Expanded Genetic Alphabet Increases Structural and Chemical Diversity of Six-Letter DNA for High-Affinity Protein-Targeting Aptamers

Expanded Genetic Alphabet Increases Structural and Chemical Diversity of Six-Letter DNA for High-Affinity Protein-Targeting Aptamers | 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 Expanded Genetic Alphabet Increases Structural and Chemical Diversity of Six-Letter DNA for High-Affinity Protein-Targeting Aptamers Osamu Nureki, Kazuhiro Sawada, Michiko Kimoto, Ken-Ichiro Matsunaga, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6296070/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The expansion of the genetic alphabet through unnatural base pairs (UBPs) enables novel biotechnologies to create biopolymers with enhanced informational and functional properties. Hydrophobic UBPs, such as Ds − Px and Ds − Pa′, exhibit high fidelity during PCR amplification to enable the generation of UB-containing DNA aptamers (XenoAptamers) with exceptional affinity and specificity for target proteins. We developed XenoAptamers containing Ds and Px/Pa′ that bind to each serotype or variant of dengue non-structural protein 1 (DENV-NS1), a key biomarker of dengue infection, with picomolar level dissociation constants. To elucidate the mechanisms of their high affinity and specificity, we solved cryo-electron microscopy (cryo-EM) structures of oligomeric NS1 − XenoAptamer complexes. Each XenoAptamer adopts a unique tertiary structure tailored to the target protein’s surficial cavity, with Ds enhancing the structural diversity by an aromatic core formation, while Pa′ fits precisely into NS1’s hydrophobic pocket, expanding and strengthening binding interactions. Rigidity in the XenoAptamers’ tertiary structures by e.g. enhancing dipole-dipole interactions between nucleobases further improved their binding affinity. These findings elucidate the molecular mechanisms underlying XenoAptamers’ specificity and highlight the potential of genetic alphabet expansion for developing antibody alternatives. This approach opens new avenues for therapeutic and diagnostic applications. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biotechnology/Nucleic-acid therapeutics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Main In 1962, Alexander Rich proposed expanding the genetic information of DNA by introducing a third base pair made of two artificial bases, augmenting the four natural bases 1 . Decades later, synthetic base pairs (unnatural base pairs, UBPs) capable of functioning in replication, transcription, and/or translation were successfully developed for genetic alphabet expansion technology 2 – 4 . While some of these UBPs have been incorporated into cellular genomes to enable the production of unnatural proteins containing synthetic amino acids, their impact extends beyond protein engineering 5 , 6 . Notably, UBPs have also advanced DNA engineering, and by introducing UBs into DNA fragments (DNA aptamers), their binding affinities and specificities for target proteins were significantly improved 7 . However, a key question remains: what molecular mechanisms enable UBs to confer such enhanced binding properties and structures to DNA aptamers? The in vitro evolution method (SELEX, S ystematic E volution of L igands by EX ponential enrichment) for generating DNA and RNA aptamers was first introduced in the early 1990s 8 , 9 . DNA aptamers gained attention as potential antibody alternatives due to their ability to be chemically synthesized with low toxicity and immunogenicity. In addition, the hydrophilicity of DNA aptamers addresses the stickiness problem of antibodies. Despite their advantages, conventional aptamers face several limitations. The hydrophilic nature of DNA restricts their interaction with hydrophobic surfaces of target proteins, and the inherent chemical and structural diversity of only four natural nucleobases is insufficient to achieve the broad and strong interactions observed in antibodies with 20 standard amino acids. This limited diversity confines the binding ability of most DNA aptamers to a dissociation constant ( K D ) in the nanomolar range, constraining their diagnostic and clinical applicabilities. Thus, various approaches, such as DNA modification, have been reported to address these DNA aptamer problems 10 – 12 . One particularly effective strategy to overcome these challenges has been to expand the genetic alphabet by introducing UBPs. By employing a hydrophobic UBP between 7-(2-thienyl)imidazo[4,5-β]pyridine (Ds) and the diol derivative of 2-nitro-4-propynylpyrrole (diol-Px) with high fidelity in replication (Fig. 1 a) 13 , we developed the ExSELEX (genetic alphabet Ex pansion for SELEX ) method. This approach enabled the creation of DNA aptamers containing Ds and Px as the 5th and 6th letters with exceptional affinity and specificity, termed XenoAptamers, achieving sub-nanomolar K D values and exhibiting over 100-fold greater affinity than conventional four-letter DNA aptamers 7 , 14 , 15 . Recently, we leveraged these advances to develop a series of XenoAptamers that specifically bind to their respective serotype or variant dengue non-structural protein 1 (NS1), with K D values ranging from 27 to 182 pM 15 (Fig. 1 b and Extended Data Fig. 1 ). Dengue fever is a worldwide infectious disease caused by the dengue virus (DENV), which belongs to the Flavivirus 16 , 17 . One of the challenges complicating dengue diagnosis, treatment, and vaccine development is the existence of four distinct serotypes of DENV (DENV1 − 4). A second infection with a different serotype causes severe and occasionally lethal dengue symptoms, such as hemorrhagic fever (DHF) and dengue shock syndrome (DSS) 18 , 19 . NS1 is universally encoded in Flavivirus genomes, and DENV NS1 is a valuable biomarker for diagnosing DENV infections and may define the severity of dengue symptoms 20 , 21 . NS1 expressed in the infected cells is secreted to the bloodstream, forming tetramers and hexamers 22 . Our XenoAptamers uniquely discriminate each serotype DENV NS1 protein, which shares 69–80% amino acid sequence homology (Extended Data Table 1) 15 . Remarkably, some of these XenoAptamers, such as AptD1 and AptD1b, can even distinguish between two major NS1 variants in DENV serotype 1 (DENV1), which share 97% sequence identity (Fig. 1 c). Using these XenoAptamers, we developed an enzyme-linked immunosorbent assay (ELISA), which can accurately identify each dengue serotype and variant in clinical samples 15 . Moreover, a competitive ELISA system employing these XenoAptamers can detect serotype-specific anti-NS1 IgG in dengue patients, providing a valuable tool for diagnosing and studying immune responses to dengue infections 23 . Even one or two Ds and Px bases in XenoAptamers markedly enhance aptamer affinities and specificities, achieving K D values in the picomolar range and facilitating DENV diagnosis beyond serotype specificity. However, it remains unclear how these two hydrophobic UBs in XenoAptamers enhance the binding affinity and specificity by ca. 1000 folds, and how XenoAptamers recognize the intricate structures of DENV NS1 protein oligomers. To elucidate the molecular basis for the precise recognition by XenoAptamers, we conducted a Cryo-EM single-particle analysis of the complexes of three NS1 protein and XenoAptamer pairs. Selection of high-affinity XenoAptamers specific for serotypes and variants of DENV-NS1 To elucidate the molecular mechanism by which XenoAptamers bind to target proteins with exceptionally high affinity and specificity, we conducted structural analysis using three XenoAptamers (AptD1, AptD1c, and AptD2) specifically bound to DENV NS1 proteins. AptD1 specifically recognizes DENV1-NS1-variant1 (v1) (Fig. 1 c, Extended Data Fig. 1 ) 15 . Additionally, in this study, we identified another XenoAptamer, AptD1c, by thoroughly analyzing the sequences in the enriched library previously obtained through ExSELEX for DENV1-NS1-v1 (Fig. 1 c). After 10 rounds of ExSELEX, the sequences of both AptD1 and AptD1c were predominant in the enriched libraries. AptD1c was shown to recognize both of DENV1-NS1-v1 and DENV1-NS1-variant2 (v2) in an electrophoretic mobility shift assay (EMSA) (Fig. 1 c). Both of AptD1 and AptD1c contain two Ds bases in their loop regions (Fig. 1 b). These findings highlight the promising potential of XenoAptamers for achieving both of strict and broad selectivities. AptD2 specifically binds to the DENV serotype 2 NS1 protein (DENV2-NS1) and contains two Ds bases and one diol-conjugated 4-propynylpyrrole-2-carbaldehyde (diol-Pa′) base in its loop region (Fig. 1 b). In the initial development of the XenoAptamer targeting DENV2-NS1, we utilized an ExSELEX library containing only two Ds bases. The diol-Px base was subsequently introduced via mutation during PCR amplification in the ExSELEX process 7 , 15 . Retaining the diol-Px base throughout ExSELEX underscores its critical role in achieving high-affinity binding. Since the Px nucleoside is unstable under the conditions required for DNA chemical synthesis, we replaced the nitro group in the diol-Px base with an aldehyde group (diol-Pa′) during the preparation of AptD2 (Fig. 1 d) 24 , 25 . Of these UBs, Ds at position 23 and diol-Pa′/Px at position 35 were identified as key contributors to the high affinity for DENV2-NS1 15 . Here, we investigated the essential moiety in diol-Pa′/Px responsible for tight binding as the sixth letter in AptD2. To this end, we prepared four AptD2 variants, in which the original diol-Pa′ was substituted with Pa′, pyrrole-2-carbaldehyde (Pa), thymine (T), and 5-propynyluridine (U′), and evaluated their binding efficiencies to DENV2-NS1 using EMSA (Fig. 1 d). Among these variants, only AptD2 containing diol-Pa′ (AptD2-diol-Pa′) and Pa′ (AptD2-Pa′) exhibited strong binding to the target. Notably, AptD2-Pa′ displayed a more pronounced shifted band compared to AptD2-diol-Pa′ (Fig. 1 d). This observation was consistent with the K D values measured by surface plasmon resonance (SPR), which demonstrated higher affinity for AptD2-Pa′ (69.5 pM) compared to AptD2-diol-Pa′ (115 pM) (Fig. 1 e). Interestingly, another propynyl derivative, AptD2-U′, as well as AptD2-Pa and AptD2-T, exhibited significantly reduced affinities. These results suggest that the combination of pyrrole and propynyl moieties of AptD2-Pa′ is critical for binding to DENV2-NS1 as the sixth letter. No chemical component like the propynylpyrrole residue is found among the four standard nucleobases or the 20 standard amino acids. Distinct tertiary structures of XenoAptamers for precise target protein discrimination Using the three XenoAptamers, we solved the cryo-EM structures of NS1 oligomer-XenoAptamer complexes at 2.5–2.8 Å resolutions (Fig. 2 and Extended Data Fig. 2 . The structures reveal that one aptamer binds to one NS1 dimer within all complexes. All XenoAptamers selectively stick to the outer surfaces of their respective NS1 dimers, tetramers, and hexamers without disrupting the NS1 oligomer formation (Fig. 2 a–i), which is achieved by their distinct tertiary structures (Fig. 2 j–l). The negatively charged backbone of the XenoAptamers interacts with the positively charged regions on the target proteins. However, respective binding sites of AptD1, AptD1c, and AptD2-Pa′ are different among NS1 proteins (Fig. 2 g–i), although 69–80% of their amino acid sequences are conserved. These XenoAptamers thus represent a novel class of polymer capable of exhibiting flexible bending and folding behaviors to accommodate the intricate architectures of target proteins: the formation of these distinct tertiary structures of respective XenoAptamers enable precise shape complementarity with