有机溶剂体系中刺激响应性RNA切割型脱氧核酶的工程化

doi:10.7503/cjcu20250010

摘要/Abstract

摘要：

引入新的识别元件对脱氧核酶(DNAzyme)进行工程化是拓展其应用范围的重要途径, 但这依赖于对其可工程化位点的系统表征. 为此, 本文对前期发现的需要有机溶剂的RNA切割型脱氧核酶（RNA-cleaving DNAzyme， RCD, 名为E3)进行工程化改造, 在其不同区域发现了多个可工程化位点, 并构建了多种刺激响应性RCD. 首先, 在可变区域将E3劈成两半, 通过设计“DNA拉链”构建多组分酶(Multiple component DNAzyme, MNAzyme)的策略, 在可变区鉴别了6个可用于工程化的位点; 接着在可变区成功设计了由单个外加的DNA序列激活的MNAzyme系统; 进一步在E3的底物结合臂发现了另一个可用于设计MNAzyme的区域, 并设计了一系列需2个DNA引发剂同时存在才能激活的三组分酶系统. 所有MNAzyme系统在35%(体积分数) DMSO和30%(体积分数)乙醇溶液中都有极高的选择性和极低的背景值, 多种MNAzyme和引发剂共存体系中的反应信号接近单个引发剂产生信号的加和. 本研究为在含有机溶剂的应用场景中构建响应性RCD探针提供了平台.

关键词: 脱氧核酶, 刺激响应性, 有机溶剂, 分子工程, 多组分酶

Abstract:

Engineering deoxyribozyme（DNAzyme） by the introduction of new recognition elements is an important approach for expanding its applications， however， this depends on the systematic characterization of the engineerable sites in the DNAzyme. In this work， we investigated the engineerable sites of an organic solvent-needing RNA- cleaving DNAzyme（RCD， named E3） previously reported by our group. We first split E3 into two parts at its variable region， and added two DNA sequences on the 5′-end of one part and 3′-end of the other part to form duplex to activate the DNAzyme. By construction of the zipper-like Multiple Component DNAzyme（MNAzyme）， we were able to identify 6 splitting sites that could be utilized for DNAzyme engineering. Based on this discovery， a DNAzyme system that was activated by an external DNA initiator was constructed by splitting E3 at its variable region and introducing two oligonucleotides at the split sites for the binding of the initiator. Furthermore， we identified several splitting sites on the substrate-binding arm of E3 as an alternative region for the design of MNAzyme. Application in both regions， an intricate three component DNAzyme system was designed to be activated only in the presence of the two specific initiators. All the MNAzyme systems exhibited extremely high selectivity and very low background both in 35%（volume fraction） DMSO and 30%（volume fraction） ethanol solvents. Moreover， in mixing of various MNAzyme systems and all the relative initiators， the cleavage products were almost the sum of that in mixing these MNAzymes in the presence of individual initiator. These results demonstrate that E3 is an excellent scaffold for constructing responsive RCD probes in applications in organic solvents-containing scenarios.

Key words: DNAzyme, Stimuli-responsive, Organic solvent, Molecular engineering, Multiple component DNAzyme

中图分类号:

O629.8

TrendMD:

贺荣荣, 游昕, 郑星, 杨梦晗, 常天俊. 有机溶剂体系中刺激响应性RNA切割型脱氧核酶的工程化. 高等学校化学学报, 2025, 46(7): 20250010.

HE Rongrong, YOU Xin, ZHENG Xing, YANG Menghan, CHANG Tianjun. Engineering Stimuli-responsive RNA-cleaving DNAzymes in Organic Cosolvents. Chem. J. Chinese Universities, 2025, 46(7): 20250010.

图/表 4

Fig.1 Investigation of the engineering sites of E3 by construction of a DNA “zipper” initiated MNAzyme（A） The secondary structure of E3. The potentially engineering sites are pointed out by arrows； a DNA sequence embedded with an RNA base（rA） was used as the substrate（5′FRQ30）； 5′FRQ30 was modified with a quencher（Q） neighboring the cleavage site and a fluorophore（F） at the 5′ end for tracking the cleavage products. （B） Schematic diagram of the DNA "zipper"-activated MNAzyme. （C） Cleavage yields of 5′FRQ30 by the DNA "zipper" activated MNAzymes which were constructed at different splitting sites. PC indicates the positive control， i.e. the cleavage of the substrate by the intact DNAzyme E3； the concentration of each component of the MNAzyme is 2500 nmol/L， and 5′FRQ30 is 250 nmol/L； the cleavage reaction was carried out at 23 ℃ for 1 h； Unclv and Clv indicate the uncleaved and the cleaved substrates， respectively； the DNA sequences used in this section are shown in Table S2 in Supporting Information of this paper.

