CN116926170A - 基于硫修饰核酸及硫修饰核酸识别蛋白的核酸检测方法 - Google Patents
基于硫修饰核酸及硫修饰核酸识别蛋白的核酸检测方法 Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
本发明提供了一种基于硫修饰核酸和硫修饰核酸识别蛋白的核酸检测方法及应用。具体地,本发明提供了一种核酸检测效应器,其与硫修饰的向导核酸联用以实现核酸检测功能,该效应器包括靶向元件和报告元件,其中所述靶向元件与所述报告元件形成一融合蛋白,所述靶向元件包含硫修饰核酸识别蛋白,所述报告元件为限制性核酸内切酶或荧光双分子互补报告系统。本发明首次将硫修饰识别蛋白与核酸检测相结合,构建了通过硫修饰向导核酸与靶标核酸匹配结合并结合硫修饰识别蛋白从而进行检测的系统。本发明具有非侵入性、特异性、通量高等优势,可在1h内完成,灵敏度达到1000拷贝以内。
Description
技术领域
本发明属于生物技术领域,涉及一种检测目标核酸的系统和方法,尤其涉及一种基于硫修饰核酸及硫修饰核酸识别蛋白的核酸检测方法及其应用。
背景技术
核酸检测适用于病原体的检测、癌症筛查、单核苷酸多态性(SNPs,singlenucleotide polymorphisms)分析、细菌耐药基因筛查等,可广泛应用于分子医学诊断、环境微生物检测、食品安全等应用领域。近年来,由病原微生物造成的安全问题涉及面广且影响日益严重,对人类的健康和财产威胁巨大。在短时间内准确快速地对病原微生物进行检测,有助于食品安全的监测以及早期诊断。
目前,基于聚合酶链式反应(polymerase chain reaction,PCR)的传统核酸检测技术仍是重要的检测手段,包括qPCR、DNA测序等。DNA测序技术准确率高,但也存在着高成本、耗时长等缺陷。qPCR不仅是快速、高灵敏的检测方法,同时也可以进行定量分析,但qPCR也依然存在着准确性低的缺陷。此外,qPCR和DNA测序技术都因为对实验仪器的依赖性,只能在特殊的实验室条件下完成,限制了其在基层实验室的广泛使用。
近年来,核酸等温扩增技术在检测中显示出良好的应用前景。核酸序列扩增(nucleic acid sequence-based amplification,NASBA)技术是针对RNA靶标在41℃条件下进行扩增的技术,可与电化学发光法等联合检测。滚环扩增(rolling circleamplification,RCA)技术是以单链环状寡核苷酸为模板进行线性合成,最终生成一条含有重复靶序列的线状单链DNA或RNA。环介导等温扩增(loop-mediated isothermalamplification,LAMP)技术是用4-6条引物在65℃条件下利用链置换DNA聚合酶快速合成靶序列,如果在体系中同时加入逆转录酶或者使用具有逆转录活性的聚合酶就可以实现对RNA的扩增,即RT-LAMP技术。重组酶聚合酶等温扩增(recombinase polymeraseamplification,RPA)模仿体内复制机制,在37℃条件下利用重组酶、单链结合蛋白和DNA聚合酶实现靶序列的指数级别扩增,反应不需要退火过程,灵敏度好且特异性高。切口酶扩增反应(nicking endonuclease amplification reaction,NEAR)技术利用切口酶识别病毒核酸的特定短序列,在56℃条件下合成单链靶序列。
最近,将等温扩增与成簇规律间隔的短回文重复序列以及相关蛋白(clusteredregularly interspaced short palindromic repeats,CRISPR associated protein,CRISPR/Cas)体系联用的核酸检测技术为建立快速有效的检测手段开辟了新方向。2017年,张锋团队在《科学》杂志发表了一项全新的核酸检测系统——SHERLOCK,在此系统中,研究人员利用Cas13a结合特异性靶RNA后会对其他RNA切割的特性,结合RPA设计了RNA荧光报告系统。该技术可以快速地在野外(非实验室)条件下完成对寨卡病毒等病原体的检测,但鉴于RNA的不稳定性,增加了操作难度。2018年,Doudna团队利用Cas12a蛋白在剪切靶向dsDNA后能非特异性切割单链DNA的活性,将其与RPA结合,开发了DETECTR技术。但是,CRIPSR系统存在脱靶问题,会导致检测出现假阳性;另外,该系统在选择靶向DNA区域时受PAM序列限制。
因此,本领域迫切需要开发一种高特异性、高灵敏度、没有序列限制、不需要复杂仪器、在多种场景都可以使用的核酸检测技术。
发明内容
本发明的目的在于提供一种基于硫修饰核酸及硫修饰核酸识别蛋白的核酸检测方法及其应用。
本发明的第一方面,提供了一种核酸检测效应器,所述核酸检测效应器包括靶向元件和报告元件,其中所述靶向元件与所述报告元件形成一融合蛋白,其中所述靶向元件包含硫修饰核酸识别蛋白。
在另一优选例中,所述核酸检测效应器在硫修饰向导核酸的引导下,可结合于待检测核酸的预定区域,并通过报告元件与分子信标接触从而产生报告信号。
在另一优选例中,所述的硫修饰向导核酸与待检测核酸,在预定区域内形成双链互补结构。
在另一优选例中,所述的硫修饰向导核酸为单链的磷硫酰化修饰核酸。
在另一优选例中,所述的硫修饰向导核酸包含磷硫酰化修饰的gDNA或gRNA。
在另一优选例中,所述gDNA或gRNA的序列长度大于10nt,小于100nt,其中含有至少一个硫修饰核苷酸。
在另一优选例中,所述gDNA或gRNA的序列长度为10-30nt。
在另一优选例中,所述的硫修饰向导核酸除了硫修饰,还可以同时带有其他化学修饰,所述化学修饰选自下组:生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA)或其组合。
在另一优选例中,所述的核酸检测效应器为单链核酸的核酸检测效应器。
在另一优选例中,所述的单链核酸为单链DNA、单链RNA、或其组合。
在另一优选例中,所述硫修饰核酸识别蛋白为硫结合结构域蛋白(SBD),其特异性识别被硫修饰的核酸。
在另一优选例中,所述的硫修饰的核酸指核酸中磷酸基团中的一个OH(O或O-)被SH(或S-)所取代和或一个=0被=S替换。
在另一优选例中,所述的硫修饰的核酸指核酸中的被修饰为或其对应异构体。
在另一优选例中,所述硫修饰核苷酸可以为一个核苷酸。
在另一优选例中,所述硫修饰核苷酸可以为N个连续的核苷酸(1≤N≤10)。
在另一优选例中,所述硫修饰核苷酸优选地为C或G。
在另一优选例中,所述硫结合结构域蛋白(SBD)结合DNA/DNA双链或DNA/RNA杂合双链。
在另一优选例中,所述硫结合结构域蛋白(SBD)包括:SBDgsu、SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu或SBD同源蛋白。
在另一优选例中,硫结合结构域蛋白(SBD)来源于选自下组的物种:GsuMcrA、MMoMcrA、ScoMcrA、SprMcrA、HgaMcrA、EcoMcrA;优选地,来源于GsuMcrA。