the respective NS1 protein surface, facilitating extremely high affinity and specific binding. All XenoAptamers bear a mini-hairpin sequence, CGCGAAGCG, at their 3′-terminus (Fig. 1 b), which provides enhanced stability against nucleases and heat 26 , 27 . Although the Cryo-EM densities of this mini-hairpin region are not visible, atomic models suggest that the mini-hairpin does not interact with the protein surface but instead protrudes outward. These findings suggest that the mini-hairpin region can be utilized as a modification site for practical XenoAptamer applications, as previously reported 26 – 28 . The fifth letter, Ds, enhances the structural diversity of DNA aptamer by facilitating unique base-stacking formations Our structural analysis revealed that the Ds bases play a pivotal role in shaping the unique and intricate tertiary structures of XenoAptamers. Notably, in three XenoAptamers studied, the Ds bases do not directly interact with the protein (Fig. 3 ). Instead, the Ds base, which consists of a 2-thienyl group and 3-deazapurine, provides a wide stacking platform to recruit surrounding and even distant nucleobases via aromatic p-stacking interactions (Fig. 3 j). Each Ds base interacts with natural bases in various orientations, a feature not observed in natural base-base stacking. The large Ds bases also allow stacking interactions with more than two natural bases, as shown in Ds20 of AptD1c and Ds23 of AptD2. In the complex between AptD1 and DENV1-NS1-v1, the Ds base at position 19 stacks with T18 and T21 (Fig. 3 b), displacing the C20 base outward (Fig. 5 d). Similarly, the Ds25 base intercalates between the T13–A26 and the non-Watson-Crick G14–G24 base pairs (Fig. 3 c). The aromatic core mediated by Ds base is also observed in AptD1c (Fig. 3 d–f). In the complex between AptD1c and DENV1-NS1-v2, Ds33 stacks with C32 and the C34–G24 pair (Fig. 3 e), while Ds20 stacks with the non-canonical G17–A38 pair, displacing T18 outward (Fig. 5 d). In the complex between AptD2 and DENV2-NS1 (Fig. 3 g–i), as previously proposed 15 , AptD2 incorporates a G-quartet structure, and Ds23 tightly stacks on the surface of the quartet formed by G24, G33, G37, and G43 (Fig. 3 h and 3 j). The hydrophobic Ds bases exhibit significantly stronger stacking interactions than natural bases. These stacking interactions promote and stabilize the diverse and unique hydrophobic core structures of XenoAptamers to allow their shape complementarity with the respective NS1 protein surface (Fig. 3 j), thereby enhancing their specificity and high binding affinity toward target proteins. Dipole moments of nucleobases are critical for base-stacking interactions. We previously demonstrated that replacing Ds bases in XenoAptamers with either of two Ds variants, 4-(2-thienyl)pyrrolo[2,3-β]pyridine (Ys) and 4-(2-thienyl)benzimidazole (Bs), significantly impacts their binding affinities (Fig. 4 a) 29 . Specifically, substituting Ds19 and Ds25 with Ys19 and Bs25 in AptD1 (AptD1-YsBs) resulted in a marked enhancement of binding affinity, improving the K D from hundreds of pM to 36 − 64 pM (Extended Data Fig. 3 C), representing a more than five-fold increase 29 . In contrast, the inverse substitution of Ds19 and Ds25 with Bs19 and Ys25 in AptD1 (AptD1-BsYs) significantly reduced the affinity to 1 − 9 nM 29 (Extended Data Fig. 3 C). Our structural analysis revealed that these drastic differences in binding affinity arise from variations in the dipole moments of Ds, Ys, and Bs. The dipole moments of these three UBs in the current structure are presented in Fig. 4 a 29 . In AptD1 (AptD1-DsDs), the dipole moment of Ds19 is oriented approximately 90 degrees relative to those of the stacked T21 and T18 bases (Fig. 4 b, Extended Data Fig. 3 ), indicating that interactions between Ds19 and T21/T18 are suboptimal due to moderate electrostatic repulsion. Similarly, the stacking of Ds25 with G24 and A26 is energetically unfavorable. In contrast, AptD1-YsBs optimizes the dipole moment orientations at both positions 19 and 25, greatly stabilizing the stacked structure (Fig. 4 b). However, in the reversed variant, AptD1-BsYs, both UBs align their dipole moments in the same direction as the neighboring bases, leading to a significant reduction in the structural integrity of the XenoAptamer and its affinity for the target protein (Fig. 4 b). These findings underscore the critical role of stacking interactions involving hydrophobic UBs with larger sizes than natural bases, as they are essential for stabilizing the unique tertiary structures of XenoAptamers. The sixth letter, Px/Pa′, directly interacts with a hydrophobic pocket in the target protein In contrast to Ds, Pa′ directly and snugly binds a deep hydrophobic pocket in its target protein (Fig. 3 i). In the complex structure of AptD2 bound to DENV2-NS1, the non-canonical pair between Ds23 and G36 stacks with the G-quartet formed by G24, G33, G37, and G43 (Fig. 3 h). As the result, the Pa′35 base moiety, adjacent to G36, protrudes outward from the XenoAptamer structure, allowing the propynyl group of Pa′35 to engage with the hydrophobic pocket of DENV2-NS1 (Fig. 3 i). This pocket is located at the concave interface between the wing and b-ladder domains (Fig. 7 b and 7 c) and is formed by Ile96, Pro244, Leu247, Ile264–Trp268, and the C-terminal residues Ser348–Val350 of DENV2-NS1 (Fig. 7 b). To our best knowledge, this structure represents the first observation of a propynyl group engaging in hydrophobic interactions with a target protein cavity. This interaction appears to be highly strong and specific, as substituting Pa′35 with T or propynyl-U (U′) significantly reduced the binding affinity of AptD2 for DENV2-NS1, as shown in Fig. 1 d. Furthermore, this structural analysis provides a clear explanation for the superior binding ability of Pa′ to diol-Pa′ (Figs. 1 d-f). Precise interactions between XenoAptamer and serotype- or variant-specific DENV-NS1 proteins The unique structures of XenoAptamers enable numerous moieties within the XenoAptamers to specifically interact with their target DENV-NS1 proteins. The schematic representations of these structures and their interactions with DENV-NS1 proteins are illustrated in Figs. 5 – 7 . AptD1 − DENV1-NS1-v1 AptD1 interacts with a pocket formed by residues Thr176 − Val178, Ile224 − Lys227, Leu237 − Ser239, Lys245, and Phe261 within the connector subdomain and β-ladder domain of the same DENV1-NS1-v1 protomer (Fig. 5 ). Specifically, the phosphate group of Ds19 forms hydrogen bonds with Thr176 (Fig. 5 d), the C20 nucleobase forms hydrogen bonds with Lys245 and Gln177 (Fig. 5 e), and the phosphate of T21 forms salt bridges with Lys227 (Fig. 5 e). In addition, G14 and G23 hydrogen bond with Glu238, and T27 forms a stacking interaction with Phe261 (Fig. 5 f). Such a wide interaction surface ensures the specificity and high affinity (pM-order) of XenoAptamers. Binding experiments with the AptD1 variants further highlighted the critical roles of most of the bases in the aptamer structure 15 . Within the pocket, Val178 and Lys227 are v1-specific residues, and are replaced by Met178 and Arg227 in the v2 variant (Extended Data Fig. 1 ). The v1-Val178 does not directly interact with AptD1, but snugly fits the phosphate group of Ds19 via van der Waals interactions (Extended Data Fig. 4 ). In contrast, the larger side-chains of Met178 and Arg227 in the v2 variant would sterically hinder the interactions with the AptD1 main chain. Therefore, AptD1 shows strict variant specificity based on just two amino acid differences in DENV1-NS1. AptD1c − DENV1-NS1-v2 AptD1c recognizes both DENV1-NS1-v1 and -v2 variants (Fig. 1 c). AptD1c interacts with a pocket formed by Thr176–Met178 and Pro226–Arg227 from one protomer (Fig. 6 d, e) and Thr209–Trp210, Trp232–Asn234, and Gln253–Tyr256 from the other protomer of the v2 variant (Fig. 6 c, f). Additionally, AptD1c interacts with another surface formed by Leu237–Ser239, Phe261–Ala265, and Asn293–Pro296 (Fig. 6 g-i). Among these residues, again only Met178 and Arg227 are specific to the v2 variant (Extended Data Fig. 1 ), explaining the broad specificity of AptD1c. The T18–T30 bases, except for Ds19, of AptD1c also participate, showing a wide interaction surface critical for the specific and high affinity for DENV1-NS1-v2 (Fig. 6 i). Instead of Met178 and Arg227 of DENV1-NS1-v2, the smaller Val178 and Lys227 residues of the v1 variant might fit better to AptD1c, which may explain why AptD1c shows higher affinity for v1 (Fig. 1 c). AptD1 and AptD1c distinguish DENV1-NS1 from DENV2-NS1, as well as DENV3- and DENV4-NS1 proteins 15 . A structural comparison between DENV1- and DENV2-NS1 reveals why these aptamers do not bind to DENV2-NS1. Within the interaction surface of DENV1-NS1-v1 for AptD1 and AptD1c, Thr176–Gln177–Val178 and Phe261 are not conserved and replaced by Asp176–Val177–Phe178 and His261 in DENV2-NS1 (Extended Data Fig. 1 ). These amino acid differences alter the spatial conformation and electrostatic interface, explaining why AptD1 and AptD1c cannot recognize DENV2-NS1. While DENV1-NS1-v1 and DENV1-NS1-v2 share around 96% amino acid sequence identity, and only ten amino acids within 352 amino acid residues of DENV NS1 are replaced. Among them, only the amino acid residue (Lys227 in v1 and Arg227 in v2) at position 227 was observed as a residue interacting with AptD1 and AptD1c. In the complex of AptD1 and DENV1-NS1-v1, the 3′- and 5′-phosphates at C20 interact with Lys227 (Fig. 5 e), which might be critical for tight binding, exhibiting the high specificity to only variant 1. On the other hand, in the two complexes of both AptD1 and AptD1c, the interactions of three amino acid residues, Gln177, Ser239, and Phe261, with the XenoAptamers were observed (Figs. 5 c and 6 i), leading to the broad specificity of AptD1c that binds to both of DENV1-NS1-v1 and -v2. AptD2 − DENV2-NS1 AptD2-Pa′ recognizes DENV2-NS1 by interacting with a pocket formed by Pro244–Leu247 and His261–Trp268 (Fig. 7 d–f). His261, Ile264, and Thr265 of the DENV2-NS1 pocket are replaced by Phe261, Thr264, and Ala265 in the DENV1-NS1 pocket, and are unfavorable for AptD2-Pa′ recognition (Fig. 7 b, d). His261 is especially critical since its imidazole side chain provides a hydrogen-bonding interaction with the backbone phosphate of A20 of AptD2-Pa′ (Fig. 7 d). Furthermore, Leu247 of DENV2-NS1 is replaced by Tyr247 in DENV1-NS1, which narrows the hydrophobic pocket described above to preclude the entrance of the Pa′ base of AptD2 (Fig. 7 b), explaining why AptD2 cannot bind DENV1-NS1. Thus, XenoAptamers strictly distinguish variants and serotypes at the single amino acid level. Discussion The cryo-EM structures of the XenoAptamer − NS1 complexes reveal the crucial roles of the Ds and Pa′ bases in achieving precise target specificity with exceptionally high affinity. The Ds base creates novel DNA structures by forming a stable hydrophobic aromatic core through unconventional base-stacking interactions, thereby enhancing the structural diversity of DNA. This increased diversity enables XenoAptamers to adapt to the positively charged surfaces of target proteins, enhancing numerous interactions at binding sites in a structure-specific manner. In contrast, the Pa′ base directly interacts with a deep hydrophobic pocket on the target protein, further augmenting the physicochemical diversity of DNA. Together, the Pa′ and Ds bases function synergistically as a “sword and shield” within XenoAptamers, driving their extraordinary binding affinity and specificity. The hydrophobic Ds and Px/Pa′ bases are intriguing as novel side chains for biopolymers, offering distinct characteristics compared to the 20 amino acid side chains found in