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Takeru Kanazu, Erika Komiya, [Daimei Miura](https://orcid.org/0009-0007-0005-9976), [Kaori Tsukakoshi](https://orcid.org/0000-0003-4779-1113), [Kazunori Ikebukuro](https://orcid.org/0000-0003-2838-0562), [Tomohiko Yamazaki](https://orcid.org/0000-0003-2136-8042), [Ryutaro Asano](https://orcid.org/0000-0001-6795-8377)

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[Turning on Protein Function Inhibited by DNA Aptamers Employing a Covalent DNA-Binding Protein](https://mdr.nims.go.jp/datasets/609b2d57-2041-4f51-8d1e-7e0c1c3c3727)

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Turning on Protein Function Inhibited by DNA Aptamers Employing a Covalent DNA-Binding ProteinTurning on Protein Function Inhibited by DNA Aptamers Employinga Covalent DNA-Binding ProteinTakeru Kanazu, Erika Komiya, Daimei Miura, Kaori Tsukakoshi, Kazunori Ikebukuro,*Tomohiko Yamazaki,* and Ryutaro Asano*Cite This: ACS Nanosci. Au 2026, 6, 201−207 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Certain DNA aptamers can serve as effectiveinhibitors, although their inhibitory action is typically unidirec-tional. Simple removal of these aptamers from their targetcomplexes could improve their application in areas such asoligonucleotide therapeutics. In the current study, we used uracil-DNA glycosylase from Mycobacterium smegmatis (UdgX), whichbinds covalently to DNA after uracil removal. A suitable site forincorporating uracil-DNA into a G-quadruplex-structured DNAaptamer was identified, and subsequent restoration of thrombinactivity previously inhibited by the DNA aptamers was examined.The addition of UdgX restored thrombin activity to nearly 100% within 1 min, demonstrating greater speed and efficacy thancomplementary strand addition targeting the thrombin aptamer. Furthermore, UdgX restored the binding capacity of the anti-VEGFantibody bevacizumab that had been inhibited by a hairpin-structured aptamer. These findings highlight the versatile potential ofUdgX to turn on protein functions inhibited by DNA aptamers.KEYWORDS: Anticoagulant, DNA aptamer, DNA aptamer remover, UdgX, uracil-DNA glycosylaseDNA aptamers have garnered considerable attention dueto their ability to bind and inhibit various substances,including ions, chemical compounds, and proteins.1−3 Forinstance, the anticoagulant thrombin aptamer binds to theblood coagulation factor thrombin, thereby inhibiting bloodcoagulation. Meanwhile, DNA aptamers can also function asdrugs for cancer4 and neurodegenerative diseases5 by bindingto and inhibiting their targets. Furthermore, unlike antibodies,DNA aptamers can be easily prepared by chemical synthesisbased on known sequences.Thus, if the DNA aptamers can be removed from theirbinding complex using a simple operation, we can turn on thetarget molecule’s function. This enhances the utility of DNAaptamers for various applications. For instance, in the case ofantithrombin aptamers that are utilized as anticoagulants, thefunction of thrombin must be rapidly restored when bleedingoccurs.6 Therefore, the development of a rapid, effective, anduniversal method for DNA aptamer removal is important toaccelerate the application of aptamer-based drugs. To this end,researchers have employed complementary strands�allowingthe aptamer to release the target by forming double-strandedDNA�and G-quadruplex (G4)-specific ligands, which recog-nize typical guanine-rich nucleic acid sequences, as inhibitorsof antithrombin aptamers.7,8 However, these operations aretime-consuming, necessitating the development of a rapid turn-on operation.Uracil-DNA glycosylase�commonly referred to as UdgX�is derived from Mycobacterium smegmatis. Distinct from otheruracil-DNA glycosylases, UdgX establishes an irreversiblecovalent glycosidic bond at the abasic site through a histidineresidue, following the recognition and excision of aberranturacil in either single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Since its initial characterization in2015, UdgX has predominantly been utilized for uracildetection.9,10 More recently, we have leveraged UdgX’s uniquecovalent DNA-binding capability to construct a versatile,reversible