在另一优选例中,所述SBD同源蛋白可结合于硫修饰寡核苷酸,并且具有一个或多个以下条件:
C1)具有硫修饰寡核苷酸(包括脱氧核糖核酸DNA或核糖核酸RNA在内的核苷酸)结合活性的蛋白结构域;
C2)与SBDgsu、SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu中任意一个蛋白的氨基酸序列的同一性≥20%(较佳地≥50%,更佳地≥70%,最佳地≥80%或≥90%);
C3)包含P-L-W基序或A-L-W基序(例如:SBDgsu中的P86-L90-W96;SBDmmo中的P75-L79-W85;SBDspr中的P79-L83-W89;SBDtcu中的A73-L77-W83)。
在另一优选例中,所述报告元件包括单分子报告元件和双分子互补报告元件。
在另一优选例中,所述单分子报告元件为限制性核酸内切酶。
在另一优选例中,所述限制性核酸内切酶特异性识别特定DNA序列;优选地,所述限制性核酸内切酶为BclI,其特异性识别5’-TGATCA-3’/5’-TGATCA-3’的DNA序列。
在另一优选例中,所述双分子互补报告元件为荧光双分子互补报告系统。
在另一优选例中,所述荧光双分子互补报告系统包括(但不限于):Fluc-N和Fluc-C、LgBiT和SmBiT、LacA和LacB。
在另一优选例中,所述报告元件选自下组:限制性核酸内切酶、荧光双分子互补报告系统、或其组合。
在另一优选例中,所述分子信标选自下组:双链DNA、荧光素酶、或其组合。
在另一优选例中,所述双链DNA含有能被限制性核酸内切酶特异性识别的序列。
在另一优选例中,所述双链DNA含有能够被BclI酶特异性识别的序列,即5’-TGATCA-3’/5’-TGATCA-3’的DNA序列。
在另一优选例中,所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团。
在另一优选例中,所述荧光基团包括(但不限于):异硫氰酸荧光素(Fluoresceinisothiocyanate,FITC)、羧基荧光素(Carboxyfluorescein,FAM)、四氯-6-羧基荧光素(Tetrachloro fluorescein,TET)、六氯-6-甲基荧光素(Hexachloro fluorescein,HEX);优选地,所述荧光基团为FAM。
在另一优选例中,所述淬灭基团包括(但不限于):BHQ-1、BHQ-2、4-(4-恶烷氨基苯偶氮)苯甲酸(DABCYL);优选地,所述淬灭基团为BHQ-1。
在另一优选例中,所述的靶向元件和/或所述报告元件各自独立地为实施例中所制备的核酸检测效应器的对应元件。
在另一优选例中,所述的靶向元件和所述报告元件通过化学键(如肽键)直接相连或通过接头连接。
在另一优选例中,所述的接头包括柔性接头和非柔性接头。
在另一优选例中,所述接头选自下组:(GGGGS)n,其中n为1-4的正整数,优选地n为2-3的正整数。
在另一优选例中,所述融合蛋白含有一个或多个SBD。
在另一优选例中,所述多个SBD可以相同也可以不同。
在另一优选例中,所述多个SBD可位于报告元件的同一侧(例如左侧或右侧),也可以位于报告元件的两侧。
在另一优选例中,所述融合蛋白的结构如下式I或I'或I”所示:
A-L-B(I)
B-L-A(I')
A-L-B-L-A(I”)
式中,
A为靶向元件SBD;
B为报告元件;
L各自独立地为无或连接肽;
各“-”独立地为化学键。
在另一优选例中,所述的化学键包括肽键、或共价键。
在另一优选例中,所述的融合蛋白的序列如SEQ ID No:6、SEQ ID No:32或SEQ IDNo:33所示。
本发明的第二方面,提供了一种核酸检测的方法,包含步骤:
(i)在硫修饰向导核酸的存在下,使得如本发明第一方面所述的核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;和
(ii)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号。
在另一优选例中,所述核酸检测效应器含有靶向元件SBD和报告元件。
在另一优选例中,所述的SBD选自下组:与SBDgsu、SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu中任意一个蛋白的氨基酸序列的同一性≥20%的SBD蛋白或其组合;优选地,所述SBD为SBDgsu。
在另一优选例中,所述报告元件包括单分子报告元件和双分子互补报告元件。
在另一优选例中,所述单分子报告元件为限制性核酸内切酶;优选地,所述限制性核酸内切酶为BclI。
在另一优选例中,所述双分子互补报告元件为荧光双分子互补报告系统。
在另一优选例中,所述硫修饰向导核酸为硫修饰的单链gDNA或gRNA。
在另一优选例中,所述的硫修饰向导核酸除了硫修饰,还可以同时带有其他化学修饰,包括(但不限于)生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA);优选地,所述硫修饰向导核酸还带有生物素修饰。
在另一优选例中,所述待检测核酸选自下组:单链DNA、单链RNA、或其组合。
在另一优选例中,所述待检测核酸通过等温扩增大量富集。
在另一优选例中,所述等温扩增包括以下方法:核酸序列扩增(nucleic acidsequence-based amplification,NASBA)技术、滚环扩增(rolling circleamplification,RCA)技术、环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术、重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)技术、切口酶扩增反应(nicking endonuclease amplification reaction,NEAR)技术。
在另一优选例中,所述分子信标选自下组:双链DNA、荧光素酶、或其组合。
在另一优选例中,所述分子信标为双链DNA。
在另一优选例中,所述双链DNA含有能被限制性核酸内切酶特异性识别的序列。
在另一优选例中,所述双链DNA含有能够被BclI酶特异性识别的序列,即5’-TGATCA-3’/5’-TGATCA-3’的DNA序列。
在另一优选例中,所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团。
在另一优选例中,所述荧光基团包括(但不限于)异硫氰酸荧光素(Fluoresceinisothiocyanate,FITC)、羧基荧光素(Carboxyfluorescein,FAM)、四氯-6-羧基荧光素(Tetrachloro fluorescein,TET)、六氯-6-甲基荧光素(Hexachloro fluorescein,HEX);优选地,所述荧光基团为FAM。