proteins. The broader π-electron system of Ds enables unique stacking interactions with various other bases and specialized structures, such as G4 quartets. The small dipole moment of the Ds base allows it to stack with any natural base at any angle, enhancing the diversity of DNA structures. In contrast, due to the large dipole moment of each natural base (Extended Data Fig. 3 A), stacking interactions between natural bases are restricted by their orientation (new Extended data Fig. XA). Additionally, as demonstrated in experiments with Ds variants of AptD1 (Fig. 3 ), stacking between Ds base and natural bases within XenoAptaemrs can further stabilize the XenoAptamer’s structure by replacing the Ds bases with Ys or Bs bases, depending on the orientation of the natural bae’s dipole moment. The hydrophobic interaction of the propynyl group of Px/Pa′ with the hydrophobic cavity of target proteins represents a highly distinctive feature. While the propynyl group has been used to enhance the stacking structures of UBPs 30 , 31 , there are no reported examples of its hydrophobic interactions with other molecules. The sole reported instance involves the generation of DNA aptamers containing 5-(1-pentynyl)-U 32 . A thienyl-imidazopyridine of Ds and a propynyl-pyrrole of Pa′ /Px might also be incorporated as novel side chain of non-standard amino acids for artificial proteins by expanding genetic code 32 , 33 . These hydrophobic properties of the unnatural Ds and Px/Pa′ bases play a critical role in significantly enhancing the affinity of DNA aptamers. Similarly, Benner’s and Tan’s teams reported the development of six-letter DNA aptamers using their hydrogen-bonded UBP, Z and P 34 , 35 . However, these UBs, with physicochemical properties closely resembling those of natural bases, did not substantially enhance the binding capabilities of the aptamers. XenoAptamers achieve high affinity by improving both the k on and k off rate parameters. The hydrophobic Ds and Pa′ bases are directly conjugated to ribose in the DNA main chain, resulting in more compact and less flexible structures. This rigidity likely contributes to the enhanced affinity by optimizing both rate parameters, thereby improving K D values. In contrast, a representative modified four-letter DNA aptamer, SOMAmer, primarily enhances the k off rates. SOMAmers incorporate modified deoxyuridines and deoxycytidines 36 , 37 , where U and C are conjugated to hydrophobic aromatic groups via linkers. Consequently, the hydrophobic residues in SOMAmers may exhibit greater flexibility compared to the rigid hydrophobic Ds and Pa′ bases in XenoAptamers. The rigid, pre-formed structures of XenoAptamers, which closely match the target shapes, may favor faster k on rates and higher target-specificity, in contrast to the induced-fit binding mechanism seen in SOMAmers and conventional aptamers, This cryo-EM structural analysis reveals that increasing the rigidity of the unique base-stacking structure by altering the dipole moment of the Ds base significantly enhances the affinity of XenoAptamers. By utilizing a Ds variant, either Ys or Bs, in which a nitrogen atom in Ds is substituted with carbon, the affinity of AptD1 was significantly modified, with AptD1-YsBs showing a dramatic improvement in XenoAptamer affinity. Our structural analysis demonstrates that adjusting the dipole moment of Ds stabilizes the base-stacking structures in AptD1. This discovery provides a rational strategy for optimizing aptamers containing UBs by post-ExSELEX. Furthermore, this dipole moment-based approach may offer a general strategy using natural base variants to enhance the functionality of a wide range of DNA/RNA-based molecules. The XenoAptamers, AptD1, AptD1c, and AptD2, exhibit unique specificities toward different serotypes or variants of DENV-NS1 proteins. Cryo-EM structural analysis reveals numerous interactions between each XenoAptamer and its corresponding NS1 protein. In previous studies, we demonstrated that many bases—both UBs and natural bases—within the XenoAptamers are critical for achieving tight binding to their targets 14 . For instance, transition mutations in any of the 15 bases within the 19-base loop region of AptD1 significantly reduced its affinity for DENV1-NS1-v1. Consequently, even minor disruptions to these interactions can severely impact binding. Each XenoAptamer, with its unique and rigid structure, may also have the ability to recognize subtle conformational differences among the variants of NS1 proteins. The high specificity of XenoAptamers is likely a result of the harmonious integration of numerous precise and sensitive interactions between XenoAptamers and their target proteins. In addition to their local interactions, electrostatic interactions between the XenoAptamers and the targets are highly significant. These interactions involve the phosphate groups in the XenoAptamers and the basic regions within the targets, which are generally non-specific and independent of the base sequences. However, in the case of XenoAptamers, their rigid structure allows them to strongly recognize the tertiary structures of the basic regions within the targets. This results in structure-specific interactions, which are believed to enhance the selectivity of the XenoAptamer. The sixth letter, Px, was accidentally introduced into AptD2 during PCR amplification in the ExSELEX procedure. Sequences containing Px were selected through ExSELEX because Px contributed to the binding. Consequently, in combination with the fifth letter, Ds, it was revealed that these UBs expand both the chemical diversity and structural diversity of DNA, enabling the creation of novel modalities of DNA molecules and increasing the success rates of high-affinity XenoAptamer generation. These results encourage us to develop a method for creating a six-letter XenoAptamer containing Ds and Px/Pa′ as alternatives to antibodies for both therapeutic and diagnostic applications. Methods Expression and purification of NS1 The plasmids encoding DENV NS1s were constructed and purified as previously reported 15 . The plasmids were transfected into Expi293F cells (Thermo Fisher), which were grown and maintained in Expi293 medium (Gibco) at 37°C, with 8% CO 2 under humidified conditions. The supernatant, containing secreted NS1, was incubated with Ni-NTA resin (Qiagen) for 30 min. The resin was washed with 15 column volumes of wash buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 20 mM imidazole), and the protein was eluted in elution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 300 mM imidazole). The protein was further purified by size-exclusion chromatography on a Superdex 200 10/300 Increase column, equilibrated in SEC buffer (PBS buffer with 1 mM MgCl 2 ). The peak fractions were collected. Synthesis of XenoAptamers and Variants XenoAptamers and their variants were chemically synthesized in-house using an H8 DNA/RNA Synthesizer (K&A Laborgerate) with conventional phosphoramidite chemistry. The phosphoramidites of natural deoxyribonucleosides, Biotin-dT, and 5′-propynyluridine (dU′) were purchased from Glen Research. The dDs 25 , dPa 24,25 , dPa′ 25,38 diol-dPa′ 15,39 , dYs, and dBs 29 phosphoramidites were synthesized in-house following previously established protocols. The synthesized DNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE). Cryo-EM sample preparation and data acquisition For the apo sample, the purified protein was concentrated to about 1–2 mg/mL. For the complex with the aptamer, the purified protein was mixed with the aptamer and incubated for 30 min. The complex was concentrated to about 1–2 mg/mL. The purified protein and concentrated complex solution were applied to freshly glow-discharged Au 200 mesh R1.2/1.3 grids (Quantifoil), using a Vitrobot Mark IV (FEI) at 4°C, with a waiting time of 10 s and a blotting time of 2 s under 100% humidity conditions. The grids were then plunge-frozen in liquid ethane and cooled to liquid nitrogen temperature. Cryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode (The University of Tokyo, Japan). Movies were recorded at a nominal magnification of 105 K, corresponding to a calibrated pixel size of 0.83Å, with a total dose of approximately 50 electrons per Å2 per 48 frames. The data were automatically acquired using the EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −1.6 μm. Image processing For all datasets, image processing was performed with RELION-3.1 40 . Beam-induced motion correction and dose weighting were performed with RELION’s implementation of the MotionCor2 algorithm 41 , and the contrast transfer function (CTF) parameters were estimated with CTFFIND-4.1.13 42 . The processed movies were transferred to and further analyzed with the cryoSPARC v3.3.2 software platform. The particles were subjected to several rounds of reference-free 2D classifications to create particle sets. The particles were curated by cryoSPARC heterogenous refinement. As for DENV2-NS1, the selected particles were subjected to 3D variability analysis 43 and the resulting maps with different conformations were used for subsequent heterogeneous refinement. After per-particle CTF refinement 44 and Bayesian polishing 45 were performed, the sets of selected particles were refined using non-uniform refinement 46 . The overall resolutions were determined according to the Fourier shell correlation (FSC) = 0.143 criterion 47 . The local resolution was estimated by cryoSPARC. Model building and validation The initial template was derived from the DENV2-NS1 dimer structure (PDB identifier: 4O6B), followed by manual model building with COOT. The model was refined using Phenix real_space_refine, with secondary structure restraints 48 . The cryo-EM density map figures were generated using UCSF ChimeraX 49 . Molecular graphics figures were prepared using CueMol (http://www.cuemol.org). Binding Analysis of XenoAptamer Variants to NS1 Binding analyses of XenoAptamer variants to NS1 were performed using electrophoretic mobility shift assays (EMSA) and surface plasmon resonance (SPR). Recombinant DENV-NS1 proteins with a C-terminal poly-histidine-tag were obtained from the Native Antigen Company (DENV2-NS1 and DENV1-NS1-v1) or produced in-house using a conventional CHO cell expression system (DENV1-NS1-v2) as described previously 15 . Both EMSA and SPR experiments were conducted according to previous protocols, with modifications 15,29 . Electrophoretic mobility shift assays (EMSA) In EMSA, each DNA was mixed with the DENV NS1 protein at the indicated final concentration in binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 2.7 mM KCl). After a 30-min incubation at 25°C, 0.25 volume of 25% glycerol containing bromophenol blue was added. The mixtures were subjected to PAGE, using a 4% polyacrylamide gel containing 5% glycerol, 1 mM MgCl 2 , and 2.7 mM KCl. The running buffer was 22.5 mM Tris-acetate (pH 7.5) or Tris-borate (pH 7.5). Band patterns were visualized using a LAS-4000 bioimaging analyzer (Fuji Film), after staining with SYBR Gold. Surface Plasmon Resonance (SPR) Dissociation constants were determined using a BIAcore T200 (Cytiva) at 25°C, with running buffer composed of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 2.7 mM KCl, and 0.05% (v/v) Tween-20. Biotinylated DNA aptamer variants were immobilized on a Sensor chip SA. The interactions between the immobilized DNA and NS1 proteins were monitored via injections (150 sec) of 1.25, 2.5, 5, 10, and 20 nM NS1 in the Kinetic Injection mode, at a flow rate of 100 μL/min. Curve fitting was performed using a 1:1 binding model with the BIAevaluation software. Declarations Acknowledgements The authors thank K. Kobayashi, H. Akasaka, T. Kobayashi and S. Omura for assistance with electron microscopy, Prof. Gota Kawai for assistance with dipole moment mapping, and the members of the O.N. laboratory for comments and discussions. Electron microscopy data were collected at the cryo-EM facility at the University of Tokyo. O.N. was supported by AMED Grant Numbers JP223fa627001 and JP19am0401005, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Numbers JP23ama121002 (support number 3272) and JP23ama121012, the Cabinet Office, Government of Japan, Public/Private R&D Investment Strategic Expansion Program (PRISM), Grant Number JPJ008000, and JST CREST Grant Number JPMJCR20E2. Author contributions K.S. performed biochemical and structural analyses with assistance from F.K.S., T.K., Y.I., and Y.K.; K.S. and F.K.S. performed model building and structural refinement; M.K., K.M., and H.P.T. performed biochemical and biophysical experiments on aptamers; K.S., M.K., I.H., and O.N. conceived the project; K.S., R.T. and Y.K. wrote the manuscript with help from all authors; I.H. and O.N. supervised the research. Data and material availability The structures of DENV-NS1 multimers and XenoAptamer complexes have been deposited in the Protein Data Bank. Materials generated in this study are available upon request. Declaration of interests M.K. and I.H. are co-founders and board members of Xenolis. O.N. is a co-founder of, board member of and a scientific advisor for Curreio. References Rich, A. Horizons of Biochemistry . (AcademicPress, 1962). Kimoto, M. & Hirao, I. Genetic alphabet expansion technology by creating unnatural base pairs. 