DNA−protein coupling module by designing anaptamer−enzyme complex that efficiently forms covalentbonds with uracil-DNA-containing aptamers.11In this study, we focus on the remarkable properties ofUdgX to develop a rapid, turn-on technology for proteinfunctions inhibited by DNA aptamers. First, we examinedwhere uracil-DNA could be incorporated into the antithrom-bin aptamer RE3112 and evaluated the aptamer’s functionality.Interestingly, certain RE31 mutants exhibited higher antico-agulant activity than the original RE31 sequence. ThrombinReceived: September 25, 2025Revised: December 13, 2025Accepted: December 15, 2025Published: December 20, 2025Letterpubs.acs.org/nanoau© 2025 The Authors. Published byAmerican Chemical Society201https://doi.org/10.1021/acsnanoscienceau.5c00143ACS Nanosci. Au 2026, 6, 201−207This article is licensed under CC-BY-NC-ND 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on June 24, 2026 at 09:36:53 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Takeru+Kanazu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Erika+Komiya"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Daimei+Miura"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kaori+Tsukakoshi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kazunori+Ikebukuro"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Tomohiko+Yamazaki"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Tomohiko+Yamazaki"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Ryutaro+Asano"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acsnanoscienceau.5c00143&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=tgr1&ref=pdfhttps://pubs.acs.org/toc/anaccx/6/2?ref=pdfhttps://pubs.acs.org/toc/anaccx/6/2?ref=pdfhttps://pubs.acs.org/toc/anaccx/6/2?ref=pdfhttps://pubs.acs.org/toc/anaccx/6/2?ref=pdfpubs.acs.org/nanoau?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acsnanoscienceau.5c00143?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://pubs.acs.org/nanoau?ref=pdfhttps://pubs.acs.org/nanoau?ref=pdfhttps://creativecommons.org/licenses/by-nc-nd/4.0/function, initially inhibited by RE31, was restored upon theintroduction of UdgX, which formed a covalent bond withuracil-DNA. The turn-on property induced by UdgX was fasterand more effective than that induced by adding acomplementary strand to RE31. Furthermore, we validatedthe restored binding capacity of the antivascular endothelialgrowth factor (VEGF) antibody, bevacizumab, previouslyinhibited by an anti-ideotype aptamer, A14#1,13 which adoptsa stem−loop structure. These findings establish that the UdgX-based turn-on technology is universally applicable, irrespectiveof aptamer structure. Moreover, this work represents the firstreport of a rapid turn-on technology for protein functioninhibited by DNA aptamers.■ INCORPORATION OF URACIL-DNA INTO ATHROMBIN-BINDING APTAMER, RE31RE31 is a thrombin-binding aptamer that exhibits anticoagu-lant activity by inhibiting the enzymatic activity of thrombin.12Considering that UdgX recognizes uracil-DNA, we firstinvestigated the introduction of uracil-DNA into RE31. Wesubstituted thymine in RE31 with uracil due to their similarstructures. Specifically, we replaced thymine on the loop ofRE31 with a uracil base, or added a uracil-DNA-containingsequence to the 3′ or 5′ end of RE31 (Table 1). All uracil-DNA-replaced or introduced mutants are referred to as RE31mutants in this study.Circular dichroism (CD) spectroscopy was used to confirmthe secondary structure of the RE31 mutants. All RE31mutants exhibited positive peaks at 245 and 295 nm and anegative peak at 265 nm, indicating that they formed anantiparallel G4 structure similar to that of the originalRE3114,15 (see Supporting Information Figure S1 for CDspectra). In contrast, some mutants with a long sequenceadded at either the 5′ or 3′ terminus�i.e., RE31_5′(AAGT-GUAGGCA), RE31_5′(GUAG), RE31_5′(TGUAG),RE31_3′(AAGTGUAGGCA), and RE31_3′(GUAG)�dem-onstrated smaller peaks at 265 nm than that of RE31. Thesesequences were predicted to form an antiparallel G4 structure;however, the introduction of longer sequences might lead toloosening of the structure.16Covalent binding of UdgX to the RE31 