在另一优选例中,所述淬灭基团包括(但不限于)BHQ-1、BHQ-2、4-(4-恶烷氨基苯偶氮)苯甲酸(DABCYL);优选地,所述淬灭基团为BHQ-1。
在另一优选例中,所述方法包括以下步骤:
(1)在硫修饰向导核酸的存在下,使得核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;
(2)使用磁珠将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;和
(3)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号;
其中,所述核酸检测效应器含有靶向元件SBD和报告元件,所述报告元件为单分子报告元件,所述单分子报告元件为限制性核酸内切酶;
所述硫修饰向导核酸除了硫修饰,还同时带有其他化学修饰,包括(但不限于)生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA);优选地,所述硫修饰向导核酸还带有生物素修饰;
所述分子信标为双链DNA,所述双链DNA含有能被限制性核酸内切酶特异性识别的序列,进一步地,所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团。
在另一优选例中,所述方法包括以下步骤:
(1)在硫修饰向导核酸的存在下,使得核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;
(2)任选地,使用磁珠将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;和
(3)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号;
其中,所述核酸检测效应器含有靶向元件SBD和报告元件,所述报告元件为双分子互补报告元件,所述双分子互补报告元件为荧光双分子互补报告系统;
所述硫修饰向导核酸除了硫修饰,任选地还同时带有其他化学修饰,包括(但不限于)生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA);
所述分子信标为荧光素酶。
在另一优选例中,所述磁珠为标记后的磁珠,且所述磁珠通过所述标记与带有其他化学修饰的硫修饰向导核酸结合,从而将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;
所述标记包括(但不限于)链霉亲和素标记、巯基标记、羧基标记、聚苯乙烯包覆标记、炔基标记、叠氮标记、环氧基标记、羟基标记;优选地,所述磁珠为链霉亲和素标记磁珠。
本发明的第三方面,提供了一种用于核酸检测的反应体系,所述的反应体系包括:
(a)如本发明第一方面所述的核酸检测效应器;
(b)硫修饰向导核酸,所述硫修饰向导核酸引导所述核酸检测效应器特异性结合于待检测核酸的预定区域;和
(c)分子信标,所述分子信标与所述核酸检测效应器接触后从而产生报告信号。
在另一优选例中,所述的检测体系还包括:(d)磁珠,所述磁珠用于将“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离。
在另一优选例中,所述的检测体系还包括:(e)缓冲液。
在另一优选例中,所述的待检测核酸为单链DNA、单链RNA、或其组合。
在另一优选例中,所述的待检测核酸通过等温扩增大量富集。
在另一优选例中,所述待检测的核酸包括来源于选自下组的待检测的核酸:植物、动物、昆虫、微生物、病毒、环境样本、临床样本或其组合。
在另一优选例中,所述的待检测的核酸是人工合成或天然存在的DNA。
在另一优选例中,所述的待检测的核酸包括野生型或突变型的DNA。
在另一优选例中,所述的待检测核酸在所述反应体系中的浓度为1×103至1×108拷贝/微升,较佳地1×104至1×107拷贝/微升。
在另一优选例中,所述的检测体系中,所述硫修饰向导核酸浓度为0.1-10nM,较佳地1-10nM,更佳地1-5nM。
在另一优选例中,所述的硫修饰向导核酸为硫修饰的gDNA或gRNA。
在另一优选例中,所述的硫修饰向导核酸除了硫修饰,还可以带有其他化学修饰,所述化学修饰选自下组:生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA)或其组合。
在另一优选例中,所述的gDNA或gRNA的长度为10-100nt,较佳地10-30nt。
在另一优选例中,所述的分子信标在所述反应体系中的浓度为160至2560nM,较佳地320至1280nM,更佳地640nM。
在另一优选例中,所述的分子信标为双链DNA或荧光素酶。
在另一优选例中,所述的磁珠的使用量为20至400μg;优选地,使用量为100μg。
在另一优选例中,所述磁珠为标记后的磁珠,且所述磁珠通过所述标记与带有其他化学修饰的硫修饰向导核酸结合,从而将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;
所述标记包括(但不限于)链霉亲和素标记、巯基标记、羧基标记、聚苯乙烯包覆标记、炔基标记、叠氮标记、环氧基标记、羟基标记;优选地,所述磁珠为链霉亲和素标记磁珠。
在另一优选例中,所述的检测体系包括:
(a1)核酸检测效应器;所述核酸检测效应器包含靶向元件SBD和单分子报告元件限制性核酸内切酶;
(b1)硫修饰向导核酸;
(c1)分子信标,所述分子信标为双链DNA,其含有能被限制性核酸内切酶特异性识别的序列,且所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团;和
(d1)磁珠。
在另一优选例中,所述的检测体系包括:
(a2)核酸检测效应器;所述核酸检测效应器包含靶向元件SBD和双分子互补报告元件,所述双分子互补报告元件为荧光双分子互补报告系统;
(b2)硫修饰向导核酸;和
(c2)分子信标,所述分子信标为荧光素酶。
本发明的第四方面,提供了一种核酸,所述的核酸编码如本发明第一方面所述的核酸检测效应器。
在另一优选例中,所述的核酸为线性序列。
在另一优选例中,所述核酸具有5'-3'(5'至3')的式II或II'结构:
P1-X1-L1-X2(II)
P1-X2-L1-X1(II');
式中,P1为第一启动子序列;
X1为硫结合结构域蛋白(SBD)的编码序列;
L1为无或连接序列的编码序列;
X2为报告元件的编码序列;
并且,各“-”独立地为化学键。
在另一优选例中,所述核酸构建物含有一个或多个SBD编码序列。
在另一优选例中,所述多个SBD编码序列可以相同也可以不同。
在另一优选例中,所述多个SBD编码序列可位于报告元件编码序列的同一侧(例如左侧或右侧),也可以位于报告元件编码序列的两侧。
本发明的第五方面,提供了一种载体,所述的载体含有如本发明第四方面所述的核酸。
在另一优选例中,所述的载体包括表达载体。
