and the presence of a 3\u0026rsquo; flanking G proved pivotal in maintaining high enantioselectivities. Notably, systematic investigation of neighboring sequences for asymmetric DNA catalysis has not been realized before this study. Having identified the optimal DNA sequence, we probed other reaction parameters. Variations in pH within the range of 6.5 to 9.5 exhibited minimal impact on conversion or enantioselectivity (\u003cstrong\u003eTable S4\u003c/strong\u003e). Remarkably, reducing catalyst loadings to as low as 0.04 mol% still resulted in full conversion and 93% ee (i.e., 2500 turn-over number or TON), outperforming any previously reported DNA-based asymmetric reactions in terms of efficiency (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD \u003cstrong\u003eand Table S5\u003c/strong\u003e). To elucidate the origins of reactivity and enantioselectivity, we conducted a series of control experiments (\u003cstrong\u003eTable S6\u003c/strong\u003e). No reaction occurred in the absence of Cu\u003csup\u003e2+\u003c/sup\u003e, and DNA was the exclusive source of chirality. Moreover, supramolecular interactions between 6,6'-dimethyl-2,2'-bipyridine (dmbpy) and the DNA AP site, intact DNA (i.e., identical sequence without an AP site), or salmon testes DNA (st-DNA) led to substantially reduced reaction rates and enantiocontrols, indicating that the DNA AP site serves as a superior covalent anchoring point compared to the supramolecular approach (\u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). Intriguingly, the DNA AP site hosted Cu\u003csup\u003e2+\u003c/sup\u003e-dmbpy in a non-covalent fashion more productively in comparison to intact DNA or st-DNA, suggesting distinct binding modes. The rate acceleration observed in the presence of DNA is likely because hydrophobic and flat indoles are preferentially enriched around DNA through hydrophobic interactions under aqueous conditions, thereby increasing the local indole concentrations near the catalytic center for rate enhancements (\u003cem\u003e20\u003c/em\u003e). Notably, the reaction rate was found to be sequence-independent with the AP-DNA-L1 system and had no correlation with observed enantioselectivities (\u003cstrong\u003eTable S7\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo investigate the general applicability of this methodology, we surveyed the substrate scope of \u0026alpha;,\u0026beta;-unsaturated acylimidazoles \u003cstrong\u003e1\u003c/strong\u003e and indoles \u003cstrong\u003e2\u003c/strong\u003e using 2.5 mol% of Cu(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e and 3.75 mol% DNA-L1 hybrid catalyst with significantly lower catalyst loadings compared to previously reported DNA catalysis systems (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). We found that indoles bearing electron-donating substituents, such as methyl or methoxy groups at the 5-position, exhibited complete conversions and delivered excellent ee values (\u003cstrong\u003e3b\u003c/strong\u003e and \u003cstrong\u003e3c\u003c/strong\u003e). The introduction of slightly electron-withdrawing halogens at the same 5-position did not impact either the conversions or the enantioselectivity (\u003cstrong\u003e3d\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e). However, the presence of a strongly electron-withdrawing cyano group at this position led to a negative effect on both conversion and enantioselectivity (\u003cstrong\u003e3h\u003c/strong\u003e). For 6-substituted indoles, we observed that various substituents at this position successfully participated in the reaction, affording the desired products with excellent conversions and ees even with an electron-withdrawing ester group (\u003cstrong\u003e3i\u003c/strong\u003e-\u003cstrong\u003em\u003c/strong\u003e). Substitutions at the 4-position of the indole scaffold led to a modest reduction in enantioselectivity (\u003cstrong\u003e3n\u003c/strong\u003e and \u003cstrong\u003e3o\u003c/strong\u003e). Next, we explored 7-substituted indoles, revealing that they smoothly reacted with \u003cstrong\u003e1a\u003c/strong\u003e, yielding full conversions and displaying excellent ees (\u003cstrong\u003e3p\u003c/strong\u003e and \u003cstrong\u003e3q\u003c/strong\u003e). While the steric hindrance imposed by 2-methylindole had a negligible effect on reactivity, it may have contributed to a slight reduction in enantioselectivity (\u003cstrong\u003e3r\u003c/strong\u003e). 