mutants wasconfirmed by SDS-PAGE (see Supporting Information FigureS2 for SDS-PAGE of RE31 and RE31 mutant). UdgX did notform covalemt binding to the original RE31 aptamer, whichdid not contain uracil-DNA (cFigure S2C1 and S2C2). Incontrast, the formation of the complex with UdgX wasconfirmed for the RE31 mutants, excluding RE31_5′(U) andRE31_3′(U). These results indicate that UdgX is capable ofbinding to uracil-DNA located in all loop regions, as well as atTable 1. Oligonucleotide Sequences for RE31, RE31 Mutants, and Complementary StrandACS Nanoscience Au pubs.acs.org/nanoau Letterhttps://doi.org/10.1021/acsnanoscienceau.5c00143ACS Nanosci. Au 2026, 6, 201−207202https://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=tbl1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=tbl1&ref=pdfpubs.acs.org/nanoau?ref=pdfhttps://doi.org/10.1021/acsnanoscienceau.5c00143?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asthe 5′ and 3′ termini of the G4-forming thrombin aptamer.Furthermore, as previously reported,10,15 our results suggestthat the presence of at least two additional nucleotides besidethe uracil base was required at the 5′ or 3′ terminal for theefficient DNA binding of UdgX.■ MEASUREMENT OF COAGULATION TIME USINGTHE RE31 MUTANTSWe investigated the effect of the RE31 mutants on coagulationactivity by measuring fibrinogen coagulation time. Figures 1A−C show the results for the uracil-DNA-replaced RE31 mutant,RE31 with uracil-DNA containing sequences introduced at the5′ end, and RE31 with uracil-DNA containing sequencesintroduced at the 3′ end, respectively. All RE31 mutantsexhibited extended coagulation times, indicating that RE31mutants with thymine replaced by uracil-DNA also possessthrombin inhibitory activity (Figure 1A−C, and SupportingInformation Figure S3A−C for Time-course measurement ofabsorbance at 380 nm monitoring fibrinogen coagulation).Interestingly, certain RE31 mutants demonstrated higherinhibitory activity; in particular, RE31_T21U showed a 1.5-fold higher activity than RE31. The replacement of T13 ofthrombin binding aptamer (TBA) (the original sequence ofRE31, Supporting Information Table S1 for Oligonucleotidesequences for TBA) with a thymine analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2-deoxyuridine, reportedly decreases thebinding affinity for thrombin,17 suggesting the importance ofT21 for RE31 binding. In addition, the replacement of uracil-DNA in antisevere acute respiratory syndrome coronavirus 2(SARS-CoV-2) nucleocapsid protein DNA aptamers to fit thebinding pocket and enhance stereocomplementarity increasedbinding affinity.18 Considering these factors, the increasedsteric complementarity between RE31_T21U and thrombinmay also have contributed to the enhanced binding affinity,which, in turn, contributed to the enhanced thrombininhibitory activity of RE31_T21U.In contrast, RE31_T11U exhibited a 0.6-fold decrease inthrombin inhibitory activity. We hypothesized that substitutingthymine with uracil, which lacks a methyl group, would alter itsmolecular structure. This structural modification may contrib-ute to diminished binding affinity for thrombin. Furthermore,prior studies have demonstrated that replacing T11 withFigure 1. (A) Coagulation time without aptamer (white), or with RE31 (gray), RE31 after the addition of UdgX (black), uracil-DNA-replacedRE31 mutant (light blue), and uracil-DNA-replaced RE31 mutant (with UdgX) (deep blue). (B) Coagulation time without aptamer (white), orwith RE31 (gray), RE31 after the addition of UdgX (black), RE31 with uracil-DNA-containing sequences introduced at the 5′ end (light green),and RE31 with uracil-DNA-containing sequences introduced at the 5′ end (with UdgX) (deep green). (C) Coagulation time without aptamer(white), or with RE31 (gray), RE31 after the addition of UdgX (black), RE31 with uracil-DNA-containing sequences introduced at the 3′ end(light orange), and RE31 with uracil-DNA-containing sequences introduced at the 3′ end (with UdgX) (deep orange). All the results arerepresented as mean ± SD (n = 3). (D) Thrombin activity recovery rate of RE31 (black), uracil-DNA-replaced RE31 (deep blue), uracil-DNA-containing sequences introduced at the 5′ end (deep green), and RE31 with uracil-DNA-containing sequences introduced at the 3′ end (deeporange). (E) A cocrystal structure of thrombin and RE31 (Protein Data Bank (PDB) ID: 5CMX).ACS Nanoscience Au pubs.acs.org/nanoau Letterhttps://doi.org/10.1021/acsnanoscienceau.5c00143ACS Nanosci. Au 2026, 6, 201−207203https://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig1&ref=pdfpubs.acs.org/nanoau?ref=pdfhttps://doi.org/10.1021/acsnanoscienceau.5c00143?