在另一优选例中,所述的载体包括真核表达载体、原核表达载体、病毒载体。
本发明的第六方面,提供了一种基因工程细胞,所述细胞含有如本发明第五方面所述的载体,或者基因组中整合有如本发明第四方面所述的核酸。
在另一优选例中,所述的细胞被所述的载体或核酸所转化或转染。
在另一优选例中,所述的细胞还被硫修饰的gDNA或gRNA或其表达载体所转染。
在另一优选例中,所述的细胞包括真核细胞、原核细胞。
在另一优选例中,所述的细胞包括细菌、酵母、哺乳动物细胞、植物细胞。
本发明的第七方面,提供了一种试剂盒,所述试剂盒包含:
(C1)第一容器,以及位于所述第一容器中的本发明第一方面所述的核酸检测效应器、或其编码序列、或含所述编码序列的载体;
(C2)第二容器,以及位于所述第二容器中的硫修饰向导核酸;和
(C3)第三容器,以及位于所述第三容器中的分子信标;
其中,所述的核酸检测效应器在硫修饰向导核酸的引导下,可结合于预定的核酸区域,形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物,在所述复合物与分子信标接触后,产生可检测的报告信号。
在另一优选例中,所述的硫修饰向导核酸包括硫修饰的gDNA、gRNA或其组合。
在另一优选例中,所述的硫修饰向导核酸除了硫修饰,还可以同时带有其他化学修饰,所述化学修饰选自下组:生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA)或其组合。
在另一优选例中,所述分子信标包括双链DNA或荧光素酶。
在另一优选例中,所述的试剂盒还包含:(C4)第四容器,以及位于第四容器中的磁珠,所述磁珠为标记后的磁珠,且所述磁珠通过所述标记与带有其他化学修饰的硫修饰向导核酸结合,从而将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离。
在另一优选例中,所述核酸检测效应器包含SBD和限制性核酸内切酶;而分子信标为双链DNA,所述双链DNA含有可被所述限制性核酸内切酶特异性识别的序列,且所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团。
在另一优选例中,所述核酸检测效应器包含SBD和荧光双分子互补报告系统;而分子信标为荧光素酶。
本发明的第八方面,提供了一种如本发明第一方面所述的核酸检测效应器的用途,用于制备核酸检测的试剂或试剂盒。
在另一优选例中,所述核酸为单链DNA、单链RNA、或其组合。
在另一优选例中,所述核酸包括来源于选自下组的待检测的核酸:植物、动物、昆虫、微生物、病毒、环境样本、临床样本或其组合。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了SBD同源蛋白的氨基酸序列比对;三角标注的是P-L-W或A-L-W基序。
图2显示了硫修饰DNA的化学结构。
图3显示了硫修饰DNA结合结构域对硫修饰核酸的结合活性。
图4显示了SBDgsu-BclI切割活性验证。
图5显示了利用SBDgsu-BclI融合蛋白进行核酸检测的流程示意图。
图6显示了用SBDgsu-BclI融合蛋白检测体系中的单链DNA。
图7显示了NEAR扩增技术联用SBDgsu-BclI检测系统用于检测单链DNA。
图8显示了NEAR扩增技术联用SBDgsu-BclI检测系统用于检测双链DNA。
图9显示了NEAR扩增技术联用SBDgsu-BclI检测系统用于检测白色念珠菌基因组DNA。
图10显示了用SBDgsu-FlucN和SBDgsu-FlucC融合蛋白进行核酸检测的流程示意图。
图11显示了用SBDgsu-FlucN和SBDgsu-FlucC融合蛋白检测体系中的单链DNA。
图12显示了RPA扩增技术联用SBDgsu-BclI检测系统用于检测单链DNA。
图13显示了RPA扩增技术联用SBDgsu-BclI检测系统用于检测白色念珠菌DNA。
具体实施方式
本发明人通过广泛而深入的研究,通过对硫修饰DNA识别结构域的研究,首次开发了一种基于硫修饰核酸及硫修饰核酸识别蛋白的靶标核酸检测方法,并提供了包含执行所述方法所需核酸检测效应器的检测体系和试剂盒。实验结果表明,采取本发明的方法成功地在1小时以内完成检测,灵敏度可达1000拷贝。在此基础上,完成了本发明。
术语
如本文所用,术语“硫修饰”也可称为“磷硫酰化修饰”,是指对本文所述的核酸检测效应器的硫修饰向导核酸进行的修饰;具体地,是指DNA或RNA磷酸骨架上非桥联氧原子被硫原子取代而发生的修饰。
如本文所用,术语“硫修饰核酸识别蛋白”,“硫结合结构域蛋白”以及“SBD”可互换使用,均指能够特异性识别带有硫修饰核酸的蛋白。
如本文所用,术语“硫修饰核酸”,或“被硫修饰的核酸”均表示核酸带有硫原子修饰;其中,所述核酸可以是RNA,也可以是DNA。
如本文所用,术语“gRNA”或“gDNA”是指可与待检测核酸的碱基互补配对的一段单链核酸序列,其包含一个或多个位点的硫修饰,引导SBD到达待检测核酸。在本发明的一个优选实施方式中,所述“gRNA”或“gDNA”还同时带有其他化学修饰,例如:生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA)等。
如本文所用,术语“分子信标”是指能够与本发明的核酸检测效应器中的报告元件相互配合(接触),从而产生可被检测到的报告信号的分子。
在本发明的一个优选实施方式中,所述分子信标为双链DNA,其包括可被核酸检测效应器中的报告元件(限制性核酸内切酶)特异性识别的序列。进一步地,所述双链DNA的5’端和3’端还分别标记有荧光基团和淬灭基团。当所述双链DNA分子与核酸检测效应器相互接触且被特异性识别时,双链DNA分子被切断,使得原本标记在5’端和3’端的荧光基团和淬灭基团彼此远离,从而发出可检测的荧光信号。
在本发明的另一个优选实施方式中,所述分子信标为荧光素酶。当所述荧光素酶与核酸检测效应器(包含荧光双分子互补报告系统作为报告元件)相互接触时,产生可检测的荧光信号。
核酸检测效应器
在本发明的一个方面,提供了一种核酸检测效应器。如本文所用,术语“本发明的核酸检测效应器”、“本发明的融合蛋白”可互换使用,均指本发明第一方面中所述的核酸检测效应器,即包含了至少一个靶向元件和报告元件的融合蛋白,所述靶向元件包含硫修饰核酸识别蛋白,而所述报告元件包括单分子报告元件和双分子互补报告元件;其中,所述核酸检测效应器在硫修饰向导核酸的引导下,可结合于待检测核酸的预定区域,并通过报告元件与分子信标接触从而产生报告信号。
在本发明的一个优选实施方式中,所述核酸检测效应器包括SBD和单分子报告元件,所述单分子报告元件可与分子信标接触后独立发挥功能并产生报告信号,而不需要要其它分子的协助。所述单分子报告元件可以为限制性核酸内切酶。在本发明的一个实施例中,构建了包括来源于GsuMcrA的SBD,即SBDgsu,和限制性核酸内切酶BclI的融合蛋白SBDgsu-BclI作为核酸检测效应器。
