核酸切割酶在病原微生物检测中的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2019-0568

摘要/Abstract

摘要： DNA是遗传信息的重要载体,其空间构象折叠性质使其具有很多的功能。利用核酸切割酶（cleaving DNAzyme）识别特定单链DNA分子并能够切割其中某条单链的性质来构建传感器,将特异性识别过程转化为凝胶电泳表征、释放荧光、比色现象的信号输出,同时能很好的和扩增反应结合来实现信号放大。核酸切割酶通过体外筛选技术获得,可以与靶物质（小分子、蛋白质,甚至整个细胞）特异性结合。由于具有制备简单,易于修饰和良好稳定性等优点,核酸切割酶被用于构建生物传感器以检测病原微生物,已应用到现场检测甚至医疗中的体内检测,结合已经成熟的检测设备血糖仪、横流层析试纸条带进行微生物检测,并广泛地应用到生物传感、食品安全、医疗在内的重要领域中。综述了近年来核酸切割酶在微生物检测中的应用,讨论了核酸切割酶在微生物检测中的切割机理和产物、靶标以及表征手段,探索核酸切割酶在微生物实际检测中的意义。对该技术的发展前景及其面临的问题进行展望,以期核酸切割酶在微生物检测领域能够更好的发展。

关键词: 核酸切割酶, 微生物, 生物传感, 信号放大, 检测

Abstract: DNA is an important carrier of genetic information,and its folding property of spatial conformation makes it have many functions. Using a RNA-cleaving DNAzyme to recognize a specific single-stranded DNA molecule and to cleave one of the single strands,a signal output sensor can be constructed,which converts the specific recognition process into the signal outputs of gel electrophoresis character,fluorescence,and colorimetric,and it can be combined with amplification reaction to achieve signal amplified. RNA-cleaving DNAzymes are obtained by in vitro screening techniques that specifically bind to target substances（such as small molecules,proteins,and even whole cells）. Due to its advantages of convenient preparation,easy modification and fine stability,RNA-cleaving DNAzymes are used to construct biosensors to detect pathogenic microorganisms,for example in-situ detection and even in-vivo detection in medical treatment. In the future,it will be combined with mature detection,for instance,equipment blood glucose meter,cross-flow chromatography strips for microbiological testing. Furthermore,it can be used in important areas such as biosensing,food safety,and medical care. Here we review the application of RNA-cleaving DNAzymes in microbial detection. In addition,we discuss the mechanism,products,targets and characterization of RNA-cleaving DNAzymes in microbial detection,as well as the significance and problems of RNA-cleaving DNAzymes in the actual detection of microorganisms. Finally,we prospect the further application of this technology,aiming at the better development of nucleic acid cleavage enzymes in microbial detection.

Key words: RNA-cleaving DNAzymes, microorganism, biosensing, signal amplification, detection

王昕, 朱龙佼, 许文涛, 翟晨, 王书雅, 黄蔚霞. 核酸切割酶在病原微生物检测中的研究进展[J]. 生物技术通报, 2020, 36(1): 182-192.

WANG Xin, ZHU Long-jiao, XU Wen-tao, ZHAI Chen, WANG Shu-ya, HUANG Wei-xia. Research Progress on RNA-cleaving DNAzyme in the Detection of Pathogens[J]. Biotechnology Bulletin, 2020, 36(1): 182-192.