1-Methylindole demonstrated remarkable reactivity, readily undergoing the desired transformation with \u003cstrong\u003e1a\u003c/strong\u003e to furnish the product in quantitative conversion and with high enantioselectivity (\u003cstrong\u003e3s\u003c/strong\u003e). Subsequently, we examined the scope of \u0026alpha;,\u0026beta;-unsaturated acylimidazoles \u003cstrong\u003e1\u003c/strong\u003e. Gratifyingly, replacing the methyl group with ethyl or propyl group, had no discernible impact on the conversions or ee values (\u003cstrong\u003e3t\u003c/strong\u003e and \u003cstrong\u003e3u\u003c/strong\u003e). With a larger cyclohexyl group, lower reactivity but higher enantioselectivity were observed (\u003cstrong\u003e3v\u003c/strong\u003e). Furthermore, we successfully employed \u0026beta;-aryl substituted \u0026alpha;,\u0026beta;-unsaturated acylimidazoles, although some aryl substitutions led to varying degrees of conversions and consistently yielded good to excellent ees (\u003cstrong\u003e3w\u003c/strong\u003e-\u003cstrong\u003eaa\u003c/strong\u003e). Finally, several functional groups in \u0026alpha;,\u0026beta;-unsaturated acylimidazoles, such as methoxy and alkenyl groups, were well tolerated, giving excellent conversions and enantioselectivities (\u003cstrong\u003e3ab\u003c/strong\u003e, \u003cstrong\u003e3ac\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn an attempt to expand the substrate scope to pyrrole derivatives, the optimal conditions and DNA sequence proved ineffective. A scrutiny of various Lewis acidic metal ions was undertaken, revealing that the scandium ion exhibited the highest reactivity. Employing the GG sequence, 65% ee was achieved with complete conversion, marking the first example of utilizing scandium in asymmetric DNA catalysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Subsequently, a systematic assessment of neighboring bases was carried out using 2-methylpyrrole as the substrate. Gratifyingly, TT sequence emerged as the optimal scaffold, resulting in an increase of ee to 82% (\u003cstrong\u003e4a\u003c/strong\u003e). Full conversions and good enantioselectivities were obtained for non-substituted pyrrole and \u003cem\u003eN\u003c/em\u003e-methylpyrrole (\u003cstrong\u003e4b\u003c/strong\u003e and \u003cstrong\u003e4c\u003c/strong\u003e). The reaction of 4,5,6,7-tetrahydroindole underwent smoothly to yield the desired product with 86% ee (\u003cstrong\u003e4d\u003c/strong\u003e). Furthermore, 2,5-disubstituted pyrroles reacted at the 3-position with excellent conversions and moderate enantioselectivities (\u003cstrong\u003e4e\u003c/strong\u003e and \u003cstrong\u003e4f\u003c/strong\u003e). It is worth highlighting that only non-substituted pyrrole had been reported in asymmetric DNA catalysis prior to our study.\u003c/p\u003e\n\u003cp\u003eSubsequently, our investigation delved into the more challenging Friedel-Crafts conjugate addition/enantioselective protonation reaction. A systematic exploration of the DNA sequences immediate adjacent to the catalyst was conducted, revealing that GC instead of GG or TT yielded a superior 80% ee and complete conversion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). This outcome outperformed both the recent DNA/RNA hybrid and the benchmark st-DNA (\u003cstrong\u003e6a\u003c/strong\u003e) (\u003cem\u003e27\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e). Intriguingly, stereoinduction was observed to be reversed with certain neighboring base pairs. 