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asunlocked nucleic acid−uracil (UNA−uracil) leads to acomplete loss of thrombin-binding ability.19 Collectively,these findings suggest that T11 plays an important role inthrombin binding. This highlights the importance of carefullyselecting the position at which the uracil-DNA substitutionsare introduced.Most RE31 mutants with uracil-DNA-containing sequencesintroduced at the 5′ or 3′ end exhibited structural changes (seeSupporting Information Figure S1 for CD spectra). Interest-ingly, among them, RE31_5′(U) alone exhibited highinhibitory activity, and the addition of a single uracil-DNA atthe 5′ end induced favorable structural changes that enhancedthrombin binding.■ TURNING ON THROMBIN FUNCTION BYREMOVING RE31 MUTANTS WITH UDGXThe turning on of thrombin activity by removal of the RE31mutants from thrombin induced by UdgX was evaluated as adecrease in fibrinogen coagulation time. The addition of UdgXresulted in a shorter coagulation time in all RE31 mutants,indicating that thrombin activity was turned on by the covalentbinding of UdgX to RE31 mutants (Figure 1A−C, S3A−C).T11U, T12U, T20U, and T21U mutants exhibited nearly100% thrombin activity recovery (Figure 1D). This was likelyattributed to the uracil-DNA substitutions being at the RE31−thrombin interaction interface, allowing UdgX to restoreactivity (Figure 1E).15 In contrast, a variant with uracil in thesecond RE31 loop�a region not directly involved in thrombininteractions�demonstrated low functional recovery. Thesefindings suggest that introducing uracil-DNA at the aptamer−target interface enables UdgX to effectively inhibit the aptamer.A turning-on effect was not observed upon the addition ofbovine serum albumin (BSA) to RE31 lacking uracil-DNAsubstitution. In contrast, the addition of UdgX to RE31induced a turning-on effect (see Supporting InformationFigure S4 for coagulation time of adding BSA and UdgX).One possible reason is that UdgX is a DNA glycosylase thatcan interact with DNA and result in the removal of RE31,although not to the same extent with the RE31 mutants, suchas T11U, T12U, T20U, and T21U.To investigate whether UdgX removes the aptamer from itstarget, we used AlphaAssay to assess the binding of RE31 andits mutant, RE31_T21U. In the absence of UdgX, both RE31and RE31_T21U were able to bind thrombin (Figure 2; KDvalue: RE31, 0.61 nM; RE31_T21U, 0.50 nM). However,when UdgX was present, RE31 retained its ability to bindthrombin (KD value: 0.53 nM), while RE31_T21U did notproduce any detectable binding signal (KD value: belowdetection limit). These results imply that adding UdgXactivates thrombin by binding with the aptamer and removingit from the target. Notably, RE31_T21U showed a lower KDfor thrombin than RE31, suggesting it has a greater affinity forthrombin. Additionally, RE31_T21U exhibited the strongestinhibitory effect on thrombin (Figure 1A). These findingssuggest that the substitution of T21 with uracil altered itsstructural complementarity with thrombin, thereby affecting itsactivity.Previously, we applied SpyCatcher-fused UdgX as a simplenucleic acid−protein linkage tool,11 revealing that the fusionwith SpyCatcher improved the expression of UdgX inEscherichia coli and its final yield after purification. Consideringthat truncated SpyCatcher (TrSC) is more structurally stablethan SpyCatcher,20 we fused it with UdgX in this study.Specifically, we used TrSC-fused UdgX as a turn-on inducer inthrombin protein complexes with DNA aptamers. This allowedus to produce a homogeneous protein with a high yield (29.2mg/L culture). We also confirmed that untagged or unfusedUdgX exhibited a similar ability to turn on thrombin (seeSupporting Information Figure S5 for coagulation times withunfused UdgX, and SpyTag-fused UdgX). Furthermore,comparison of the turn-on activity of a construct in whichUdgX was fused with a 13-amino-acid SpyTag (AHIVMV-DAYKPTK) revealed no difference relative to unfused UdgXor UdgX. This suggests that UdgX’s turn-on activity remainsunchanged even after fusion, expanding its potentialapplications.