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Extended Data Extended Data Table 1 is not available with this version. Additional Declarations Yes there is potential Competing Interest. M.K. and I.H. are co-founders and board members of Xenolis. O.N. is a co-founder of, board member of and a scientific advisor for Curreio. Supplementary Files Figsup.pdf Extended Figures 1-4 Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Nature Communications → 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. 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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-6296070","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":441862612,"identity":"c622737a-a27b-4a69-8bc5-d793a2f16e85","order_by":0,"name":"Osamu Nureki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYJACxgYDmwQ4TwJKMxPQkgbTYkCsFobDmFpwAnOJ3IcPZxScz5NvYH/AzMPwR05yRvIBhh81DOzmOLRYzkg3NtxgcLvY4ACPAVCLgbG0RFoCY88xBmbLBuxaDG6ksUk+MLiduIGBh4GZ959B4jyJHAMG3gYGZoMDOLWw/3xgcC5xPsRhEC2Mf/FrYWPcYHAgseEAA9hhibOBWpjx2nLmGbPkDIPkYoPDPAYH5zAYG0v2PEs4LHNMArdfjqcxfuz5Y5cn397+8MEbBjk5iePJBx++qbFJxhViCACMOohLBBJADIlkA4Ja4IAfotOOBC2jYBSMglEwvAEAunlRe60Pqs0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1813-7008","institution":"University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Osamu","middleName":"","lastName":"Nureki","suffix":""},{"id":441862613,"identity":"d41b68cb-365d-4ddb-84f4-7c428f52395b","order_by":1,"name":"Kazuhiro Sawada","email":"","orcid":"https://orcid.org/0000-0002-7582-2354","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Sawada","suffix":""},{"id":441862614,"identity":"bb06a5da-bab8-44cc-a76c-996923585ec5","order_by":2,"name":"Michiko Kimoto","email":"","orcid":"","institution":"Xenolis Pte. 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Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Ichiro","middleName":"","lastName":"Hirao","suffix":""}],"badges":[],"createdAt":"2025-03-24 13:57:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6296070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6296070/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67486-x","type":"published","date":"2025-12-16T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80690438,"identity":"6a17ecac-4466-47db-a536-83645eaf3086","added_by":"auto","created_at":"2025-04-16 05:08:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":492711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDENV-NS1 targeting unnatural-base aptamers (XenoAptamers).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Unnatural bases contained in XenoAptamers. The Ds−diol-Px pair is utilized in PCR as a third base pair in the ExSELEX process.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Secondary structures of three XenoAptamers targeting DENV1-NS1-v1 (AptD1), DENV1-NS1-v1 and -v2 (AptD1c) and DENV2-NS1 (AptD2).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Binding analysis of anti-DEN1-NS1-variants (v1 and v2) by an electrophoretic mobility shift assay (EMSA) in the presence of 3M urea at 30℃.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Binding analysis of AptD2 derivatives with substituted Pa'35 variant bases by EMSA.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee, f\u003c/strong\u003e) K\u003csub\u003eD\u003c/sub\u003e values of AptD2 and its derivatives (AptD2-Pa′ (e) and AptD2-Pa′\u003csub\u003e(diol) \u0026nbsp;\u003c/sub\u003e(f) ) determined by SPR.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/3cc95fb822fafe2c98759bb1.png"},{"id":80691983,"identity":"eb8abb58-80d7-41fc-88d7-85482fef9667","added_by":"auto","created_at":"2025-04-16 05:40:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1392258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM density maps and structures of XenoAptamers bound to DENV NS1s.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea-c\u003c/strong\u003e) Cryo-EM density maps of AptD1 with DENV1-NS1-v1 (a), AptD1c with DENV1-NS1-v2 (b), and AptD2 with DENV2-NS1 (c).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed–i\u003c/strong\u003e) Cryo-EM structures of XenoAptamers with their respective NS1 proteins. In the figures, only one XenoAptamer and one dimer of NS1 are shown. The electrostatic surface potential models of NS1 show the XenoAptamers’ recognition of the positively charged surfaces of NS1 proteins (g\u003cstrong\u003e–\u003c/strong\u003ei).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ej\u003c/strong\u003e) Cryo-EM structures of XenoAptamers, in which the unnatural bases Ds and Pa′are shown in yellow and black, respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/b44703ab15d9310f41dd455f.png"},{"id":80690439,"identity":"bd4f62d4-dcc9-4449-b1e1-ec75e121fc1e","added_by":"auto","created_at":"2025-04-16 05:08:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1691678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUnnatural bases of XenoAptamers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea–c\u003c/strong\u003e) The overall and focused structures of AptD1 and the two Ds bases: Ds19 (b) and Ds25 (c).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed–f\u003c/strong\u003e) The overall and focused structures of AptD1c and the two Ds bases: Ds33(e) and Ds20 (f).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg–i\u003c/strong\u003e) The overall and focused structures of AptD1 and Ds and Pa' bases: Ds23 (h) and Pa′35 (i).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ej\u003c/strong\u003e) The structures of Ds bases and their stacking bases in each XenoAptamer.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/2256b65cdd1f50b76a9de3db.png"},{"id":80690442,"identity":"e37da725-f0da-4087-b00c-37e5caba5be9","added_by":"auto","created_at":"2025-04-16 05:08:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":722303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUnnatural base Ds forms an aromatic hydrophobic core with surrounding bases and stabilizes the XenoAptamer structures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Dipole moments of Ds and its variant Ys and Bs (Extended Data Fig. 3A).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Structures of unnatural bases at positions 19 and 25 in AptD1-DsDs and its variants, YsBs and BsYs. The red-blue bar indicates the dipole moment of each base.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/4b37647c5531b50584ae7379.png"},{"id":80690444,"identity":"8e78a3be-4497-4003-87d8-46f5cc284fcf","added_by":"auto","created_at":"2025-04-16 05:08:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":757646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDENV1-NS1-v1 recognition of AptD1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea, b\u003c/strong\u003e) Overall structure of DENV1-NS1-v1 complexed with AptD1 (green). In the XenoAptamer structures, the unnatural Ds bases are shown in orange. One protomer of NS1 protein is colored dark blue (b-roll domain), orange (connector subdomain), yellow (wing domain), and pink (b-ladder domain), while the other is shown in light colors. The residues interacting with AptD1 are highlighted in red.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) The modeled structure of AptD1. The interactions within the aptamer are shown by solid lines, and those between the aptamer and NS1 are shown by dotted lines. The residues that interact with the nucleic acids through their main chains are shown in parentheses. The nucleic acids forming stacking interactions with Ds are highlighted in orange.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed–f\u003c/strong\u003e) DENV1-NS1-v1 recognition of AptD1 in the positively-charged cleft (d, e) and at the outer surface (f).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/99fbd94449698aa749945245.png"},{"id":80691303,"identity":"f9b8bbce-ca4d-420b-b6eb-28abc059a879","added_by":"auto","created_at":"2025-04-16 05:32:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1441099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDENV1-NS1-v2 recognition of AptD1c.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea, b\u003c/strong\u003e) Overall structure (a) and cleft-focused structure (b) of DENV1-NS1-v2 with AptD1c (green). In the XenoAptamer structures, the unnatural bases Ds are shown in orange. One protomer of NS1 protein is colored dark blue (b-roll domain), orange (connector subdomain), yellow (wing domain), and pink (b-ladder domain), while the other is shown in light colors. The residues interacting with AptD1 are highlighted in red.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec–h\u003c/strong\u003e) DENV1-NS1-v1 recognition of AptD1c in the positively-charged cleft (c\u003cstrong\u003e–\u003c/strong\u003ef) and at the outer surface (g, h).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ei\u003c/strong\u003e) The modeled structure of AptD1c. The interactions within the aptamer are shown by solid lines, and those between the aptamer and NS1 are shown by dotted lines. The residues that interact with the nucleic acids through their main chains are shown in parentheses. The nucleic acids forming stacking interactions with Ds are highlighted in orange.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/441effd8ec5fd832eb8ac48a.png"},{"id":80691305,"identity":"8ed5f33e-94ad-401e-9cfe-0f8f7abecdec","added_by":"auto","created_at":"2025-04-16 05:32:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1124362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDENV2-NS1 recognition of AptD2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Overall structure of the DENV2-NS1 complex with AptD2 (green). In the XenoAptamer structures, the unnatural bases Ds and Pa′ are shown in orange and black.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb–d\u003c/strong\u003e) DENV2-NS1 recognition of AptD2.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee, f\u003c/strong\u003e) Pa′35 recognizes the hydrophobic pocket.