参考文献

[1] Nayak M, Kotian A, Marathe S, et al.Detection of microorganisms using biosensors—A smarter way towards detection techniques[J]. Biosensors and Bioelectronics, 2009, 25(4):661-667.
[2] Abubakar I, Irvine L, Aldus CF, et al.A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food[J]. Health Technology Assessment(Winchester, England), 2007, 11(36):1-216.
[3] Principles of bacterial detection:biosensors, recognition receptors and microsystems[M]. Springer Science & Business Media, 2008.
[4] Call DR.Challenges and opportunities for pathogen detection using DNA microarrays[J]. Crit Rev Microbiol, 2005, 31(2):91-99.
[5] Lazcka O, Del Campo FJ, Munoz FX.Pathogen detection:A perspective of traditional methods and biosensors[J]. Biosensors and Bioelectronics, 2007, 22(7):1205-1217.
[6] Velusamy V, Arshak K, Korostynska O, et al.An overview of foodb-orne pathogen detection:In the perspective of biosensors[J]. Biotechnology Advances, 2010, 28(2):232-254.
[7] Yang L, Bashir R.Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria[J]. Biotechnology Advances, 2008, 26(2):135-150.
[8] Arlett JL, Myers EB, Roukes ML.Comparative advantages of mechanical biosensors[J]. Nat Nanotechnol, 2011, 6(4):203.
[9] Skottrup PD, Nicolaisen M, Justesen AF.Towards on-site pathogen detection using antibody-based sensors[J]. Biosensors and Bioelectronics, 2008, 24(3):339-348.
[10] 董文娟, 王保亚, 彭文雯, 等. 索氏梭菌在艰难梭菌感染实验室检测中的影响分析[J]. 北京医学, 2019(3):202-205.
[11] 孙淑娟. Development of FICA and UPLC-MS/MS for Simultaneous Determination of Aflatoxin B1 and Zearalenone in Chinese herbal medicines[D]. 北京:中国农业大学, 2018.
[12] Brandon DL, Adams LM.Milk matrix effects on antibody binding analyzed by enzyme-linked immunosorbent assay and biolayer interferometry[J]. J Agric Food Chem, 2015, 63(13):3593-3598.
[13] 梁华丽, 方毅. 简析食品微生物检测技术进展[J]. 食品工程, 2015(1):22-24.
[14] Ali MM, Aguirre SD, Lazim H, et al.Fluorogenic DNAzyme probes as bacterial indicators[J]. Angew Chem Int Ed Engl, 2011, 50(16):3751-3754.
[15] Li D, Song S, Fan C.Target-responsive structural switching for nucleic acid-based sensors[J]. Accounts of Chemical Research, 2010, 43(5):631-641.
[16] Ellington AD, Szostak JW.In vitro selection of RNA molecules that bind specific ligands[J]. Nature, 1990, 346(6287):818.
[17] Qi L, Zhao Y, Yuan H, et al.Amplified fluorescence detection of mercury(II)ions(Hg²+)using target-induced DNAzyme cascade with catalytic and molecular beacons[J]. Analyst, 2012, 137(12):2799-2805.
[18] Gotrik MR, Feagin TA, Csordas AT, et al.Advancements in aptamer discovery technologies[J]. Accounts of Chemical Research, 2016, 49(9):1903-1910.
[19] Hui CY, Liu M, Li Y, et al.A paper sensor printed with multifunctional bio/nano materials[J]. Angew Chem Int Ed Engl, 2018, 57(17):4549-4553.
[20] 马芬, 张成孝. 增强型铅离子电化学发光传感器的研究[J]. 分析化学, 2009, 37(A02):107.
[21] Li J, Lu Y.A highly sensitive and selective catalytic DNA biosensor for lead ions[J]. J Am Chem Soc, 2000, 122(42):10466-10467.
[22] Liu J, Lu Y.Improving fluorescent DNAzyme biosensors by combining inter-and intramolecular quenchers[J]. Analytical Chemistry, 2003, 75(23):6666-6672.
[23] 戢太云, 徐鲁荣, 周培. 核酸荧光探针检测铅离子的研究[J]. 分析测试学报, 2010(1):51-54.
[24] Liu J, Lu Y.A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles[J]. J Am Chem Soc, 2003, 125(22):6642-6643.
[25] Liu D, Daubendiek SL, Zillman MA, et al.Rolling circle DNA synthesis:small circular oligonucleotides as efficient templates for DNA polymerases[J]. J Am Chem Soc, 1996, 118(7):1587-1594.
[26] Wei W, Jia LY.Progress in aptamer screening methods[J]. Chinese J Anal Chem, 2009, 37(3):454-460.
[27] Pichon V, Brothier F, Combès A.Aptamer-based-sorbents for sample treatment—a review[J]. Analytical and Bioanalytical Chemistry, 2015, 407(3):681-698.
[28] Du F, Guo L, Qin Q, et al.Recent advances in aptamer-functionalized materials in sample preparation[J]. TrAC Trends in Analytical Chemistry, 2015, 67:134-146.