■ COMPARISON OF THE THROMBIN ACTIVITYRECOVERY RATES BETWEEN THECOMPLEMENTARY STRAND AND UDGXConsidering the therapeutic application of aptamers asanticoagulants, a key factor for an optimal drug candidate isthe ability to rapidly restore thrombin activity by efficientaptamer removal. To date, complementary strands have beenused to remove aptamers. Therefore, we compared therecovery rates of thrombin activity following aptamer removalvia UdgX with those following removal of the complementarystrand, using RE31_T21U as the aptamer model. Thecomplementary strand (final concentration 100 nM) resultedin approximately 13% recovery after 1 min, increasing to 73%at 10 min postaddition. Notably, only approximately 56%removal was observed at 1 min, even at 1000 nM of thecomplementary strand. Conversely, UdgX (final concentration100 nM) achieved nearly complete recovery�approximately100%�within 1 min (Figure 3A). Wakui et al. sought toenhance the DNA aptamer removal rate by extending the DNAaptamer and complementary strand by 10 bases;6 however, thismodification resulted in an increase to 83%, which was lowerthan the rate achieved by UdgX in the current study. UdgXcarries a positive charge at its DNA-binding site (seeSupporting Information Figure S6 for Surface electrificationof UdgX) and forms covalent bonds with DNA, which isbelieved to contribute to effective aptamer removal. Unlike thecomplementary strand, the positive charge facilitates inter-actions with DNA via electrostatic and covalent binding,thereby preventing dissociation. Overall, UdgX can turn on theactivity inhibited by DNA aptamers more efficiently thancomplementary strands. Thus, UdgX is expected to rapidly andeffectively inhibit such as anticoagulant aptamers.Figure 2. Alpha assay of RE31 (orange) and RE31_T21U (blue).Their binding was not inhibited in the absence of UdgX.Contrastingly, the addition of UdgX inhibited the binding ofRE31_T21U.ACS Nanoscience Au pubs.acs.org/nanoau Letterhttps://doi.org/10.1021/acsnanoscienceau.5c00143ACS Nanosci. Au 2026, 6, 201−207204https://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig2&ref=pdfpubs.acs.org/nanoau?ref=pdfhttps://doi.org/10.1021/acsnanoscienceau.5c00143?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asFigure 3. (A) Thrombin activity recovery rate of UdgX (Final concentration 100 nM: red), complementary strand (Final concentration 1000 nM:deep blue), and complementary strand (Final concentration 100 nM: light blue). All the results are represented as mean ± SD (n = 3). (B)Coagulation time in serum without aptamer (white), or with RE31_T21U (gray), RE31_T21U plus UdgX (red), and RE31_T21U pluscomplementary strand (blue). The reactions were performed in buffer supplemented with serum at a final concentration of 40%.Figure 4. Determination of the aptamer removal effect of UdgX via aptamer blotting. BSA was used as the negative control and NeutrAvidin-HRPfor detection. (A) Schematic diagram turn-on technology for Bevacizumab employing UdgX. (B) Determination of the aptamer removal effect ofUdgX via aptamer blotting. BSA was used as the negative control, and NeutrAvidin-HRP for detection. (C) Signal intensities quantified at eachUdgX concentration using ImageJ.ACS Nanoscience Au pubs.acs.org/nanoau Letterhttps://doi.org/10.1021/acsnanoscienceau.5c00143ACS Nanosci. Au 2026, 6, 201−207205https://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?fig=fig4&ref=pdfpubs.acs.org/nanoau?ref=pdfhttps://doi.org/10.1021/acsnanoscienceau.5c00143?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asNext, to demonstrate the potential of UdgX in practicalutility, fibrinogen coagulation reactions were performed inserum. In serum at a final concentration of 40%, adding RE31T21U prolonged fibrinogen coagulation times (Figure 3B andSupporting Information Figure S7 for Effect of serum onfibrinogen coagulation reaction). Introducing UdgX reducedcoagulation time, whereas the complementary strand had noeffect�potentially due to DNase degradation. These resultsindicate that the turning on effects of UdgX are retained inserum.