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) The modeled structure of AptD2. The interactions within the aptamer are shown by solid lines, and those between the aptamer and NS1 are shown by dotted lines. The residues that interact with the nucleic acids through their main chains are shown in parentheses. The nucleic acids forming stacking interactions with Ds are highlighted in orange.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh\u003c/strong\u003e) G-quadruplex structures of AptD2.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/301b7b1f7125f648738720a9.png"},{"id":100863289,"identity":"3c668b2c-fd39-42e5-9673-b4034613e1dd","added_by":"auto","created_at":"2026-01-22 08:06:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9016778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/9cc2ee97-19df-444d-97ad-4303bc24808a.pdf"},{"id":80690448,"identity":"d9e72ced-0c3f-4f5d-99c7-81926703e5be","added_by":"auto","created_at":"2025-04-16 05:08:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2927068,"visible":true,"origin":"","legend":"Extended Figures 1-4","description":"","filename":"Figsup.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6296070/v1/62aafb4f8df5c42e2d87df4c.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nM.K. and I.H. are co-founders and board members of Xenolis. O.N. is a co-founder of, board member of and a scientific advisor for Curreio.","formattedTitle":"Expanded Genetic Alphabet Increases Structural and Chemical Diversity of Six-Letter DNA for High-Affinity Protein-Targeting Aptamers","fulltext":[{"header":"Main","content":"\u003cp\u003eIn 1962, Alexander Rich proposed expanding the genetic information of DNA by introducing a third base pair made of two artificial bases, augmenting the four natural bases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Decades later, synthetic base pairs (unnatural base pairs, UBPs) capable of functioning in replication, transcription, and/or translation were successfully developed for genetic alphabet expansion technology\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. While some of these UBPs have been incorporated into cellular genomes to enable the production of unnatural proteins containing synthetic amino acids, their impact extends beyond protein engineering\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, UBPs have also advanced DNA engineering, and by introducing UBs into DNA fragments (DNA aptamers), their binding affinities and specificities for target proteins were significantly improved\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, a key question remains: what molecular mechanisms enable UBs to confer such enhanced binding properties and structures to DNA aptamers?\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e evolution method (SELEX, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eystematic \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eE\u003c/span\u003evolution of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eL\u003c/span\u003eigands by \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eEX\u003c/span\u003eponential enrichment) for generating DNA and RNA aptamers was first introduced in the early 1990s\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. DNA aptamers gained attention as potential antibody alternatives due to their ability to be chemically synthesized with low toxicity and immunogenicity. In addition, the hydrophilicity of DNA aptamers addresses the stickiness problem of antibodies. Despite their advantages, conventional aptamers face several limitations. The hydrophilic nature of DNA restricts their interaction with hydrophobic surfaces of target proteins, and the inherent chemical and structural diversity of only four natural nucleobases is insufficient to achieve the broad and strong interactions observed in antibodies with 20 standard amino acids. This limited diversity confines the binding ability of most DNA aptamers to a dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) in the nanomolar range, constraining their diagnostic and clinical applicabilities. Thus, various approaches, such as DNA modification, have been reported to address these DNA aptamer problems\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne particularly effective strategy to overcome these challenges has been to expand the genetic alphabet by introducing UBPs. By employing a hydrophobic UBP between 7-(2-thienyl)imidazo[4,5-β]pyridine (Ds) and the diol derivative of 2-nitro-4-propynylpyrrole (diol-Px) with high fidelity in replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, we developed the ExSELEX (genetic alphabet \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eEx\u003c/span\u003epansion for \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSELEX\u003c/span\u003e) method. This approach enabled the creation of DNA aptamers containing Ds and Px as the 5th and 6th letters with exceptional affinity and specificity, termed XenoAptamers, achieving sub-nanomolar K\u003csub\u003eD\u003c/sub\u003e values and exhibiting over 100-fold greater affinity than conventional four-letter DNA aptamers\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRecently, we leveraged these advances to develop a series of XenoAptamers that specifically bind to their respective serotype or variant dengue non-structural protein 1 (NS1), with \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values ranging from 27 to 182 pM\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Dengue fever is a worldwide infectious disease caused by the dengue virus (DENV), which belongs to the Flavivirus\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. One of the challenges complicating dengue diagnosis, treatment, and vaccine development is the existence of four distinct serotypes of DENV (DENV1\u0026thinsp;\u0026minus;\u0026thinsp;4). A second infection with a different serotype causes severe and occasionally lethal dengue symptoms, such as hemorrhagic fever (DHF) and dengue shock syndrome (DSS)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. NS1 is universally encoded in Flavivirus genomes, and DENV NS1 is a valuable biomarker for diagnosing DENV infections and may define the severity of dengue symptoms\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. NS1 expressed in the infected cells is secreted to the bloodstream, forming tetramers and hexamers\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur XenoAptamers uniquely discriminate each serotype DENV NS1 protein, which shares 69\u0026ndash;80% amino acid sequence homology (Extended Data Table\u0026nbsp;1)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Remarkably, some of these XenoAptamers, such as AptD1 and AptD1b, can even distinguish between two major NS1 variants in DENV serotype 1 (DENV1), which share 97% sequence identity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Using these XenoAptamers, we developed an enzyme-linked immunosorbent assay (ELISA), which can accurately identify each dengue serotype and variant in clinical samples\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Moreover, a competitive ELISA system employing these XenoAptamers can detect serotype-specific anti-NS1 IgG in dengue patients, providing a valuable tool for diagnosing and studying immune responses to dengue infections\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEven one or two Ds and Px bases in XenoAptamers markedly enhance aptamer affinities and specificities, achieving \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values in the picomolar range and facilitating DENV diagnosis beyond serotype specificity. However, it remains unclear how these two hydrophobic UBs in XenoAptamers enhance the binding affinity and specificity by ca. 1000 folds, and how XenoAptamers recognize the intricate structures of DENV NS1 protein oligomers. To elucidate the molecular basis for the precise recognition by XenoAptamers, we conducted a Cryo-EM single-particle analysis of the complexes of three NS1 protein and XenoAptamer pairs.\u003c/p\u003e\n\u003ch3\u003eSelection of high-affinity XenoAptamers specific for serotypes and variants of DENV-NS1\u003c/h3\u003e\n\u003cp\u003eTo elucidate the molecular mechanism by which XenoAptamers bind to target proteins with exceptionally high affinity and specificity, we conducted structural analysis using three XenoAptamers (AptD1, AptD1c, and AptD2) specifically bound to DENV NS1 proteins. AptD1 specifically recognizes DENV1-NS1-variant1 (v1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, in this study, we identified another XenoAptamer, AptD1c, by thoroughly analyzing the sequences in the enriched library previously obtained through ExSELEX for DENV1-NS1-v1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). After 10 rounds of ExSELEX, the sequences of both AptD1 and AptD1c were predominant in the enriched libraries. AptD1c was shown to recognize both of DENV1-NS1-v1 and DENV1-NS1-variant2 (v2) in an electrophoretic mobility shift assay (EMSA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Both of AptD1 and AptD1c contain two Ds bases in their loop regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These findings highlight the promising potential of XenoAptamers for achieving both of strict and broad selectivities.\u003c/p\u003e \u003cp\u003eAptD2 specifically binds to the DENV serotype 2 NS1 protein (DENV2-NS1) and contains two Ds bases and one diol-conjugated 4-propynylpyrrole-2-carbaldehyde (diol-Pa\u0026prime;) base in its loop region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the initial development of the XenoAptamer targeting DENV2-NS1, we utilized an ExSELEX library containing only two Ds bases. The diol-Px base was subsequently introduced via mutation during PCR amplification in the ExSELEX process\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Retaining the diol-Px base throughout ExSELEX underscores its critical role in achieving high-affinity binding. Since the Px nucleoside is unstable under the conditions required for DNA chemical synthesis, we replaced the nitro group in the diol-Px base with an aldehyde group (diol-Pa\u0026prime;) during the preparation of AptD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Of these UBs, Ds at position 23 and diol-Pa\u0026prime;/Px at position 35 were identified as key contributors to the high affinity for DENV2-NS1\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we investigated the essential moiety in diol-Pa\u0026prime;/Px responsible for tight binding as the sixth letter in AptD2. To this end, we prepared four AptD2 variants, in which the original diol-Pa\u0026prime; was substituted with Pa\u0026prime;, pyrrole-2-carbaldehyde (Pa), thymine (T), and 5-propynyluridine (U\u0026prime;), and evaluated their binding efficiencies to DENV2-NS1 using EMSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Among these variants, only AptD2 containing diol-Pa\u0026prime; (AptD2-diol-Pa\u0026prime;) and Pa\u0026prime; (AptD2-Pa\u0026prime;) exhibited strong binding to the target. Notably, AptD2-Pa\u0026prime; displayed a more pronounced shifted band compared to AptD2-diol-Pa\u0026prime; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This observation was consistent with the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values measured by surface plasmon resonance (SPR), which demonstrated higher affinity for AptD2-Pa\u0026prime; (69.5 pM) compared to AptD2-diol-Pa\u0026prime; (115 pM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Interestingly, another propynyl derivative, AptD2-U\u0026prime;, as well as AptD2-Pa and AptD2-T, exhibited significantly reduced affinities. These results suggest that the combination of pyrrole and propynyl moieties of AptD2-Pa\u0026prime; is critical for binding to DENV2-NS1 as the sixth letter. No chemical component like the propynylpyrrole residue is found among the four standard nucleobases or the 20 standard amino acids.