[29] Gopinath SCB, Awazu K, Fons P, et al.A sensitive multilayered structure suitable for biosensing on the BioDVD platform[J]. Analytical Chemistry, 2009, 81(12):4963-4970.
[30] Cao X, Li S, Chen L, et al.Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus[J]. Nucleic Acids Res, 2009, 37(14):4621-4628.
[31] Morrison D, Rothenbroker M, Li Y.DNAzymes:selected for applications[J]. Small Methods, 2018, 2(3):1700319.
[32] Schlosser K, Li Y.Biologically inspired synthetic enzymes made from DNA[J]. Chemistry & Biology, 2009, 16(3):311-322.
[33] Silverman SK.In vitro selection, characterization, and application of deoxyribozymes that cleave RNA[J]. Nucleic Acids Res, 2005, 33(19):6151-6163.
[34] Wolfe M, Ali M, Tram K, et al.A DNAzyme-based colorimetric paper sensor for Helicobacter pylori[J]. Angew Chem Int Ed Engl, 2019, 58(29):9907-9911.
[35] Schlosser K, Li Y.A versatile endoribonuclease mimic made of DNA:characteristics and applications of the 8-17 RNA cleaving DNAzyme[J]. ChemBioChem, 2010, 11(7):866-879.
[36] He S, Qu L, Shen Z, et al.Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe[J]. Analytical Chemistry, 2014, 87(1):569-577.
[37] Shen Z, Wu Z, Chang D, et al.A catalytic DNA activated by a specific strain of bacterial pathogen[J]. Angew Chem Int Ed Engl, 2016, 55(7):2431-2434.
[38] Li Y.Advancements in using reporter DNAzymes for identifying pathogenic bacteria at speed and with convenience[J]. Future Microbiology, 2011, 6(9):973-976.
[39] DeStefano JJ, Cristofaro JV. Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity[J]. Nucleic Acids Res, 2006, 34(1):130-139.
[40] Vivekananda J, Kiel JL.Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay[J]. Laboratory Investigation, 2006, 86(6):610.
[41] Shen Y, Brennan JD, Li Y.Characterizing the secondary structure and identifying functionally essential nucleotides of pH6DZ1, a fluorescence-signaling and RNA-cleaving deoxyribozyme[J]. Biochemistry, 2005, 44(36):12066-12076.
[42] Chiuman W, Li Y.Evolution of high-branching deoxyribozymes from a catalytic DNA with a three-way junction[J]. Chemistry & Biology, 2006, 13(10):1061-1069.
[43] Chiuman W, Li Y.Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores[J]. J Mol Biol, 2006, 357(3):748-754.
[44] Liu M, Zhang Q, Li Z, et al.Programming a topologically constrained DNA nanostructure into a sensor[J]. Nature Communications, 2016, 7:12074.
[45] Ali MM, Li Y.Colorimetric sensing by using allosteric-DNAzyme-coupled rolling circle amplification and a peptide nucleic acid-organic dye probe[J]. Angewandte Chemie, 2009, 121(19):3564-3567.
[46] Xiao SJ, Hu PP, Li YF, et al.Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor[J]. Talanta, 2009, 79(5):1283-1286.
[47] Kuai H, Zhao Z, Mo L, et al.Circular bivalent aptamers enable in vivo stability and recognition[J]. J Am Chem Soc, 2017, 139(27):9128-9131.
[48] Dong H, Han L, Wu ZS, et al.Biostable aptamer rings conjugated for targeting two biomarkers on circulating tumor cells in vivo with great precision[J]. Chem Mater, 2017, 29(24):10312-10325.
[49] Fire A, Xu SQ.Rolling replication of short DNA circles[J]. Proc Natil Acad Sci USA, 1995, 92(10):4641-4645.
[50] Liu M, Hui CY, Zhang Q, et al.Target-induced and equipment-free DNA amplification with a simple paper device[J]. Angew Chem Int Ed Engl, 2016, 55(8):2709-2713.
[51] Carrasquilla C, Little JRL, Li Y, et al.Patterned paper sensors printed with long-chain DNA aptamers[J]. Chemistry-A European Journal, 2015, 21(20):7369-7373.
[52] Liu M, Zhang Q, Chang D, et al.A DNAzyme feedback amplification strategy for biosensing[J]. Angew Chem Int Ed Engl, 2017, 56(22):6142-6146.
[53] Chiuman W, Li Y.Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores[J]. J Mol Biol, 2006, 357(3):748-754.