■ EVALUATION OF THE EFFECT OF TURNING ONTARGET ACTIVITY USING A STEM−LOOP TYPEBEVACIZUMAB-BINDING APTAMERTo demonstrate how protein functions suppressed by DNAaptamers can be reactivated using a versatile process, anotheraptamer was analyzed. We previously developed a bevacizu-mab-binding aptamer, A14#1,13 that possesses a hairpinstructure. In contrast, the RE31 aptamer features a G-quadruplex (G4) structure�a typical conformation formedby guanine-rich nucleic acid sequences�and interacts withthrombin through this G4 configuration.12 While A14#1 bindsbevacizumab via its loop within the step−loop structure, bothaptamers use distinct structures and modes for target binding.To explore further, we substituted a thymine base near thebinding site of A14#1 with a uracil, generating the variantA14#1_T12U (see Supporting Information Table S1 forOligonucleotide sequences for A14#1, and A14#1_T12U), andcompared its bevacizumab-binding ability using aptamerblotting. No differences were observed in binding strengthbetween A14#1 and A14#1_T12U when UdgX was absent(Figure 4). However, in the presence of UdgX, chemilumi-nescence at the bevacizumab spot disappeared, indicating thatUdgX inhibited A14#1_T12U binding. This inhibition wasobserved quantitatively at UdgX concentrations from 0 to 20μM, but not with A14#1.Typically, aptamer binding is disrupted by complementarystrands designed to destabilize secondary structures, such ashairpins or G4 structures.21,22 However, designing suchcomplementary strands for each aptamer can be challenging.The findings of the current study indicate that UdgX candisrupt aptamer binding with hairpin or G4 structures bysubstituting thymine with uracil-DNA, without significantlyaffecting the aptamer’s binding ability. In conclusion, UdgXeffectively removes DNA aptamer binding across variousstructures, making it a valuable tool for reactivating proteinsinhibited by DNA aptamers.■ CONCLUSIONSWe have developed a rapid turn-on technology for restoringprotein functions inhibited by DNA aptamers, using the DNA-binding protein, UdgX. When thrombin was inhibited by anantithrombin aptamer with a G-quadruplex structure, addingUdgX restored its function more quickly and effectively thansimply introducing a complementary strand. Remarkably,UdgX retained efficacy in serum. The approach was alsobroadly applicable, as demonstrated with an anti-ideotypeaptamer featuring a hairpin structure. While issues such asdelivery, activation timing, and immunogenicity associatedwith UdgX require further investigation, this technologyenables DNA aptamers to undergo reversible reactions,potentially expanding their utility in applications likeoligonucleotide therapeutics.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143.CD spectra of RE31 and RE31 mutants, SDS-PAGE ofRE31 mutant and UdgX, Time-course measurement ofabsorbance at 380 nm monitoring fibrinogen coagu-lation, Coagulation time of only RE31 and RE31 withUdgX or BSA, Coagulation times with unfused UdgX orSpyTag-fused UdgX, Surface electrification of UdgX,Effect of serum on fibrinogen coagulation reaction,Table of oligonucleotide sequences for TBA, A14#1, andA14#1_T12U, Table of TrSC-fused UdgX proteinsequence, and Material and method of this paper(PDF)■ AUTHOR INFORMATIONCorresponding AuthorsRyutaro Asano − Department of Biotechnology and LifeScience, Graduate School of Engineering, Tokyo University ofAgriculture and Technology, Koganei, Tokyo 184-8588,Japan; orcid.org/0000-0001-6795-8377;Email: ryutaroa@cc.tuat.ac.jpTomohiko Yamazaki − Research Center for Macromoleculesand Biomaterials, National Institute for Material Sciences(NIMS), Tsukuba, Ibaraki 305-0047, Japan; orcid.org/0000-0003-2136-8042; Email: yamazaki.tomohiko@nims.go.jpKazunori Ikebukuro − Department of Biotechnology and LifeScience, Graduate School of Engineering, Tokyo University ofAgriculture and Technology, Koganei, Tokyo 