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDistinct tertiary structures of XenoAptamers for precise target protein discrimination\u003c/h2\u003e \u003cp\u003eUsing the three XenoAptamers, we solved the cryo-EM structures of NS1 oligomer-XenoAptamer complexes at 2.5\u0026ndash;2.8 \u0026Aring; resolutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The structures reveal that one aptamer binds to one NS1 dimer within all complexes. All XenoAptamers selectively stick to the outer surfaces of their respective NS1 dimers, tetramers, and hexamers without disrupting the NS1 oligomer formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;i), which is achieved by their distinct tertiary structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej\u0026ndash;l). The negatively charged backbone of the XenoAptamers interacts with the positively charged regions on the target proteins. However, respective binding sites of AptD1, AptD1c, and AptD2-Pa\u0026prime; are different among NS1 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg\u0026ndash;i), although 69\u0026ndash;80% of their amino acid sequences are conserved. These XenoAptamers thus represent a novel class of polymer capable of exhibiting flexible bending and folding behaviors to accommodate the intricate architectures of target proteins: the formation of these distinct tertiary structures of respective XenoAptamers enable precise shape complementarity with the respective NS1 protein surface, facilitating extremely high affinity and specific binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll XenoAptamers bear a mini-hairpin sequence, CGCGAAGCG, at their 3\u0026prime;-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), which provides enhanced stability against nucleases and heat\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Although the Cryo-EM densities of this mini-hairpin region are not visible, atomic models suggest that the mini-hairpin does not interact with the protein surface but instead protrudes outward. These findings suggest that the mini-hairpin region can be utilized as a modification site for practical XenoAptamer applications, as previously reported\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe fifth letter, Ds, enhances the structural diversity of DNA aptamer by facilitating unique base-stacking formations\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur structural analysis revealed that the Ds bases play a pivotal role in shaping the unique and intricate tertiary structures of XenoAptamers. Notably, in three XenoAptamers studied, the Ds bases do not directly interact with the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Instead, the Ds base, which consists of a 2-thienyl group and 3-deazapurine, provides a wide stacking platform to recruit surrounding and even distant nucleobases via aromatic p-stacking interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Each Ds base interacts with natural bases in various orientations, a feature not observed in natural base-base stacking. The large Ds bases also allow stacking interactions with more than two natural bases, as shown in Ds20 of AptD1c and Ds23 of AptD2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the complex between AptD1 and DENV1-NS1-v1, the Ds base at position 19 stacks with T18 and T21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), displacing the C20 base outward (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Similarly, the Ds25 base intercalates between the T13\u0026ndash;A26 and the non-Watson-Crick G14\u0026ndash;G24 base pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The aromatic core mediated by Ds base is also observed in AptD1c (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026ndash;f). In the complex between AptD1c and DENV1-NS1-v2, Ds33 stacks with C32 and the C34\u0026ndash;G24 pair (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), while Ds20 stacks with the non-canonical G17\u0026ndash;A38 pair, displacing T18 outward (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In the complex between AptD2 and DENV2-NS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u0026ndash;i), as previously proposed\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, AptD2 incorporates a G-quartet structure, and Ds23 tightly stacks on the surface of the quartet formed by G24, G33, G37, and G43 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). The hydrophobic Ds bases exhibit significantly stronger stacking interactions than natural bases. These stacking interactions promote and stabilize the diverse and unique hydrophobic core structures of XenoAptamers to allow their shape complementarity with the respective NS1 protein surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej), thereby enhancing their specificity and high binding affinity toward target proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDipole moments of nucleobases are critical for base-stacking interactions. We previously demonstrated that replacing Ds bases in XenoAptamers with either of two Ds variants, 4-(2-thienyl)pyrrolo[2,3-β]pyridine (Ys) and 4-(2-thienyl)benzimidazole (Bs), significantly impacts their binding affinities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Specifically, substituting Ds19 and Ds25 with Ys19 and Bs25 in AptD1 (AptD1-YsBs) resulted in a marked enhancement of binding affinity, improving the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e from hundreds of pM to 36\u0026thinsp;\u0026minus;\u0026thinsp;64 pM (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), representing a more than five-fold increase\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In contrast, the inverse substitution of Ds19 and Ds25 with Bs19 and Ys25 in AptD1 (AptD1-BsYs) significantly reduced the affinity to 1\u0026thinsp;\u0026minus;\u0026thinsp;9 nM\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Our structural analysis revealed that these drastic differences in binding affinity arise from variations in the dipole moments of Ds, Ys, and Bs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe dipole moments of these three UBs in the current structure are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In AptD1 (AptD1-DsDs), the dipole moment of Ds19 is oriented approximately 90 degrees relative to those of the stacked T21 and T18 bases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that interactions between Ds19 and T21/T18 are suboptimal due to moderate electrostatic repulsion. Similarly, the stacking of Ds25 with G24 and A26 is energetically unfavorable. In contrast, AptD1-YsBs optimizes the dipole moment orientations at both positions 19 and 25, greatly stabilizing the stacked structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, in the reversed variant, AptD1-BsYs, both UBs align their dipole moments in the same direction as the neighboring bases, leading to a significant reduction in the structural integrity of the XenoAptamer and its affinity for the target protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These findings underscore the critical role of stacking interactions involving hydrophobic UBs with larger sizes than natural bases, as they are essential for stabilizing the unique tertiary structures of XenoAptamers.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe sixth letter, Px/Pa′, directly interacts with a hydrophobic pocket in the target protein\u003c/h3\u003e\n\u003cp\u003eIn contrast to Ds, Pa\u0026prime; directly and snugly binds a deep hydrophobic pocket in its target protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). In the complex structure of AptD2 bound to DENV2-NS1, the non-canonical pair between Ds23 and G36 stacks with the G-quartet formed by G24, G33, G37, and G43 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). As the result, the Pa\u0026prime;35 base moiety, adjacent to G36, protrudes outward from the XenoAptamer structure, allowing the propynyl group of Pa\u0026prime;35 to engage with the hydrophobic pocket of DENV2-NS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). This pocket is located at the concave interface between the wing and b-ladder domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) and is formed by Ile96, Pro244, Leu247, Ile264\u0026ndash;Trp268, and the C-terminal residues Ser348\u0026ndash;Val350 of DENV2-NS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). To our best knowledge, this structure represents the first observation of a propynyl group engaging in hydrophobic interactions with a target protein cavity. This interaction appears to be highly strong and specific, as substituting Pa\u0026prime;35 with T or propynyl-U (U\u0026prime;) significantly reduced the binding affinity of AptD2 for DENV2-NS1, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed. Furthermore, this structural analysis provides a clear explanation for the superior binding ability of Pa\u0026prime; to diol-Pa\u0026prime; (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePrecise interactions between XenoAptamer and serotype- or variant-specific DENV-NS1 proteins\u003c/h3\u003e\n\u003cp\u003eThe unique structures of XenoAptamers enable numerous moieties within the XenoAptamers to specifically interact with their target DENV-NS1 proteins. The schematic representations of these structures and their interactions with DENV-NS1 proteins are illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAptD1\u0026thinsp;\u0026minus;\u0026thinsp;DENV1-NS1-v1\u003c/strong\u003e \u003cp\u003eAptD1 interacts with a pocket formed by residues Thr176\u0026thinsp;\u0026minus;\u0026thinsp;Val178, Ile224\u0026thinsp;\u0026minus;\u0026thinsp;Lys227, Leu237\u0026thinsp;\u0026minus;\u0026thinsp;Ser239, Lys245, and Phe261 within the connector subdomain and β-ladder domain of the same DENV1-NS1-v1 protomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, the phosphate group of Ds19 forms hydrogen bonds with Thr176 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), the C20 nucleobase forms hydrogen bonds with Lys245 and Gln177 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), and the phosphate of T21 forms salt bridges with Lys227 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In addition, G14 and G23 hydrogen bond with Glu238, and T27 forms a stacking interaction with Phe261 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Such a wide interaction surface ensures the specificity and high affinity (pM-order) of XenoAptamers. Binding experiments with the AptD1 variants further highlighted the critical roles of most of the bases in the aptamer structure\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Within the pocket, Val178 and Lys227 are v1-specific residues, and are replaced by Met178 and Arg227 in the v2 variant (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The v1-Val178 does not directly interact with AptD1, but snugly fits the phosphate group of Ds19 via van der Waals interactions (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, the larger side-chains of Met178 and Arg227 in the v2 variant would sterically hinder the interactions with the AptD1 main chain. Therefore, AptD1 shows strict variant specificity based on just two amino acid differences in DENV1-NS1.