5-Substituted indoles bearing methyl,\u0026nbsp;methoxy, fluoro, and morpholino groups reacted smoothly with full conversions and good to excellent ee values (\u003cstrong\u003e6b\u003c/strong\u003e-\u003cstrong\u003ee\u003c/strong\u003e). Substituents at other positions were also well-tolerated, affording the desired products with quantitative conversions and high ee values (\u003cstrong\u003e6f\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e). Importantly, the newly devised DNA catalyst demonstrated significantly enhanced efficacy compared to the recent DNA/RNA hybrid or st-DNA given that considerably higher catalyst loadings are required in the latter cases. Taken together, we have successfully demonstrated that the new chemoenzymatic conjugation strategy yielded superior DNA catalysts for benchmark enantioselective Friedel-Crafts alkylation and protonation reactions in terms of both stereoselectivity and reactivity.\u003c/p\u003e\n\u003cp\u003eFollowing the successful demonstration of establishing point chirality through the newly developed conjugation strategy, we questioned whether this strategy could be extended to controlling axial chirality\u0026mdash;an aspect yet unrealized in DNA and elusive in enzyme catalysis (\u003cem\u003e38\u003c/em\u003e). To address this, we embarked on an investigation into atroposelective Friedel-Crafts alkylation between \u0026alpha;,\u0026beta;-unsaturated acylimidazole \u003cstrong\u003e7\u003c/strong\u003e and \u003cem\u003eN\u003c/em\u003e-aryl pyrrole \u003cstrong\u003e8a\u003c/strong\u003e with the aim of constructing axial chiral C‒N bonds \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. Systematic sequence evaluation yielded promising results, achieving a 76% ee with the TT sequence when utilizing 1 mol% of Cu(OAc)\u003csub\u003e2\u003c/sub\u003e at room temperature in a single screen (\u003cstrong\u003eTable S8\u003c/strong\u003e). Subsequent enhancement of the enantioselectivity to 92% was achieved by increasing the catalyst loading to 5 mol% and conducting the reaction at 5\u0026deg;C. Notably, the stereoinduction exhibited a pronounced dependency on neighboring sequences, with several sequences yielding opposite enantioselectivities. Under the optimized conditions, an exploration of the substrate scope for \u003cem\u003eN\u003c/em\u003e-aryl pyrroles was conducted. Ortho iso-propyl, methyl, propyl, or trifluoromethyl substituted \u003cem\u003eN\u003c/em\u003e-aryl pyrrole smoothly underwent Friedel-Crafts alkylation, yielding good to excellent conversions and excellent ee values (\u003cstrong\u003e9a\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e). Halogen-substituted substrates were also effectively employed, yielding the desired products in high conversion and displaying high ee values (\u003cstrong\u003e9e\u003c/strong\u003e-\u003cstrong\u003eg\u003c/strong\u003e). Furthermore, \u003cem\u003eN\u003c/em\u003e-naphthyl 2-methylpyrrole readily reacted with \u003cstrong\u003e7\u003c/strong\u003e, resulting in the product with 90% ee (\u003cstrong\u003e9h\u003c/strong\u003e). Phenyl groups fused with 6- or 5-membered alkyl rings proved sufficiently hindered to induce axial chirality, furnishing the product in 87% ee or 84% ee, respectively (\u003cstrong\u003e9i\u003c/strong\u003e, \u003cstrong\u003e9j\u003c/strong\u003e). A series of functional groups, such as vinyl, cyano, and nitro groups, were well tolerated, affording the desired products with good stereocontrols. It is worth noting that this study marks the first example of atroposelective DNA catalysis.\u003c/p\u003e\n\u003cp\u003eThe structural diversity of small molecular catalysts covalently attached to DNA has been largely constrained by phosphoramidite chemistry-based solid-phase synthesis primarily due to its limited accessibility and poor functional group tolerance. Consequently, a vast majority of catalytic modalities has remained incompatible with DNA catalysis. In order to expand the scope\u0026nbsp;of asymmetric DNA catalysis to include more previously challenging catalytic modalities, we embarked on the synthesis of a library of DNA-small molecule hybrid catalysts bearing a wide array of unprotected functional groups using the newly established conjugation strategy. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Small molecules decorated with amino, hydroxyl, or carboxyl groups could be readily transformed into corresponding oxyamine or hydrazide for subsequent conjugation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition to the introduction of four bidentate bipyridine ligands, we successfully attached tridentate terpyridine ligands through a hydroxyl or a carboxyl group to DNA in good yields (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, L1 and L2). Monodentate phosphine ligands, including a carboxyl-modified RuPhos, were effectively introduced to DNA for a broad spectrum of transition-metal-catalyzed transformations (L7-9). A secondary amine, a valuable organo-catalyst for enamine catalysis, was seamlessly incorporated into DNA without the need for protecting groups (L10). Subsequently, we incorporated a versatile organo-catalyst TEMPO to DNA without compromising the integrity of the oxygen radical (L11). A carboxyl-functionalized 4-dimethylaminopyridine (DMAP) was conjugated to DNA with moderate yield (L12) to potentially facilitate DNA-catalyzed acyl transfer reactions. Pyridoxamine, one form of vitamin B6 utilized as a biomimetic catalyst for asymmetric catalysis, was successfully installed, bearing free hydroxyl and amino groups (L13). Another organo-catalyst, thiourea, was introduced onto DNA with good yield (L14). The inclusion of free sulfonic acid aimed to enable potential Br\u0026oslash;nsted acid DNA catalysis was realized in decent yield (L15). Lastly, a redox-active nicotinamide adenine dinucleotide hydrogen (NADH) derivative was introduced into DNA for potential DNA photocatalysis (L16). All these small-molecule catalysts were incorporated into a 17-mer hpDNA. To demonstrate the robustness of this conjugation strategy, a 44-mer DNA aptamer capable of adopting a three-dimensional structure was selected (\u003cem\u003e37\u003c/em\u003e). A panel of small-molecule catalysts, each possessing distinctive structures, were successfully incorporated at predetermined positions with satisfactory yields. Notably, this methodology allows for the programmable placement of small-molecule catalysts at virtually any position within DNA without compromising their efficacy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Taken together, this chemoenzymatic conjugation strategy exhibits the capacity to effectively couple virtually any DNA sequence with small molecules harboring diverse functional groups and features simple non-chromatographic purification.\u003c/p\u003e\n\u003cp\u003eThrough the integration of UDG-mediated DNA AP site generation with bioorthogonal conjugation, we have showcased the development of a myriad of DNA catalysts for asymmetric catalysis in a high-throughput manner, thereby overcoming challenges related to accessibility, functional group tolerance, and low-throughput issues. The utilization of DNA AP site-based conjugation has revealed a significantly broader scope with superior efficiency and selectivity across all investigated reactions. Additionally, the first DNA-based atroposelective catalysis has been realized with excellent stereocontrol. For all reactions, the catalyst optimization process has been dramatically expedited with HTS, yielding satisfactory results in a matter of a few days. Last but not the least, a diverse array of catalysts has been effectively immobilized on DNA, thereby affording prospects for a multitude of catalytic modalities for asymmetric catalysis previously unattainable.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Anh Tu\u0026acirc;n Phan at Nanyang Technological University for the assistance of MALDI-TOF analysis. We thank Prof. Ye Zhu at National University of Singapore for providing the precursor of L9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational University of Singapore start-up grant A-0008363-00-00 (R.-Y.Z.)