在本发明的另一个优选实施方式中,所述核酸检测效应器包括SBD和双分子互补报告元件,所述双分子互补报告元件分别与一个SBD相连或同时与至少一个SBD蛋白相连。所述双分子互补报告元件中的两个分子相互配合,与分子信标接触后产生报告信号。在本发明的一个实施例中,构建了包括来源于GsuMcrA的SBD,即SBDgsu,和荧光互补双分子Fluc-N和Fluc-C的融合蛋白,SBDgsu-FlucN和SBDgsu-FlucC作为核酸检测效应器。
硫结合结构域蛋白(SBD)
硫结合结构域蛋白简称SBD。在本发明中,进行了融合蛋白的构建,SBD作为靶向元件,将本发明的核酸检测效应器带到待检测核酸的预定区域。
SBD可以位于融合蛋白的N端或C端。融合蛋白中也可以存在多个SBD结构域,比如SBD-报告元件-SBD的形式,以提高检测效率。此外,SBD和报告元件还可以有多种排列组合方式,如SBD-SBD-报告元件,报告元件-SBD-SBD、报告元件-SBD-报告元件等等。
在本发明中,代表性的所述硫结合结构域蛋白(SBD)包括:SBDgsu、SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu或SBD同源蛋白。
一些代表性的SBD同源蛋白的氨基酸序列比对结果见图1。
硫修饰的核酸
在本发明中,所述的硫修饰的核酸指核酸中磷酸基团中的一个OH(O或O-)被SH(或S-)所取代和或一个=0被=S替换。
在另一优选例中,所述的硫修饰的核酸指核酸中的被修饰为或其对应异构体。
一种代表性的硫修饰的核酸的结构如图2所示。
硫修饰核酸结合结构域对某些硫修饰核酸的结合活性如图3所示。
本发明的核酸检测方法、检测体系和试剂盒
本发明还提供了一种采用本发明的核酸检测效应器进行核酸检测的方法、检测体系和试剂盒。
总体上,本发明的核酸检测方法包含步骤:(i)在硫修饰向导核酸的存在下,使得本发明第一方面所述的核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;和(ii)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号。
在本发明的一个优选实施方式中,所述方法包括以下步骤:
(1)在硫修饰向导核酸的存在下,使得核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;
(2)使用磁珠将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;和
(3)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号;
其中,所述核酸检测效应器含有靶向元件SBD和报告元件,所述报告元件为单分子报告元件,所述单分子报告元件为限制性核酸内切酶;
所述硫修饰向导核酸除了硫修饰,还同时带有其他化学修饰,包括(但不限于)生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA);优选地,所述硫修饰向导核酸还带有生物素修饰;
所述分子信标为为双链DNA,所述双链DNA含有能被限制性核酸内切酶特异性识别的序列,进一步地,所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团。
使用该检测方法的时,配备包含以下组分的检测体系或使用包含以下组分的试剂盒:
(a1)核酸检测效应器;所述核酸检测效应器包含靶向元件SBD和单分子报告元件限制性核酸内切酶;
(b1)硫修饰向导核酸;和
(c1)分子信标,所述分子信标为双链DNA,其含有能被限制性核酸内切酶特异性识别的序列,且所述双链DNA在5’端和3’端分别标记荧光基团和淬灭基团;和
(d1)磁珠,所述磁珠为标记后的磁珠,且所述磁珠通过所述标记与带有其他化学修饰的硫修饰向导核酸结合,从而将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;
所述标记包括(但不限于)链霉亲和素标记、巯基标记、羧基标记、聚苯乙烯包覆标记、炔基标记、叠氮标记、环氧基标记、羟基标记;优选地,所述磁珠为链霉亲和素标记磁珠。
在本发明的另一个优选实施方式中,所述方法包括以下步骤:
(1)在硫修饰向导核酸的存在下,使得核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;
(2)任选地,使用磁珠将所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物与未形成复合物的待测核酸分离;和
(3)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号;
其中,所述核酸检测效应器含有靶向元件SBD和报告元件,所述报告元件为双分子互补报告元件,所述双分子互补报告元件为荧光双分子互补报告系统;
所述硫修饰向导核酸除了硫修饰,任选地还同时带有其他化学修饰,包括(但不限于)生物素修饰、氨基修饰、氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA);
所述分子信标为荧光素酶。
使用该检测方法的时,配备包含以下组分的检测体系或使用包含以下组分的试剂盒:
(a2)核酸检测效应器;所述核酸检测效应器包含靶向元件SBD和双分子互补报告元件,所述双分子互补报告元件为荧光双分子互补报告系统;
(b2)硫修饰向导核酸;和
(c2)分子信标,所述分子信标为荧光素酶。
本发明的主要优点在于:
(1)快速:在测试条件准备好的情况下,从拿到样品,到拿到检测结果只需约1小时。
(2)高效:本发明具有极高的灵敏度,可以检测到1000拷贝/微升的靶标DNA。
(3)简单:没有特殊复杂的步骤。
(4)多用途:本发明应用于检测病原微生物、癌症筛查、基因突变、细菌耐药基因检测等应用场景。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条例,例如J.Sambrook等人,分子克隆实验指南(第四版)(科学出版社有限责任公司,2017)中所述的条件,或按照产品制造商提供的产品说明书中所述条件。实施例中所用的材料、试剂、仪器等,如无特殊说明,均可从商业途径得到。
实施例1
SBDgsu-BclI融合蛋白纯化及活性验证
1.1SBDgsu-BclI融合蛋白表达载体构建
通过基因合成,将BclI合成到大肠杆菌表达载体pBAD-Myc-HisA,并添加EcoRI和KpnI酶切位点,合成序列如下(SEQ ID NO:1):
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其中,下划线部分为KpnI和EcoRI酶切位点,斜体部分为BclI序列。
以上述质粒为模板使用如下引物扩增获得片段1:
BclI-F:
CTGGAAGAACAAGCCCATCTGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGCAGCCGAACCCGAAATTT(SEQ ID NO:2)
BclI-R:CAGTGGTGGTGGTGGTGGTGCTCGAGTCATTTATAATACTGATTCA(SEQ ID NO:3)
以含有SBDgsu序列的质粒作为模板,使用如下引物扩增获得片段2:
SBDgsu-F:AGCAAATGGGTCGCGGATCCGAATTCATGACCAGTCTGACCCCAAG(SEQ ID NO:4)
SBDgsu-R:
AAATTTCGGGTTCGGCTGCATGCTGCCGCCGCCGCCGCTGCCGCCGCCGCCCAGATGGGCTTGTTCTTCCAG(SEQ ID NO:5)
PCR完成后,使用胶回收试剂盒(OMEGA)对片段1和2进行回收。再在同一体系中以这两个片段为模板,以引物SBDgsu-F和BclI-R进行扩增获得片段3。PCR完成后利用同源重组试剂盒(诺唯赞)将PCR产物构建至商业表达载体pET28a上,并测序验证。
SBDgsu-BclI融合蛋白氨基酸序列如SEQ ID NO:6所示:
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMTSLTPSLKLFSSLSRAPGAVWTEATRRKAPHKPLLLLAVLDLVHRGVITSPFIAVSGDLVELNELFNLYWRRVVPLGLTSSIAFPFSRLSREPFWELVPQPGIAITDAVINNTSSVSYLRKYALGAKLDDGLFRVMQSGEGREALREALLLSCFSADAAALLREQSVINREAFDYSRVLEEQAHLGGGGSGGGGSMQPNPKFINKSSAFWAYAKLLSEQLGYSKDGVVISYSEAQARAKLKKLGINVKEGIFKDVLRYLKYRAELLNKHKDYLMDVEEARKYFQVALKQHQQNNYTCKLPLNKQKNEKKDYAYFTCIINIIAETELRYFANNNGLVYGKDIYFDDNPMNLSYILNFNRELEGIMSRRFDGAFPSTVNPILIWEIKEYYYTTTFGSRIADGVYETQLDGYEIKTIREETNKNIQHIYFIDDYNTWWNMGKSYLCRIIDMLHMGLVDEVIMGKEVFERWPQILRAVLNQYYK(SEQ IDNO:6)
1.2SBDgsu-BclI融合蛋白的表达
在大肠杆菌宿主BL21(DE3)中进行SBDgsu-BclI融合蛋白的异源表达。挑取携带表达载体的单克隆至LB液体培养基中培养,培养条件为37℃、220rpm,培养至OD600=0.6-0.8时添加IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度为0.2mM。再在16℃、220rpm培养条件下培养20小时诱导蛋白表达。
1.3SBDgsu-BclI融合蛋白的纯化。
菌液裂解:培养结束后,收集菌体,弃上清,将菌体重悬于50mL的Ni柱Buffer A(20mM Tris-HCl pH 8.0,300mM NaCl,50mM imidazole)中,使用预冷的细胞破碎仪破碎菌体,细胞裂解液于4℃,10000rpm离心1h,取上清,用0.45μm滤膜过滤后冰上放置。
Ni柱纯化:用Ni柱Buffer A(20mM Tris-HCl pH 8.0,300mM NaCl,50mMimidazole)平衡Ni亲和层析柱(GE),将细胞裂解液上样至Ni亲和层析柱上,用Ni柱BufferA冲洗20个柱体积后,再用Ni柱Buffer B(20mM Tris-HCl pH 8.0,300mM NaCl,500mMimidazole)冲洗2个柱体积并收集
肝素柱纯化:用肝素柱Buffer A(20mM Tris-HCl pH 8.0,50mM NaCl,1mM DTT)平衡Heparin亲和层析柱(GE),将Ni柱纯化得到的蛋白稀释后上样,用肝素柱Buffer A冲洗5个柱体积后,将肝素柱Buffer A与Buffer B(20mM Tris-HCl pH 8.0,1000mM NaCl,1mMDTT)混合,洗脱时Buffer B含量线性升高,SBDgsu-BclI融合蛋白大约在30%-60%B浓度区间内被洗脱下来。全部过程在AKTA纯化系统(GE)上进行。
分子筛纯化:用分子筛Buffer(20mM Tris-HCl pH 8.0,150mM NaCl)平衡分子筛(GE),将肝素柱纯化得到的蛋白上样,用分子筛Buffer以1mL/min的流速等梯度冲洗1.1个柱体积,SBDgsu-BclI融合蛋白大约在13-16min被冲洗下来。全部过程在AKTA纯化系统(GE)上进行。
蛋白定量与保存:收集样品,使用超滤管浓缩后,用Bradford蛋白浓度测定试剂盒进行定量,保存到-80℃冰箱中。
1.4SBDgsu-BclI融合蛋白活性验证
检测切割活性的反应底物为只含有一个BclI酶切位点的质粒pET28a。将pET28a转化至E.coli JM110中,挑取平板上单克隆至LB液体培养基中过夜培养,培养条件为37℃、220rpm。培养后提取质粒pET28a。首先在50mM Tris-HCl(pH 7.5)、10mM MgCl2、100mMNaCl、0.02%Triton X-100的反应缓冲液里混合200ng pET28a及2.5、5、10、20、40、80、160ng的SBDgsu-BclI融合蛋白。混合样品在50℃反应30分钟后,在0.8%琼脂糖胶中进行电泳检测,电泳条件为120V,30分钟。电泳结束后,琼脂糖胶用EB核酸染料显色。
如图4所示,SBDgsu-BclI融合蛋白能够切割质粒pET28a。
实施例2
用SBDgsu-BclI融合蛋白检测体系中的单链DNA
2.1单链DNA制备
单链DNA序列如下所示:
T1:AGAGTAAGTAGTTCGCCAGTTAATAGTTTG(SEQ ID NO:7)
T2:ACTGAGATACCTACAGCGTGAGCTATGAGA(SEQ ID NO:8)
用去离子水溶解后,配置成浓度约为100μM的母液。使用nanodrop对模板定量,并用去离子水将模板稀释成50μM、5μM、500nM、50nM浓度梯度。
2.2引物及信号DNA设计
检测使用的gDNA序列如下所示:
gDNA-1:B-CAAACTATTAACTGGCGsAACTACTTACTCT(SEQ ID NO:9)
其中,s代表硫修饰,B代表生物素(Biotin)基团。用去离子水溶解后,配置成浓度为100μM的母液。
信号DNA(signal-DNA)中包含了BclI内切酶的识别位点,由两条引物退火形成,序列如下:
Signal DNA-F:FAM-GCTGATCATG-Q(SEQ ID NO:10)
Signal DNA-R:CATGATCAGC(SEQ ID NO:11)