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAptD1c\u0026thinsp;\u0026minus;\u0026thinsp;DENV1-NS1-v2\u003c/strong\u003e \u003cp\u003eAptD1c recognizes both DENV1-NS1-v1 and -v2 variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). AptD1c interacts with a pocket formed by Thr176\u0026ndash;Met178 and Pro226\u0026ndash;Arg227 from one protomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e) and Thr209\u0026ndash;Trp210, Trp232\u0026ndash;Asn234, and Gln253\u0026ndash;Tyr256 from the other protomer of the v2 variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, f). Additionally, AptD1c interacts with another surface formed by Leu237\u0026ndash;Ser239, Phe261\u0026ndash;Ala265, and Asn293\u0026ndash;Pro296 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-i). Among these residues, again only Met178 and Arg227 are specific to the v2 variant (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), explaining the broad specificity of AptD1c. The T18\u0026ndash;T30 bases, except for Ds19, of AptD1c also participate, showing a wide interaction surface critical for the specific and high affinity for DENV1-NS1-v2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). Instead of Met178 and Arg227 of DENV1-NS1-v2, the smaller Val178 and Lys227 residues of the v1 variant might fit better to AptD1c, which may explain why AptD1c shows higher affinity for v1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003eAptD1 and AptD1c distinguish DENV1-NS1 from DENV2-NS1, as well as DENV3- and DENV4-NS1 proteins\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. A structural comparison between DENV1- and DENV2-NS1 reveals why these aptamers do not bind to DENV2-NS1. Within the interaction surface of DENV1-NS1-v1 for AptD1 and AptD1c, Thr176\u0026ndash;Gln177\u0026ndash;Val178 and Phe261 are not conserved and replaced by Asp176\u0026ndash;Val177\u0026ndash;Phe178 and His261 in DENV2-NS1 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These amino acid differences alter the spatial conformation and electrostatic interface, explaining why AptD1 and AptD1c cannot recognize DENV2-NS1.\u003c/p\u003e \u003cp\u003eWhile DENV1-NS1-v1 and DENV1-NS1-v2 share around 96% amino acid sequence identity, and only ten amino acids within 352 amino acid residues of DENV NS1 are replaced. Among them, only the amino acid residue (Lys227 in v1 and Arg227 in v2) at position 227 was observed as a residue interacting with AptD1 and AptD1c. In the complex of AptD1 and DENV1-NS1-v1, the 3\u0026prime;- and 5\u0026prime;-phosphates at C20 interact with Lys227 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), which might be critical for tight binding, exhibiting the high specificity to only variant 1. On the other hand, in the two complexes of both AptD1 and AptD1c, the interactions of three amino acid residues, Gln177, Ser239, and Phe261, with the XenoAptamers were observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ei), leading to the broad specificity of AptD1c that binds to both of DENV1-NS1-v1 and -v2.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAptD2\u0026thinsp;\u0026minus;\u0026thinsp;DENV2-NS1\u003c/strong\u003e \u003cp\u003eAptD2-Pa\u0026prime; recognizes DENV2-NS1 by interacting with a pocket formed by Pro244\u0026ndash;Leu247 and His261\u0026ndash;Trp268 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u0026ndash;f). His261, Ile264, and Thr265 of the DENV2-NS1 pocket are replaced by Phe261, Thr264, and Ala265 in the DENV1-NS1 pocket, and are unfavorable for AptD2-Pa\u0026prime; recognition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, d). His261 is especially critical since its imidazole side chain provides a hydrogen-bonding interaction with the backbone phosphate of A20 of AptD2-Pa\u0026prime; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Furthermore, Leu247 of DENV2-NS1 is replaced by Tyr247 in DENV1-NS1, which narrows the hydrophobic pocket described above to preclude the entrance of the Pa\u0026prime; base of AptD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), explaining why AptD2 cannot bind DENV1-NS1. Thus, XenoAptamers strictly distinguish variants and serotypes at the single amino acid level.\u003c/p\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe cryo-EM structures of the XenoAptamer\u0026thinsp;\u0026minus;\u0026thinsp;NS1 complexes reveal the crucial roles of the Ds and Pa\u0026prime; bases in achieving precise target specificity with exceptionally high affinity. The Ds base creates novel DNA structures by forming a stable hydrophobic aromatic core through unconventional base-stacking interactions, thereby enhancing the structural diversity of DNA. This increased diversity enables XenoAptamers to adapt to the positively charged surfaces of target proteins, enhancing numerous interactions at binding sites in a structure-specific manner. In contrast, the Pa\u0026prime; base directly interacts with a deep hydrophobic pocket on the target protein, further augmenting the physicochemical diversity of DNA. Together, the Pa\u0026prime; and Ds bases function synergistically as a \u0026ldquo;sword and shield\u0026rdquo; within XenoAptamers, driving their extraordinary binding affinity and specificity.\u003c/p\u003e \u003cp\u003eThe hydrophobic Ds and Px/Pa\u0026prime; bases are intriguing as novel side chains for biopolymers, offering distinct characteristics compared to the 20 amino acid side chains found in proteins. The broader π-electron system of Ds enables unique stacking interactions with various other bases and specialized structures, such as G4 quartets. The small dipole moment of the Ds base allows it to stack with any natural base at any angle, enhancing the diversity of DNA structures. In contrast, due to the large dipole moment of each natural base (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), stacking interactions between natural bases are restricted by their orientation (new Extended data Fig. XA). Additionally, as demonstrated in experiments with Ds variants of AptD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), stacking between Ds base and natural bases within XenoAptaemrs can further stabilize the XenoAptamer\u0026rsquo;s structure by replacing the Ds bases with Ys or Bs bases, depending on the orientation of the natural bae\u0026rsquo;s dipole moment.\u003c/p\u003e \u003cp\u003eThe hydrophobic interaction of the propynyl group of Px/Pa\u0026prime; with the hydrophobic cavity of target proteins represents a highly distinctive feature. While the propynyl group has been used to enhance the stacking structures of UBPs \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, there are no reported examples of its hydrophobic interactions with other molecules. The sole reported instance involves the generation of DNA aptamers containing 5-(1-pentynyl)-U\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A thienyl-imidazopyridine of Ds and a propynyl-pyrrole of Pa\u0026prime; /Px might also be incorporated as novel side chain of non-standard amino acids for artificial proteins by expanding genetic code\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese hydrophobic properties of the unnatural Ds and Px/Pa\u0026prime; bases play a critical role in significantly enhancing the affinity of DNA aptamers. Similarly, Benner\u0026rsquo;s and Tan\u0026rsquo;s teams reported the development of six-letter DNA aptamers using their hydrogen-bonded UBP, Z and P\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, these UBs, with physicochemical properties closely resembling those of natural bases, did not substantially enhance the binding capabilities of the aptamers.\u003c/p\u003e \u003cp\u003eXenoAptamers achieve high affinity by improving both the \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e rate parameters. The hydrophobic Ds and Pa\u0026prime; bases are directly conjugated to ribose in the DNA main chain, resulting in more compact and less flexible structures. This rigidity likely contributes to the enhanced affinity by optimizing both rate parameters, thereby improving K\u003csub\u003eD\u003c/sub\u003e values. In contrast, a representative modified four-letter DNA aptamer, SOMAmer, primarily enhances the \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e rates. SOMAmers incorporate modified deoxyuridines and deoxycytidines\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, where U and C are conjugated to hydrophobic aromatic groups via linkers. Consequently, the hydrophobic residues in SOMAmers may exhibit greater flexibility compared to the rigid hydrophobic Ds and Pa\u0026prime; bases in XenoAptamers. The rigid, pre-formed structures of XenoAptamers, which closely match the target shapes, may favor faster \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e rates and higher target-specificity, in contrast to the induced-fit binding mechanism seen in SOMAmers and conventional aptamers,\u003c/p\u003e \u003cp\u003eThis cryo-EM structural analysis reveals that increasing the rigidity of the unique base-stacking structure by altering the dipole moment of the Ds base significantly enhances the affinity of XenoAptamers. By utilizing a Ds variant, either Ys or Bs, in which a nitrogen atom in Ds is substituted with carbon, the affinity of AptD1 was significantly modified, with AptD1-YsBs showing a dramatic improvement in XenoAptamer affinity. Our structural analysis demonstrates that adjusting the dipole moment of Ds stabilizes the base-stacking structures in AptD1. This discovery provides a rational strategy for optimizing aptamers containing UBs by post-ExSELEX. Furthermore, this dipole moment-based approach may offer a general strategy using natural base variants to enhance the functionality of a wide range of DNA/RNA-based molecules.\u003c/p\u003e \u003cp\u003eThe XenoAptamers, AptD1, AptD1c, and AptD2, exhibit unique specificities toward different serotypes or variants of DENV-NS1 proteins. Cryo-EM structural analysis reveals numerous interactions between each XenoAptamer and its corresponding NS1 protein. In previous studies, we demonstrated that many bases\u0026mdash;both UBs and natural bases\u0026mdash;within the XenoAptamers are critical for achieving tight binding to their targets\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. For instance, transition mutations in any of the 15 bases within the 19-base loop region of AptD1 significantly reduced its affinity for DENV1-NS1-v1. Consequently, even minor disruptions to these interactions can severely impact binding. Each XenoAptamer, with its unique and rigid structure, may also have the ability to recognize subtle conformational differences among the variants of NS1 proteins.\u003c/p\u003e \u003cp\u003eThe high specificity of XenoAptamers is likely a result of the harmonious integration of numerous precise and sensitive interactions between XenoAptamers and their target proteins. In addition to their local interactions, electrostatic interactions between the XenoAptamers and the targets are highly significant. These interactions involve the phosphate groups in the XenoAptamers and the basic regions within the targets, which are generally non-specific and independent of the base sequences. However, in the case of XenoAptamers, their rigid structure allows them to strongly recognize the tertiary structures of the basic regions within the targets. This results in structure-specific interactions, which are believed to enhance the selectivity of the XenoAptamer.