基于双重猝灭分子信标及核酸染料Hoechst 33258对单链核酸的双色定量检测
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摘要
利用鸟嘌呤(G)碱基和有机猝灭基团Black Hole Quencher 1 (BHQ-1)对荧光基团6-羧基荧光素(6-Carboxyfluorescein group,FAM)的双重猝灭作用构建了一种结构简单的双重猝灭分子信标,结合核酸染料Hoechst 33258,以艾滋病毒RNA片段的反转录序列(33个碱基)为目标DNA,建立了一种高灵敏单链核酸(ssDNA)的双色荧光定量检测方法。此分子信标中,荧光基团及有机猝灭基团分别设计为FAM和BHQ-1,分子信标的茎的碱基全部设计为C-G碱基对,环的碱基序列设计为目标DNA的互补序列,与BHQ-1相连接的为3个带有G碱基的核苷酸。没有目标DNA时,分子信标呈茎-环结构,荧光基团FAM与有机猝灭基团BHQ-1及G碱基距离很近,在BHQ-1及G碱基的双重猝灭下,FAM的荧光很弱;另外,分子信标的茎的碱基全部是C-G碱基对,不能与核酸染料Hoechst 33258相结合,因此Hoechst 33258的荧光也很弱。当有目标DNA存在时,分子信标的环与目标DNA杂交形成双链,茎-环结构被破坏,FAM远离猝灭基团BHQ-1及G碱基,其荧光得到恢复;同时,核酸染料Hoechst 33258与双链DNA中的A-T碱基对相结合,其荧光显著增强。根据荧光基团FAM及核酸染料Hoechst 33258荧光增强的程度可实现对ssDNA的定量检测。在优化的条件下,目标DNA的浓度在0.05～8.0 nmol/L范围内时,FAM和Hoechst 33258的总荧光强度(ΔI_T)与目标DNA的浓度(C)具有良好的线性关系,回归方程为ΔI_T=192.2C+115.08 (R~2=0.9938),检出限为20 pmol/L (3σ,n=9)。此方法操作简单、灵敏度高、选择性好、检出限低。
A double-quenching molecular beacons(MB) was designed based on dual quenching of organic quencher Black Hole Quencher 1(BHQ-1) and guanine(G) bases to fluorophore 6-carboxyfluorescein group(FAM). A dual color fluorescence quantitative detection method for specific single-stranded DNA sequences has been developed based on MB and nucleic acid dye hoechst 33258. It is demonstrated by a reverse transcription oligonucleotide sequence(target DNA, 33 bases) of RNA fragment of human immunodeficiency virus(HIV) as model systems. In this MB, FAM and BHQ-1 are respectively selected as fluorophore and organic quencher, three continuous nucleotides with G base are connected with BHQ-1, the bases of the stem in MB is completely designed as C-G base pairs, and the base sequence of the loop in MB is designed as the complementary sequence of target DNA. In the absence of target DNA, the MBs is the stem-loop structure, the FAM is close to BHQ-1 and G bases, the fluorescence of FAM is dually quenched by BHQ-1 and G bases, and the fluorescence of FAM is very weak. In addition, the C-G base pairs of MB cannot be combined with Hoechst 33258, so the fluorescence of Hoechst 33258 is also very weak. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strand structure, the stem-loop structure of MB is destroyed, and the FAM is separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. At the same time, the nucleic acid dye Hoechst 33258 binds to the A-T base pair in double-stranded DNA, and its fluorescent signal is significantly enhanced. Thus, a dual color fluorescence quantitative detection for the target DNA can be realized through the fluorescence enhancement of FAM and Hoechst 33258. Under optimized conditions, the total fluorescence intensities of FAM and Hoechst 33258 exhibit a good linear dependence on concentration of target DNA in the range of 0.05-8.0 nmol/L, and the regression equation is ΔI_T=192.2C + 115.08(R~2=0.9938) with detection limit of 20 pmol/L(3σ, n=9). The method has simple operation, high sensitivity, good selectivity and low detection limit.