[54] Shen Y, Chiuman W, Brennan JD, et al.Catalysis and rational engineering of trans-acting pH6DZ1, an RNA-cleaving and fluorescence-signaling deoxyribozyme with a four-way junction structure[J]. ChemBioChem, 2006, 7(9):1343-1348.
[55] Ali MM, Kandadai SA, Li Y.Characterization of pH3DZ1—An RNA-cleaving deoxyribozyme with optimal activity at pH 3[J]. Canadian Journal of Chemistry, 2007, 85(4):261-273.
[56] Chiuman W, Li Y.Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates[J]. Nucleic Acids Res, 2006, 35(2):401-405.
[57] Shen Z, Wu Z, Chang D, et al.A catalytic DNA activated by a specific strain of bacterial pathogen[J]. Angew Chem Int Ed Engl, 2016, 55(7):2431-2434.
[58] Yousefi H, Ali MM, Su HM, et al.Sentinel wraps:Real-time monitoring of food contamination by printing dnazyme probes on food packaging[J]. ACS Nano, 2018, 12(4):3287-3294.
[59] He S, Qu L, Shen Z, et al.Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe[J]. Analytical Chemistry, 2014, 87(1):569-577.
[60] Di Giusto DA, Wlassoff WA, Gooding JJ, et al.Proximity extension of circular DNA aptamers with real-time protein detection[J]. Nucleic Acids Res, 2005, 33(6):e64-e64.
[61] Yang L, Fung CW, Cho EJ, et al.Real-time rolling circle amplification for protein detection[J]. Analytical Chemistry, 2007, 79(9):3320-3329.
[62] Zhou L, Ou LJ, Chu X, et al.Aptamer-based rolling circle amplification:a platform for electrochemical detection of protein[J]. Analytical Chemistry, 2007, 79(19):7492-7500.
[63] Cheglakov Z, Weizmann Y, Basnar B, et al.Diagnosing viruses by the rolling circle amplified synthesis of DNAzymes[J]. Organic & Biomolecular Chemistry, 2007, 5(2):223-225.
[64] Tian Y, He Y, Mao C.Cascade signal amplification for DNA detection[J]. ChemBioChem, 2006, 7(12):1862-1864.
[65] Zhao W, Ali MM, Brook MA, et al.Rolling-circle-amplifikation:anwendungen in der nanotechnologie und in der biodetektion mit funktionellen nucleinsäuren[J]. Angewandte Chemie, 2008, 120(34):6428-6436.
[66] Zhao W, Ali MM, Brook MA, et al.Rolling circle amplification:applications in nanotechnology and biodetection with functional nucleic acids[J]. Angew Chem Int Ed Engl, 2008, 47(34):6330-6337.
[67] Kushon SA, Jordan JP, Seifert JL, et al.Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins[J]. J Am Chem Soc, 2001, 123(44):10805-10813.
[68] Komiyama M, Ye S, Liang X, et al.PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection[J]. J Am Chem Soc, 2003, 125(13):3758-3762.
[69] Dilek I, Madrid M, Singh R, et al.Effect of PNA backbone modifications on cyanine dye binding to PNA-DNA duplexes investigated by optical spectroscopy and molecular dynamics simulations[J]. J Am Chem Soc, 2005, 127(10):3339-3345.
[70] Wilhelmsson LM, Nordén B, Mukherjee K, et al.Genetic screening using the colour change of a PNA-DNA hybrid-binding cyanine dye[J]. Nucleic Acids Res, 2002, 30(2):e3-e3.
[71] Xiang Y, Lu Y.Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets[J]. Nature Chemistry, 2011, 3(9):697.
[72] Du Y, Pothukuchy A, Gollihar JD, et al.Coupling sensitive nucleic acid amplification with commercial pregnancy test strips[J]. Angew Chem Int Ed Engl, 2017, 56(4):992-996.
[73] Liu M, Zhang Q, Kannan B, et al.Self-assembled functional DNA superstructures as high-density and versatile recognition elements for printed paper sensors[J]. Angewandte Chemie, 2018, 130(38):12620-12623.
[74] Lv Y, Hu R, Zhu G, et al.Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers[J]. Nature Protocols, 2015, 10(10):1508.
[75] Wolfe M, Ali M, Tram K, et al.A DNAzyme-based Colorimetric Paper Sensor for Helicobacter pylori[J]. Angew Chem Int Ed Engl, 2019.
[76] Liu M, Yin Q, Chang Y, et al.In vitro selection of circular DNA aptamers for biosensing applications[J]. Angew Chem Int Ed Engl, 2019, 58(24):8013-8017.
[77] Carrasquilla C, Kapteyn E, Li Y, et al.Sol-Gel-derived biohybrid materials incorporating long-Chain DNA aptamers[J]. Angew Chem Int Ed Engl, 2017, 56(36):10686-10690.