184-8588,Japan; orcid.org/0000-0003-2838-0562; Email: ikebu@cc.tuat.ac.jpAuthorsTakeru Kanazu − Department of Biotechnology and LifeScience, Graduate School of Engineering, Tokyo University ofAgriculture and Technology, Koganei, Tokyo 184-8588,Japan; Research Center for Macromolecules and Biomaterials,National Institute for Material Sciences (NIMS), Tsukuba,Ibaraki 305-0047, JapanErika Komiya − Department of Biotechnology and LifeScience, Graduate School of Engineering, Tokyo University ofAgriculture and Technology, Koganei, Tokyo 184-8588,Japan; Research Center for Macromolecules and Biomaterials,National Institute for Material Sciences (NIMS), Tsukuba,Ibaraki 305-0047, JapanDaimei Miura − Department of Biotechnology and LifeScience, Graduate School of Engineering, Tokyo University ofAgriculture and Technology, Koganei, Tokyo 184-8588,Japan; Institute of Global Innovation Research, TokyoUniversity of Agriculture and Technology, Fuchu, Tokyo 183-8538, Japan; orcid.org/0009-0007-0005-9976Kaori Tsukakoshi − Department of Chemistry, Faculty ofScience Division I, Tokyo University of Science, Shinjuku-ku,Tokyo 162-8601, Japan; orcid.org/0000-0003-4779-1113Complete contact information is available at:ACS Nanoscience Au pubs.acs.org/nanoau Letterhttps://doi.org/10.1021/acsnanoscienceau.5c00143ACS Nanosci. Au 2026, 6, 201−207206https://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsnanoscienceau.5c00143?goto=supporting-infohttps://pubs.acs.org/doi/suppl/10.1021/acsnanoscienceau.5c00143/suppl_file/ng5c00143_si_001.pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Ryutaro+Asano"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0001-6795-8377mailto:ryutaroa@cc.tuat.ac.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Tomohiko+Yamazaki"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0003-2136-8042https://orcid.org/0000-0003-2136-8042mailto:yamazaki.tomohiko@nims.go.jpmailto:yamazaki.tomohiko@nims.go.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kazunori+Ikebukuro"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0003-2838-0562mailto:ikebu@cc.tuat.ac.jpmailto:ikebu@cc.tuat.ac.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Takeru+Kanazu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Erika+Komiya"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Daimei+Miura"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0009-0007-0005-9976https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kaori+Tsukakoshi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0003-4779-1113https://orcid.org/0000-0003-4779-1113pubs.acs.org/nanoau?ref=pdfhttps://doi.org/10.1021/acsnanoscienceau.5c00143?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://pubs.acs.org/10.1021/acsnanoscienceau.5c00143Author ContributionsTakeru Kanazu: Methodology, Investigation, Validation,Formal Analysis, Writing�original draft. Erika Komiya:Methodology, Investigation. Daimei Miura: Methodology,Validation, Supervision, Writing�review and editing. KaoriTsukakoshi: Conceptualization, Methodology. Kazunori Ike-bukuro: Supervision, Validation, Writing�review and editing.Tomohiko Yamazaki: Supervision, Conceptualization, Valida-tion, Writing�review and editing, Funding Acquisition.Ryutaro Asano: Supervision, Validation, Writing�review andediting, Funding Acquisition. All the authors participated inthe discussion and coordination of the manuscript submission.All the authors have read and approved the final version of thismanuscript.FundingThis study was supported by a Grants-in-Aid for ScientificResearch from the Japan Society for the Promotion of Science[JSPS; grant number 21K18321] and the National Institute forMaterials Science Joint Research Hub Program.NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSWe thank Dr. Shinya Hattori, Ms. Tomoyo Umezawa, and Dr.Miwako Shobo [National Institute for Materials Science(NIMS)] for their excellent technical assistance. Part of thiswork was conducted at the NIMS Molecule and MaterialSynthesis Platform, supported by the Nanotechnology Plat-form Program of the Ministry of Education, Culture, Sports,Science, and Technology (MEXT), Japan. 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