\u003c/p\u003e\n\u003cp\u003eNational University of Singapore white space funding A-0008363-01-00 (R.-Y.Z.)\u003c/p\u003e\n\u003cp\u003eAcademic Research Fund Tier 1 A-8000476-00-00 (R.-Y.Z.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.-Y.Z. directed the research. R.-Y.Z. conceived the work and designed the experiments. J.S. and Z.L. conducted most experiments on DNA catalysts syntheses, optimization and substrate scope. K.K.Y.K., Q.S., A.F., P.M.L.T., T.-K.K., X.W. and L.F. helped J.S. and Z.L. with above mentioned experiments. R.-Y.Z. wrote the manuscript with the input from J.S. and others.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References and Notes","content":"\u003col\u003e\n\u003cli\u003eBlay, V., Tolani, B., Ho, S. P., Arkin, M. R. High-throughput screening: today\u0026apos;s biochemical and cell-based approaches. \u003cem\u003eDrug. Discov. Today.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1807\u0026ndash;1821 (2020).\u003c/li\u003e\n\u003cli\u003eJ\u0026auml;kel, C., Paciello, R. High-throughput and parallel screening methods in asymmetric hydrogenation. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 2912\u0026ndash;2942 (2006).\u003c/li\u003e\n\u003cli\u003eWagen, C. C., McMinn, S. E., Kwan, E. E., Jacobsen, E. N. Screening for generality in asymmetric catalysis. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e610\u003c/strong\u003e, 680\u0026ndash;686 (2022).\u003c/li\u003e\n\u003cli\u003eRein, J. \u003cem\u003eet al\u003c/em\u003e. Generality-oriented optimization of enantioselective aminoxyl radical catalysis. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e380\u003c/strong\u003e, 706\u0026ndash;712 (2023).\u003c/li\u003e\n\u003cli\u003eYang, Y., Arnold, F. H. Navigating the unnatural reaction space: directed evolution of heme proteins for selective carbene and nitrene transfer. \u003cem\u003eAcc. Chem. Res.\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 1209\u0026ndash;1225 (2021).\u003c/li\u003e\n\u003cli\u003eDrienovsk\u0026aacute;, I., Roelfes, G. Expanding the enzyme universe with genetically encoded unnatural amino acids. \u003cem\u003eNat. Catal.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 193\u0026ndash;202 (2020).\u003c/li\u003e\n\u003cli\u003eDavis, H. J., Ward, T. R. Artificial metalloenzymes: Challenges and opportunities. \u003cem\u003eACS Cent. Sci.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1120\u0026ndash;1136 (2019).\u003c/li\u003e\n\u003cli\u003eWang, Y. \u003cem\u003eet al\u003c/em\u003e. Directed evolution: Methodologies and applications. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, 12384\u0026ndash;12444 (2021).\u003c/li\u003e\n\u003cli\u003eRoelfes, G., Feringa, B. L. DNA-based asymmetric catalysis. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 3230\u0026ndash;3232 (2005).\u003c/li\u003e\n\u003cli\u003eSilverman, S. K. DNA as a versatile chemical component for catalysis, encoding, and stereocontrol. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 7180\u0026ndash;7201 (2010).\u003c/li\u003e\n\u003cli\u003ePark, S., Sugiyama, H. DNA-based hybrid catalysts for asymmetric organic synthesis. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e49\u003c/strong\u003e, 3870\u0026ndash;3878 (2010).\u003c/li\u003e\n\u003cli\u003eRioz-Mart\u0026iacute;nez, A., Roelfes, G. DNA-based hybrid catalysis. \u003cem\u003eCurr. Opin. Chem. Biol.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 80\u0026ndash;87 (2015).\u003c/li\u003e\n\u003cli\u003eAubert, S., Duchemin, N., Zhang, J.-L., Smietana, M., Arseniyadis, S. DNA-based asymmetric catalysis: Past, present and future. \u003cem\u003eTopics in Enantioselective Catalysis\u003c/em\u003e, pp. 1\u0026ndash;30 (2022).\u003c/li\u003e\n\u003cli\u003eRoelfes, G., Boersma, A. J., Feringa, B. L. Highly enantioselective DNA-based catalysis. \u003cem\u003eChem. Commun.\u003c/em\u003e 635\u0026ndash;637 (2006).\u003c/li\u003e\n\u003cli\u003eCoqui\u0026egrave;re, D., Feringa, B. L., Roelfes, G. DNA-based catalytic enantioselective Michael reactions in water. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 9308\u0026ndash;9311 (2007).\u003c/li\u003e\n\u003cli\u003eBoersma, A. J., Feringa, B. L., Roelfes, G. Enantioselective Friedel-Crafts reactions in water using a DNA-based catalyst. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 3346\u0026ndash;3348 (2009).\u003c/li\u003e\n\u003cli\u003eBoersma, A. J. \u003cem\u003eet al\u003c/em\u003e. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 991\u0026ndash;995 (2010).\u003c/li\u003e\n\u003cli\u003eOelerich, J., Roelfes, G. \u003cem\u003eChem. Sci.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 2013\u0026ndash;2017 (2013).\u003c/li\u003e\n\u003cli\u003eAmirbekyan, K. \u003cem\u003eet al.\u003c/em\u003e Design, synthesis, and binding affinity evaluation of Hoechst 33258 derivatives for the development of sequence-specific DNA-based asymmetric catalysts. \u003cem\u003eACS Catal. \u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 3096\u0026ndash;3105 (2016).\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Fern\u0026aacute;ndez, A., Megens, R. P., Villarino, L., Roelfes, G. DNA-accelerated copper catalysis of Friedel-Crafts conjugate addition/enantioselective protonation reactions in water. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 16308\u0026ndash;16314 (2016).\u003c/li\u003e\n\u003cli\u003eGuo, J. \u003cem\u003eet al.\u003c/em\u003e A localized enantioselective catalytic site on short DNA sequences and their amphiphiles. \u003cem\u003eJACS Au\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 483\u0026ndash;491 (2022).\u003c/li\u003e\n\u003cli\u003eRopartz, L., Meeuwenoord, N. J., van der Marel, G. A., van Leeuwen, P. W. N. M., Slawin, A. M. Z., Kamer, P. C. J. \u003cem\u003eChem. Commun. \u003c/em\u003e1556\u0026ndash;1558 (2007).\u003c/li\u003e\n\u003cli\u003eOltra, N. S., Roelfes, G. Modular assembly of novel DNA-based catalysts. \u003cem\u003eChem. Commun.\u003c/em\u003e 6039\u0026ndash;6041 (2008).\u003c/li\u003e\n\u003cli\u003eFournier, P., Fiammengo, R., J\u0026auml;schke, A. Allylic amination by a DNA\u0026ndash;diene\u0026ndash;iridium(I) hybrid catalyst. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 4426\u0026ndash;4429 (2009).\u003c/li\u003e\n\u003cli\u003eGjonaj, L., Roelfes, G. Novel catalyst design by using Cisplatin to covalently anchor catalytically active copper complexes to DNA. \u003cem\u003eChemCatChem.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1718\u0026ndash;1721 (2013).\u003c/li\u003e\n\u003cli\u003ePark, S. \u003cem\u003eet al.\u003c/em\u003e Development of DNA-based hybrid catalysts through direct ligand incorporation: Toward understanding of DNA-based asymmetric catalysis. \u003cem\u003eACS Catal.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 4070\u0026ndash;4073 (2014).\u003c/li\u003e\n\u003cli\u003eMansot, J. \u003cem\u003eet al.\u003c/em\u003e A rational quest for selectivity through precise ligand-positioning in tandem DNA-catalysed Friedel\u0026ndash;Crafts alkylation/asymmetric protonation. \u003cem\u003eChem. Sci.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2875\u0026ndash;2881 (2019).\u003c/li\u003e\n\u003cli\u003eDuchemin, N. \u003cem\u003eet al\u003c/em\u003e. New benchmark in DNA-based asymmetric catalysis: prevalence of modified DNA/RNA hybrid systems. \u003cem\u003eJACS Au.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 1910\u0026ndash;1917 (2022).\u003c/li\u003e\n\u003cli\u003eMatteucci, M. D., Caruthers, M. H. Synthesis of deoxyoligonucleotides on a polymer support. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e103\u003c/strong\u003e, 3185\u0026ndash;3191 (1981).\u003c/li\u003e\n\u003cli\u003eKim, Y. J., Wilson, D. M. III. Overview of base excision repair biochemistry. \u003cem\u003eCurr. Mol. Pharmacol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 3\u0026ndash;13 (2012).\u003c/li\u003e\n\u003cli\u003eSchormann, N., Ricciardi, R., Chattopadhyay, D. Uracil-DNA glycosylases-structural and functional perspectives on an essential family of DNA repair enzymes. \u003cem\u003eProtein Sci.