Fig.2 Engineering of a one⁃DNA⁃tape responsive MNAzyme system（A） Schematic diagram of the one-DNA-tape responsive MNAzyme. P1 and P2 indicate the two parts of the MNAzyme. （B） The selective response of the MNAzyme to a DNA initiator. M（X） indicates a MNAzyme that is corresponding to a certain initiator（X）； NC indicate the cleavage of 5′FRQ30 by the MNAzyme without the presence of initiator； the concentration of the substrate is 250 nmol/L； the initiator and the relative MNAzyme system are set at 2500 nmol/L. （C） The cleavage of 5′FRQ30 by a mixture of 5 MNAzymes in the presence of various initiators. "+" indicates the addition of the initiator， and "－" indicates without the addition of the initiator； the insertion was the dPAGE image of （C）； the cleavage reaction was carried out by the addition of 5 MNAzymes（each of 50 nmol/L） in the presence of the individual initiator at 50 nmol/L or all the 5 initiators at each of 50 nmol/L at 23 ℃ for 1 h； these DNA sequences are shown in Table S4 in Supporting Information of this paper.

Fig.3 Engineering a two⁃DNA⁃tape responsive MNAzyme system（A） The potential engineering sites on the substrate-binding arm of E3. The signatures of I， ⅡI， and Ⅲ between C28 and T31 indicate the tested splitting sites. （B） The kinetics of the MNAzyme system constructed by splitting of E3 at its substrate-binding arm. A DNA sequence T1 was employed as the initiator； 2500 nmol/L of T1 and the MNAzyme and 250 nmol/L of 5′FRQ30 were used in the cleavage reaction. （C） Illustration of a two-DNA-tape responsive MNAzyme system. E3 was split into three parts at the binding-arm and the variable region， and the complimentary sequences of the initiators were introduced on the terminus for construction of three partzymes（P1， P2 and P3）. （D） Kinetics of the two-DNA-tape responsive MNAzymes constructed at the combination of various spitting sites. Two DNA sequences E6 and F2 were used as initiators； the MNAzyme and the initiators were set at 250 nmol/L， and 5 ′FRQ30 was 125 nmol/L for the cleavage reactions； UM： unmeasurable； the specific sequences of NA are shown in Table S3 and in Supporting Information S5 of this paper.

Fig.4 Selectivity of the two⁃DNA⁃tape⁃responsive DNAzyme systems（A） Selectivity of the two-DNA-tape-responsive MNAzymes. The initiator groups used in the MNAzyme systems were E1+E6（from the E gene of SARS-Cov-2）， N4+N10（from the N gene of SARS-Cov-2）， NA2+NA3（from the genome of influenza H3N2）， and HA2+HA4（from the genome of influenza H7N9）； NC indicate the cleavage of 5′FRQ30 by the MNAzyme without the addition of any initiator； 2500 nmol/L initiators and MNAzymes and 250 mol/L 5′FRQ30 were used in the cleavage reactions. （B） The cleavage of 5′FRQ30 by a mixture of 4 MNAzymes in the presence of various initiator groups. "+" indicates the addition of the initiator group， and "-" indicates without the addition of the initiator； the insertion was the dPAGE image of （B）； the cleavage reaction was carried out by the addition of the 4 MNAzymes（each of 50 nmol/L） in the presence of the individual initiator group at 50 nmol/L or all the 4 initiator groups at each of 50 nmol/L at 23 ℃ for 1 h； see Table S5 in Supporting Information of this paper for these sequences.