A double-quenching molecular beacons(MB) was designed based on dual quenching of organic quencher Black Hole Quencher 1(BHQ-1) and guanine(G) bases to fluorophore 6-carboxyfluorescein group(FAM). A dual color fluorescence quantitative detection method for specific single-stranded DNA sequences has been developed based on MB and nucleic acid dye hoechst 33258. It is demonstrated by a reverse transcription oligonucleotide sequence(target DNA, 33 bases) of RNA fragment of human immunodeficiency virus(HIV) as model systems. In this MB, FAM and BHQ-1 are respectively selected as fluorophore and organic quencher, three continuous nucleotides with G base are connected with BHQ-1, the bases of the stem in MB is completely designed as C-G base pairs, and the base sequence of the loop in MB is designed as the complementary sequence of target DNA. In the absence of target DNA, the MBs is the stem-loop structure, the FAM is close to BHQ-1 and G bases, the fluorescence of FAM is dually quenched by BHQ-1 and G bases, and the fluorescence of FAM is very weak. In addition, the C-G base pairs of MB cannot be combined with Hoechst 33258, so the fluorescence of Hoechst 33258 is also very weak. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strand structure, the stem-loop structure of MB is destroyed, and the FAM is separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. At the same time, the nucleic acid dye Hoechst 33258 binds to the A-T base pair in double-stranded DNA, and its fluorescent signal is significantly enhanced. Thus, a dual color fluorescence quantitative detection for the target DNA can be realized through the fluorescence enhancement of FAM and Hoechst 33258. Under optimized conditions, the total fluorescence intensities of FAM and Hoechst 33258 exhibit a good linear dependence on concentration of target DNA in the range of 0.05-8.0 nmol/L, and the regression equation is ΔI_T=192.2C + 115.08(R~2=0.9938) with detection limit of 20 pmol/L(3σ, n=9). The method has simple operation, high sensitivity, good selectivity and low detection limit.
引文
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22 Han D,Wei C Y.Talanta,2018,181:24-31
23 Xiang D S,Zheng A H,Luo M,Ji X H,He Z K.Sci.China Chem.,2013,56(3):380-386
2 Niu X H,He W Y,Song B,Ou Z H,Fan D,Chen Y C,Fan Y,Sun X F.J.Biol.Chem.,2016,291(32):16576-16585
3 Li P P,Liu X P,Mao C J,Jin B K,Zhu J J.Anal.Chim.Acta,2019,1048:42-49
4 Sani N D M,Heng L Y,Marugan R S P M,Rajab N F.Food Chem.,2018,269:503-510
5 Loescher S,Groeer S,Walther A.Angew.Chem.Int.Edit.,2018,57(33):10436-10448
6 Qian C,Wang R,Wu C,Wang L,Ye Z,Wu J,Ji F.Anal.Chim.Acta,2018,1040:105-111
7 Zhang Z,Xiang X,Huang F H,Zheng M M,Xia X Y,Han L.Sens.Actuators B,2018,273:159-166
8 Amirbekyan K,Duchemin N,Benedetti E,Joseph R,Colon A,Markarian S A,Bethge L,Vonhoff S,Klussmann S,Cossy J,Vasseur J J,Arseniyadis S,Smietana M.ACS Catal.,2016,6(5):3096-3105
9 Xiang D S,Zhai K,Sang Q Z,Shi B A,Yang X H.Anal.Sci.,2017,33(3):275-279
10 Anuradha,Alam M S,Chaudhury N K.Chem.Pharm.Bull.,2010,58(11):1447-1454
11 Shahabadi N,Shiri F,Norouzibazaz M,Falah A.Nucleos.Nucleot.Nucl.,2018,37(3):125-146
12 XIANG Dong-Shan,ZHAI Kun,XIANG Wen-Jun,WANG Lian-Zhi.Chinese J.Anal.Chem.,2014,42(8):1210-1214向东山,翟琨,向文军,王联芝.分析化学,2014,42(8):1210-1214
13 Xiang D S,Zhou G H,Luo M,Ji X H,He Z K.Analyst,2012,137:3787-3793
14 Adegoke O,Park E Y.Nanoscale Res.Lett.,2016,11(1):523-528
15 Oh H J,Kim J,Park H,Chung S,Hwang D W,Lee D S.Biosens.Bioelectron.,2019,126:647-656
16 Xiang D S,Zhai K,Xiang W J,Wang L Z.Talanta,2014,129:249-253
17 Hu H,Zhang J Y,Ding Y,Zhang X F,Xu K L,Hou X D,Wu P.Anal.Chem.,2017,89(9):5101-5106
18 ZHAI Kun,LI Feng-Quan,SHI Bo-An,XIANG Dong-Shan.Chinese J.Anal.Chem.,2017,45(10):1462-1466翟琨,李奉权,史伯安,向东山.分析化学,2017,45(10):1462-1466
19 Hu J,Zheng P C,Jiang J H,Shen G L,Yu R Q,Liu G K.Analyst,2010,135:1084-1089
20 Zhang P,Beck T,Tan W H.Angew.Chem.Int.Edit.,2001,40(2),402-405
21 Mao X,Xu H,Zeng Q,Zeng L,Liu G.Chem.Commun.2009,21:3065-3067
22 Han D,Wei C Y.Talanta,2018,181:24-31
23 Xiang D S,Zheng A H,Luo M,Ji X H,He Z K.Sci.China Chem.,2013,56(3):380-386