其中,下划线序列代表BclI识别序列,FAM代表荧光基团,Q代表淬灭基团。当荧光基团与淬灭基团相靠近时,没有荧光信号的产生。若signal-DNA在SBDgsu-BclI作用下被切断,荧光基团和淬灭基团彼此远离,则荧光基团释放出荧光信号,可被检测。
2.3检测方法
上述DNA中,T1可以与gDNA-1在碱基互补配对的作用下形成双链硫修饰DNA,为本实施例的实验组T1/g1,T2与gDNA-1无法形成双链DNA,为本实施例的对照组T2/g1。在去离子水中加入终浓度为100mM的NaCl、5μM的gDNA-1,再分别加入5μM、500nM、50nM、5nM的T1或T2,95℃条件下处理5分钟,随后缓慢降至室温。
取1μL T1/g1或T2/g1,在1mL结合缓冲液中与10μL链霉亲和素磁珠(10mg/mL,Purimag)、2pmol SBDgsu-BclI融合蛋白混合30分钟。结合缓冲液为:20mM Tris-HCl pH8.0,1M NaCl,1mM EDTA,0.02%Triton X-100。
使用磁力架分离磁珠与液体,向磁珠中加入85μL反应缓冲液(50mM Tris-HCl pH7.5、10mM MgCl2、100mM NaCl、0.02%Triton X-100)以及终浓度640nM的signal-DNA,随后转移至96孔板中,在50℃测量10分钟内体系荧光值变化。对荧光值进行线性回归,再对斜率进行显著性分析。
上述检测过程的示意图如图5所示。
2.4检测结果
如图6所示,在不使用扩增时,基于SBDgsu-BclI的检测系统所能达到的检测极限为50nM模板DNA。
实施例3
NEAR扩增技术联合SBDgsu-BclI融合蛋白检测系统用于检测靶标DNA
3.1靶标DNA的准备
靶标DNA序列如下所示:
Target F1:
ATCCGCCTCCATCCAGTGAGTCCGGCGTTGCCGGGAAGCTAGAGTAAGTAGAACGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGC(SEQ ID NO:12)
Target R1:
GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGTTCTACTTACTCTAGCTTCCCGGCAACGCCGGACTCACTGGATGGAGGCGGAT(SEQ ID NO:13)
用去离子水溶解后,配置成浓度为100μM的母液。在去离子水中加入终浓度为10nM的Target F1、Target R1以及终浓度为100mM的NaCl。95℃条件下处理5分钟,随后缓慢降至室温形成双链DNA模板。使用nanodrop对单链DNA模板Target F1和双链DNA模板定量,并用去离子水将模板稀释成101、102、103、104、105、106、107、108、109、1010、1011浓度梯度。
3.2硫修饰向导DNA(gDNA-2)、扩增引物以及信号DNA的设计
根据靶标序列,设计与之互补配对的带有硫修饰的gDNA,序列如下:
gDNA-2:B-CAAACTATTAACTGGCGsTTCTACTTACTCT(SEQ ID NO:14)
其中,s代表硫修饰位点,B代表生物素基团。用去离子水溶解后,配置成浓度为100μM的母液。
根据目标序列,设计NEAR扩增引物NF1、NR1和保险引物BF1、BR1,序列如下:
NF1:GCTTCTTACTGAGTCCGGCGTTGCCGGGAAGCT(SEQ ID NO:15)
NR1:GCCGTGTATAGAGTCCGTACAAACTATTAACTGG(SEQ ID NO:16)
BF1:TCCGCCTCCATCC(SEQ ID NO:17)
BR1:GCAATGGCAACA(SEQ ID NO:18)
信号DNA的设计及预处理与实施例2一致。
3.3检测方法
首先,在25μL NEAR扩增混合反应液(0.75U Nt.Bst NBI、1.2U Bst 3.0DNA聚合酶、625μM dNTP、3mM MgSO4、10mM(NH4)2SO4、100mM KCl、50mM NaCl、40mM Tris-HCl pH8.4、0.1%Triton X-100)加入终浓度为800nM NF1、100nM NR1、100nM BF1、100nM BR1、5pmol gDNA-2以及不同浓度的单链DNA模板Target F1或双链DNA模板。混合样品在56℃扩增30分钟,随后缓慢降温至室温。
在1mL结合缓冲液中,将样品与10μL链霉亲和素磁珠(10mg/mL,Purimag)、2pmolSBDgsu-BclI融合蛋白混合15分钟。结合缓冲液为:20mM Tris-HCl pH8.0,1M NaCl,1mMEDTA,0.02%Triton X-100。
使用磁力架分离磁珠与液体,洗杂后向磁珠中加入85μL反应缓冲液(50mM Tris-HCl pH 7.5、10mM MgCl2、100mM NaCl、0.02%Triton X-100)以及终浓度640nM的signal-DNA,随后转移至96孔板中,在50℃测量10分钟内体系荧光值变化。对荧光值进行线性回归,再对斜率进行显著性分析。
3.4检测结果
如图7和图8所示,与等温扩增NEAR结合,使用SBDgsu-BclI检测ssDNA和dsDNA靶标所能达到的检测极限均为1000拷贝/微升。
实施例4
NEAR扩增技术联合SBDgsu-BclI融合蛋白检测系统用于白色念珠菌基因组DNA
4.1白色念珠菌基因组DNA的准备
利用酵母基因组DNA提取试剂盒(天根)从白色念珠菌SC5314中提取基因组DNA,提取后,在0.8%琼脂糖胶中进行电泳检测,电泳条件为120V,30分钟。电泳结束后,琼脂糖胶用EB核酸染料显色。检测成功的白色念珠菌基因组DNA为实验组。利用类似的方法制备的幽门螺杆菌基因组DNA为阴性对照组。
4.2硫修饰向导DNA(gDNA-3)、扩增引物以及信号DNA的设计
根据白色念珠菌毒力基因SAP2的DNA序列,设计互补配对的带有硫修饰的gDNA以及对应的扩增引物,序列如下:
gDNA-3:B-CAGCATCTGGAGsAATTAAGATAAAGTGAAT(SEQ ID NO:19)
NF2:AAAAAACAAGGAGTCATTGCTAAGAATGCTT(SEQ ID NO:20)
NR2:GCCGTGTATAGAGTCCGTAGTGGCAGCATCTGGAG(SEQ ID NO:21)
BF2:GTCCCTGTCACTT(SEQ ID NO:22)
BR2:CCGAAAATTATTTGT(SEQ ID NO:23)
其中,s代表硫修饰位点,B代表生物素基团。用去离子水溶解后,配置成浓度为100μM的母液。
信号DNA的设计及预处理与实施例2一致。
4.3检测方法