参考文献 29

[1]	Breaker R. R.， Joyce G. F.， Chem. Biol.， 1994， 1（4）， 223—229
[2]	Sreedhara A.， Li Y. F.， Breaker R. R.， J. Am. Chem. Soc.， 2004， 126（11）， 3454—3460
[3]	Mcconnell E. M.， Cozma I.， Mou Q. B.， Brennan J. D.， Li Y. F.， Chem. Soc. Rev.， 2021， 50（16）， 8954—8994
[4]	Borggräfe J.， Victor J.， Rosenbach H.， Viegas A.， Gertzen C. G. W.， Wuebben C.， Kovacs H.， Gopalswamy M.， Riesner D.， Steger G.， Schiemann O.， Gohlke H.， Span I.， Etzkorn M.， Nature， 2022， 601（7891）， 144—149
[5]	Liu H. H. Yu X.， Chen Y. Q.， Zhang J.， Wu B. X.， Zheng L. N.， Haruehanroengra P.， Wang R.， Li S. H.， Lin J. Z.， Li J. X.， Sheng J.， Huang Z.， Ma J. B.， Gan J. H.， Nat. Commun.， 2017， 8（1）， 2006
[6]	Tong Z. X.， Hu Q. Q.， Gu H. Z.， Chem. J. Chinese Universities， 2020， 41（11）， 2345—2355
	佟宗轩，胡沁沁，顾宏周. 高等学校化学学报， 2020， 41（11）， 2345—2355
[7]	Mao Y.， Qu H.， Zheng L.， Chem. J. Chinese Universities， 2021， 42（11）， 3445—3456
	毛瑜，瞿昊，郑磊. 高等学校化学学报， 2021， 42（11）， 3445—3456
[8]	Wang Q.， He Y. Q.， Wang F. A.， Chem. J. Chinese Universities， 2021， 42（11）， 3334—3356
	王庆，何雨秋，王富安. 高等学校化学学报， 2021， 42（11）， 3334—3356
[9]	Huang Z. M.， Wang X. N.， Wu Z. K.， Jiang J. H.， Chem. Asian J.， 2022， 17（6）， e202101414
[10]	Safdar S.， Lammertyn J.， Spasic D.， Trends Biotechnol.， 2020， 38（12）， 1343—1359
[11]	Guerrier-Takada C.， Gardiner K.， Marsh T.， Pace N.， Altman S.， Cell， 1983， 35（3）， 849—857
[12]	Zhou Q. B.， Zhang G. X.， Wu Y. P.， Zhang Q.， Liu Y.， Chang Y. Y.， Liu M.， J. Am. Chem. Soc.， 2023， 145（39）， 21370—21377
[13]	Chang D. R.， Zakaria S.， Samani S. E.， Chang Y. Y.， Filipe C. D. M.， Soleymani L.， Brennan J. D.， Liu M.， Li Y. F.， Acc. Chem. Res.， 2021， 54（18）， 3540—3549
[14]	Liu M.， Chang D. R.， Li Y. F.， Acc. Chem. Res.， 2017， 50（9）， 2273—2283
[15]	Hu Q. Q.， Tong Z. X.， Yalikong A.， Ge L. P.， Shi Q.， Du X. Y.， Wang P.， Liu X. Y.， Zhan W. Q.， Gao X.， Sun D.， Fu T.， Ye D.， Fan C. H.， Liu J.， Zhong Y. S.， Jiang Y. Z.， Gu H. Z.， Nat. Chem.， 2024， 16（1）， 122—131
[16]	Gerasimova Y. V.， Kolpashchikov D. M.， Chem. Biol.， 2010， 17（2）， 104—106
[17]	Yang K. F.， Schuder D. N.， Ngor A. K.， Chaput J. C.， J. Am. Chem. Soc.， 2022， 144（26）， 11685—11692
[18]	Kozlowski H. N.， Mohamed M. A. A.， Kim J.， Bell N. G.， Zagorovsky K.， Mubareka S.， Chan W. C. W.， Small Struct.， 2021， 2（8）， 2100034
[19]	Wang Y. Y.， Wang Y.， Song D. F.， Sun X.， Li Z.， Chen J. Y.， Yu H. Y.， Nat. Chem.， 2022， 14（3）， 350—359
[20]	Fokina A. A.， Stetsenko D. A， François J. C.， Expert Opin. Biol. Ther.， 2015， 15（5）， 689—711
[21]	Cao Y. R.， Zhang H. Q.， Le X. C.， Anal. Chem.， 2021， 93（47）， 15712—15719
[22]	Mokany E.， Bone S. M.， Young P. E.， Doan T. B.， Todd A. V.， J. Am. Chem. Soc.， 2010， 132（3）， 1051—1059
[23]	Yang M.， Xie Y. S.， Zhu L. J.， Li X. Y.， Xu W. T.， ACS Catal.， 2024， 14（21）， 16392—16422
[24]	Chang T. J.， He S. S.， Amini R.， Li Y. F.， ChemBioChem， 2021， 22（14）， 2368—2383
[25]	Chang T. J.， Li G. P.， Chang D. R.， Amini R.， Zhu X. N.， Zhao T. Q.， Gu J.， Li Z. P.， Li Y. F.， Angew. Chem. Int. Ed.， 2023， 62（42）， e202310941
[26]	Cui L.， Li S. H.， Zheng X.， Chang T. J.， Bing T.， Chem. J. Chinese Universities， 2024， 45（4）， 20240004
	崔力，李素慧，郑星，常天俊，邴涛. 高等学校化学学报， 2024， 45（4）， 20240004
[27]	Wang D. Y.， Lai B. H. Y.， Feldman A. R.， Sen D.， Nucleic Acids Res.， 2002， 30（8）， 1735—1742
[28]	Ruble B. K.， Richards J. L.， Cheung-Lau J. C.， Dmochowski I. J.， Inorg. Chim. Acta， 2012， 380， 386—391
[29]	Fozouni P.， Son S. M.， Derby M. D. D.， Knott G. J.， Gray C. N.， D′Ambrosio M. V.， Zhao C. Y.， Switz N. A.， Kumar G. R.， Stephens S. I.， Boehm D.， Tsou C. L.， Shu J.， Bhuiya A.， Armstrong M.， Harris A. R.， Chen P. Y.， Osterloh J. M.， Meyer⁃Franke A.， Joehnk B.， Walcott K.， Sil A.， Langelier C.， Pollard K. S.， Crawford E. D.， Puschnik A. S.， Phelps M.， Kistler A.， DeRisi J. L.， Doudna J. A.， Fletcher D. A.， Ott M.， Cell， 2021， 184（2）， 323—333