\u003c/p\u003e \u003cp\u003eThe sixth letter, Px, was accidentally introduced into AptD2 during PCR amplification in the ExSELEX procedure. Sequences containing Px were selected through ExSELEX because Px contributed to the binding. Consequently, in combination with the fifth letter, Ds, it was revealed that these UBs expand both the chemical diversity and structural diversity of DNA, enabling the creation of novel modalities of DNA molecules and increasing the success rates of high-affinity XenoAptamer generation. These results encourage us to develop a method for creating a six-letter XenoAptamer containing Ds and Px/Pa\u0026prime; as alternatives to antibodies for both therapeutic and diagnostic applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eExpression and purification of NS1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plasmids encoding DENV NS1s were constructed and purified as previously reported\u003csup\u003e15\u003c/sup\u003e. The plasmids were transfected into Expi293F cells (Thermo Fisher), which were grown and maintained in Expi293 medium (Gibco) at 37°C, with 8% CO\u003csub\u003e2\u003c/sub\u003e under humidified conditions. The supernatant, containing secreted NS1, was incubated with Ni-NTA resin (Qiagen) for 30 min. The resin was washed with 15 column volumes of wash buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 20 mM imidazole), and the protein was eluted in elution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 300 mM imidazole). The protein was further purified by size-exclusion chromatography on a Superdex 200 10/300 Increase column, equilibrated in SEC buffer (PBS buffer with 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e). The peak fractions were collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of XenoAptamers and Variants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXenoAptamers and their variants were chemically synthesized in-house using an H8 DNA/RNA Synthesizer (K\u0026amp;A Laborgerate) with conventional phosphoramidite chemistry. The phosphoramidites of natural deoxyribonucleosides, Biotin-dT, and 5′-propynyluridine (dU′) were purchased from Glen Research. The dDs\u003csup\u003e25\u003c/sup\u003e, dPa\u003csup\u003e24,25\u003c/sup\u003e, dPa′\u003csup\u003e25,38\u003c/sup\u003e diol-dPa′\u003csup\u003e15,39\u003c/sup\u003e, dYs, and dBs\u003csup\u003e29\u003c/sup\u003e phosphoramidites were synthesized in-house following previously established protocols. The synthesized DNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM sample preparation and data acquisition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the apo sample, the purified protein was concentrated to about 1–2 mg/mL. For the complex with the aptamer, the purified protein was mixed with the aptamer and incubated for 30 min. The complex was concentrated to about 1–2 mg/mL. The purified protein and concentrated complex solution were applied to freshly glow-discharged Au 200 mesh R1.2/1.3 grids (Quantifoil), using a Vitrobot Mark IV (FEI) at 4°C, with a waiting time of 10 s and a blotting time of 2 s under 100% humidity conditions. The grids were then plunge-frozen in liquid ethane and cooled to liquid nitrogen temperature.\u003c/p\u003e\n\u003cp\u003eCryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode (The University of Tokyo, Japan). Movies were recorded at a nominal magnification of 105 K, corresponding to a calibrated pixel size of 0.83Å, with a total dose of approximately 50 electrons per Å2 per 48 frames. The data were automatically acquired using the EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −1.6 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor all datasets, image processing was performed with RELION-3.1\u003csup\u003e40\u003c/sup\u003e. Beam-induced motion correction and dose weighting were performed with RELION’s implementation of the MotionCor2 algorithm\u003csup\u003e41\u003c/sup\u003e, and the contrast transfer function (CTF) parameters were estimated with CTFFIND-4.1.13\u003csup\u003e42\u003c/sup\u003e. The processed movies were transferred to and further analyzed with the cryoSPARC v3.3.2 software platform. The particles were subjected to several rounds of reference-free 2D classifications to create particle sets. The particles were curated by cryoSPARC heterogenous refinement. As for DENV2-NS1, the selected particles were subjected to 3D variability analysis\u003csup\u003e43\u003c/sup\u003e and the resulting maps with different conformations were used for subsequent heterogeneous refinement. After per-particle CTF refinement\u003csup\u003e44\u003c/sup\u003e and Bayesian polishing\u003csup\u003e45\u003c/sup\u003e were performed, the sets of selected particles were refined using non-uniform refinement\u003csup\u003e46\u003c/sup\u003e. The overall resolutions were determined according to the Fourier shell correlation (FSC) = 0.143 criterion\u003csup\u003e47\u003c/sup\u003e. The local resolution was estimated by cryoSPARC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial template was derived from the DENV2-NS1 dimer structure (PDB identifier: 4O6B), followed by manual model building with COOT. The model was refined using Phenix real_space_refine, with secondary structure restraints\u003csup\u003e48\u003c/sup\u003e. The cryo-EM density map figures were generated using UCSF ChimeraX\u003csup\u003e49\u003c/sup\u003e. Molecular graphics figures were prepared using CueMol (http://www.cuemol.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBinding Analysis of XenoAptamer Variants to NS1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBinding analyses of XenoAptamer variants to NS1 were performed using electrophoretic mobility shift assays (EMSA) and surface plasmon resonance (SPR). Recombinant DENV-NS1 proteins with a C-terminal poly-histidine-tag were obtained from the Native Antigen Company (DENV2-NS1 and DENV1-NS1-v1) or produced in-house using a conventional CHO cell expression system (DENV1-NS1-v2) as described previously\u003csup\u003e15\u003c/sup\u003e. Both EMSA and SPR experiments were conducted according to previous protocols, with modifications\u003csup\u003e15,29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophoretic mobility shift assays (EMSA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn EMSA, each DNA was mixed with the DENV NS1 protein at the indicated final concentration in binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2.7 mM KCl). After a 30-min incubation at 25°C, 0.25 volume of 25% glycerol containing bromophenol blue was added. The mixtures were subjected to PAGE, using a 4% polyacrylamide gel containing 5% glycerol, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 2.7 mM KCl. The running buffer was 22.5 mM Tris-acetate (pH 7.5) or Tris-borate (pH 7.5). Band patterns were visualized using a LAS-4000 bioimaging analyzer (Fuji Film), after staining with SYBR Gold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface Plasmon Resonance (SPR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDissociation constants were determined using a BIAcore T200 (Cytiva) at 25°C, with running buffer composed of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2.7 mM KCl, and 0.05% (v/v) Tween-20. Biotinylated DNA aptamer variants were immobilized on a Sensor chip SA. The interactions between the immobilized DNA and NS1 proteins were monitored via injections (150 sec) of 1.25, 2.5, 5, 10, and 20 nM NS1 in the Kinetic Injection mode, at a flow rate of 100 μL/min. Curve fitting was performed using a 1:1 binding model with the BIAevaluation software.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank K. Kobayashi, H. Akasaka, T. Kobayashi and S. Omura for assistance with electron microscopy, Prof. Gota Kawai for assistance with dipole moment mapping, and the members of the O.N. laboratory for comments and discussions. Electron microscopy data were collected at the cryo-EM facility at the University of Tokyo. O.N. was supported by AMED Grant Numbers JP223fa627001 and JP19am0401005, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Numbers JP23ama121002 (support number 3272) and JP23ama121012, the Cabinet Office, Government of Japan, Public/Private R\u0026amp;D Investment Strategic Expansion Program (PRISM), Grant Number JPJ008000, and JST CREST Grant Number JPMJCR20E2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.S. performed biochemical and structural analyses with assistance from F.K.S., T.K., Y.I., and Y.K.; K.S. and F.K.S. performed model building and structural refinement; M.K., K.M., and H.P.T. performed biochemical and biophysical experiments on aptamers; K.S., M.K., I.H., and O.N. conceived the project; K.S., R.T. and Y.K. wrote the manuscript with help from all authors; I.H. and O.N. supervised the research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and material availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structures of DENV-NS1 multimers and XenoAptamer complexes have been deposited in the Protein Data Bank. Materials generated in this study are available upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.K. and I.H. are co-founders and board members of Xenolis. O.N. is a co-founder of, board member of and a scientific advisor for Curreio.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRich, A. \u003cem\u003eHorizons of Biochemistry\u003c/em\u003e. (AcademicPress, 1962).\u003c/li\u003e\n\u003cli\u003eKimoto, M. \u0026amp; Hirao, I. Genetic alphabet expansion technology by creating unnatural base pairs. \u003cem\u003eChem Soc Rev\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 7602\u0026ndash;7626 (2020).\u003c/li\u003e\n\u003cli\u003eMalyshev, D. A. \u0026amp; Romesberg, F. E. 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Hydrophobic UBPs, such as Ds\u0026thinsp;\u0026minus;\u0026thinsp;Px and Ds\u0026thinsp;\u0026minus;\u0026thinsp;Pa\u0026prime;, exhibit high fidelity during PCR amplification to enable the generation of UB-containing DNA aptamers (XenoAptamers) with exceptional affinity and specificity for target proteins. We developed XenoAptamers containing Ds and Px/Pa\u0026prime; that bind to each serotype or variant of dengue non-structural protein 1 (DENV-NS1), a key biomarker of dengue infection, with picomolar level dissociation constants. To elucidate the mechanisms of their high affinity and specificity, we solved cryo-electron microscopy (cryo-EM) structures of oligomeric NS1\u0026thinsp;\u0026minus;\u0026thinsp;XenoAptamer complexes. Each XenoAptamer adopts a unique tertiary structure tailored to the target protein\u0026rsquo;s surficial cavity, with Ds enhancing the structural diversity by an aromatic core formation, while Pa\u0026prime; fits precisely into NS1\u0026rsquo;s hydrophobic pocket, expanding and strengthening binding interactions. Rigidity in the XenoAptamers\u0026rsquo; tertiary structures by e.g. enhancing dipole-dipole interactions between nucleobases further improved their binding affinity. These findings elucidate the molecular mechanisms underlying XenoAptamers\u0026rsquo; specificity and highlight the potential of genetic alphabet expansion for developing antibody alternatives. This approach opens new avenues for therapeutic and diagnostic applications.\u003c/p\u003e","manuscriptTitle":"Expanded Genetic Alphabet Increases Structural and Chemical Diversity of Six-Letter DNA for High-Affinity Protein-Targeting Aptamers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 05:08:29","doi":"10.21203/rs.3.rs-6296070/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5bedf02b-3edb-4238-a31d-48d00c4d5d42","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47052218,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":47052219,"name":"Biological sciences/Biotechnology/Nucleic-acid therapeutics"}],"tags":[],"updatedAt":"2026-01-22T08:06:37+00:00","versionOfRecord":{"articleIdentity":"rs-6296070","link":"https://doi.org/10.1038/s41467-025-67486-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-16 05:00:00","publishedOnDateReadable":"December 16th, 2025"},"versionCreatedAt":"2025-04-16 05:08:29","video":"","vorDoi":"10.1038/s41467-025-67486-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67486-x","workflowStages":[]},"version":"v1","identity":"rs-6296070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6296070","identity":"rs-6296070","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}