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1667\u0026ndash;1685 (2014).\u003c/li\u003e\n\u003cli\u003eK\u0026ouml;lmel, D. K., Kool, E. T. Oximes and hydrazones in bioconjugation: Mechanism and catalysis. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 10358\u0026ndash;10376 (2017).\u003c/li\u003e\n\u003cli\u003eAtamna, H., Cheung, T., Ames, B. N. A method for detecting abasic sites in living cells: age-dependent changes in base excision repair. \u003cem\u003eProc. Natl. Acad. Sci. U S A.\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 686\u0026ndash;691 (2000).\u003c/li\u003e\n\u003cli\u003eKojima, N., Takebayashi, T., Mikami, A., Ohtsuka, E., Komatsu, Y. Construction of highly reactive probes for abasic site detection by introduction of an aromatic and a guanidine residue into an aminooxy group. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 13208\u0026ndash;13209 (2009).\u003c/li\u003e\n\u003cli\u003eWilson, D. L., Kool, E. T. Ultrafast oxime formation enables efficient fluorescence light-up measurement of DNA base excision. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 19379\u0026ndash;19388 (2019).\u003c/li\u003e\n\u003cli\u003eYoshizawa, S., Kawai, G., Watanabe, K., Miura, K. Hirao, I. GNA trinucleotide loop sequences producing extraordinarily stable DNA minihairpins. \u003cem\u003eBiochemistry.\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 4761\u0026ndash;4767 (1997).\u003c/li\u003e\n\u003cli\u003eNakatsuka, N. \u003cem\u003eet al.\u003c/em\u003e Aptamer\u0026ndash;field-effect transistors overcome Debye length imitations for small-molecule sensing. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e362\u003c/strong\u003e, 319\u0026ndash;324 (2018).\u003c/li\u003e\n\u003cli\u003eWatts, O. F. B., Berreur, J., Collins, B. S. L., Clayden, J. Biocatalytic enantioselective synthesis of atropisomers. \u003cem\u003eAcc. Chem. Res.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 3362\u0026ndash;3375 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3941689/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3941689/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOptimizing catalysts through high-throughput screen for asymmetric catalysis is challenging due to the difficulty associated with assembling a library of catalyst analogues in a timely fashion. Here, we repurpose DNA excision repair and integrate it with bioorthogonal conjugation to construct a diverse array of DNA hybrid catalysts for highly accessible and high-throughput asymmetric DNA catalysis, enabling dramatically expedited catalyst optimization process, superior reactivity and selectivity, as well as the first atroposelective DNA catalysis. The bioorthogonality of this conjugation strategy ensures exceptional tolerance towards diverse functional groups, thereby facilitating the facile construction of 42 DNA hybrid catalysts bearing various unprotected functional groups. This unique feature holds the potential to enable catalytic modalities in asymmetric DNA catalysis that were previously deemed unattainable.\u003c/p\u003e","manuscriptTitle":"Merging DNA repair with bioorthogonal conjugation enables accessible and superior asymmetric DNA catalysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 08:45:08","doi":"10.21203/rs.3.rs-3941689/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa26c6d9-fe0c-4e57-bee5-0939530366c6","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29117236,"name":"Physical sciences/Chemistry/Catalysis/Asymmetric catalysis"},{"id":29117237,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":29117238,"name":"Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology"}],"tags":[],"updatedAt":"2024-03-05T08:45:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-05 08:45:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3941689","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3941689","identity":"rs-3941689","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}