首先,在25μL NEAR扩增混合反应液(0.75U Nt.Bst NBI、1.2U Bst 3.0DNA聚合酶、625μM dNTP、3mM MgSO4、10mM(NH4)2SO4、100mM KCl、50mM NaCl、40mM Tris-HCl pH8.4、0.1%Triton X-100)中加入终浓度为800nM NF2、100nM NR2、100nM BF2、100nM BR2、5pmol gDNA-3以及白色念珠菌或幽门螺杆菌基因组DNA。混合样品在56℃扩增30分钟,随后缓慢降温至室温。
在1mL结合缓冲液中,将样品与10μL链霉亲和素磁珠(10mg/mL,Purimag)、2pmolSBDgsu-BclI融合蛋白混合15分钟。结合缓冲液为:20mM Tris-HCl pH 8.0,1M NaCl,1mMEDTA,0.02%Triton X-100。
使用磁力架分离磁珠与液体,洗杂后再向磁珠中加入85μL反应缓冲液(50mMTris-HCl pH 7.5、10mM MgCl2、100mM NaCl、0.02%Triton X-100)以及终浓度为640nM的signal-DNA,随后转移至96孔板中,在50℃测量10分钟内体系荧光值变化。对荧光值进行线性回归,再对斜率进行显著性分析。
4.4检测结果
如图9所示,NEAR扩增技术联合SBDgsu-BclI检测系统可以特异性检测白色念珠菌基因组DNA。
实施例5
SBDgsu-FlucN和SBDgsu-FlucC融合蛋白纯化
5.1SBDgsu-FlucN和SBDgsu-FlucC融合蛋白表达载体的构建
以含SBDgsu表达质粒和Fluc表达质粒作为模板,使用扩增引物如下:
SBDgsu-FC:
AAAGGTGGCAAGATCGCGGTGGGCGGCGGCGGCAGCGGAGGAGGAGGAAGTACCAGTCTGACCCCAAGTCTG(SEQ ID No:24)
SBDgsu-FN:tgtattttcagggcgaattcACCAGTCTGACCCCAAGTCTGA(SEQ ID No:25)
SBDgsu-RC:acggagctcgaattcggatccTCACAGATGGGCTTGTTCTTCCAG(SEQ ID No:26)
SBDgsu-RN:
CTTAATGTTTTTCGCATCTTCACTTCCTCCTCCTCCGCTGCCGCCGCCGCCCAGATGGGCTTGTTCTTCCAG(SEQ ID No:27)
FlucN-F:
CTGGAAGAACAAGCCCATCTGGGCGGCGGCGGCAGCGGAGGAGGAGGAAGTGAAGATGCGAAAAACATTAAG(SEQ ID No:28)
FlucN-R:cctgcaggtcgactctagaggatccACCGTCTTTATCAATCAGCG(SEQ ID No:29)
FlucC-F:tgccacgaggaagccatatgAGCGGTTACGTGAACAACCC(SEQ ID No:30)
FlucC-R:
CAGACTTGGGGTCAGACTGGTACTTCCTCCTCCTCCGCTGCCGCCGCCGCCCACCGCGATCTTGCCACCTTT(SEQ ID No:31)
引物中小写字母为与载体匹配序列,大写字母为与融合蛋白匹配序列。PCR完成后利用同源重组试剂盒(诺唯赞)将PCR产物构建至表达载体pMAL和pET28a上,并测序验证。SBDgsu-FlucN、SBDgsu-FlucC融合蛋白氨基酸序列分别如SEQ ID NO:32和SEQ ID NO:33所示。
SBDgsu-FlucN氨基酸序列:
MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISENLYFQGEFTSLTPSLKLFSSLSRAPGAVWTEATRRKAPHKPLLLLAVLDLVHRGVITSPFIAVSGDLVELNELFNLYWRRVVPLGLTSSIAFPFSRLSREPFWELVPQPGIAITDAVINNTSSVSYLRKYALGAKLDDGLFRVMQSGEGREALREALLLSCFSADAAALLREQSVINREAFDYSRVLEEQAHLGGGGSGGGGSEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGGSSRVDLQAKLHHHHHH(SEQ ID NO:32)
SBDgsu-FlucC氨基酸序列:
MGSSHHHHHHSSGLVPRGSHMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVGGGGSGGGGSTSLTPSLKLFSSLSRAPGAVWTEATRRKAPHKPLLLLAVLDLVHRGVITSPFIAVSGDLVELNELFNLYWRRVVPLGLTSSIAFPFSRLSREPFWELVPQPGIAITDAVINNTSSVSYLRKYALGAKLDDGLFRVMQSGEGREALREALLLSCFSADAAALLREQSVINREAFDYSRVLEEQAHL(SEQID NO:33)
5.2SBDgsu-FlucN、SBDgsu-FlucC融合蛋白的诱导表达及纯化
首先,将SBDgsu-FlucN、SBDgsu-FlucC融合蛋白表达载体转化进E.coli BL21(DE3)宿主,在LB液体培养基中培养,培养条件为37℃、220rpm,培养至OD600=0.6-0.8时添加IPTG至终浓度为0.2mM,在16℃、220rpm培养条件下培养20小时培养结束后,收集菌体裂解物的上清液,依次利用镍离子亲和层析柱(GE)、肝素亲和层析柱(GE)纯化融合蛋白,并用Bradford蛋白浓度测定试剂盒进行定量,保存到-80℃冰箱中。
实施例6
用SBDgsu-FlucN和SBDgsu-FlucC融合蛋白检测体系中的单链DNA
6.1靶标单链DNA制备
靶标单链DNA序列选取白色念珠菌毒力基因SAP2的一段序列,如下所示:
Target F2:CTTATATAAAGTACCTTGAGATGAAGATCCATCACCATAACCAATTTTGA(SEQ IDNO:34)
用去离子水溶解后,配置成浓度约为100μM的母液。
6.2硫修饰向导DNA(gDNA-4和gDNA-5)的设计
gDNA-4:ATTGGTTATGsGTGAT(SEQ ID NO:35)
gDNA-5:TCAAGsGTACTTTATA(SEQ ID NO:36)
其中,s代表硫修饰位点。用去离子水溶解后,配置成浓度为100μM的母液。
6.3检测方法
在25μL体系中加入10pmol的gDNA-4和gDNA-5,然后添加不同浓度的模板,进行退火。退火后在体系中加入终浓度为100nM的SBDgsu-FlucN和SBDgsu-FlucC融合蛋白,室温下放置10min,然后加入萤光素酶底物。检测实时荧光。
上述检测过程的示意图如图10所示。
6.4检测结果
结果如图11所示。在没有模板添加的情况下,几乎没有荧光信号产生。随着模板添加浓度的增加,荧光信号逐渐升高。在本实施例中,最低检测极限为5nM。
实施例7
RPA扩增技术联合SBDgsu-Fluc检测系统用于检测靶标DNA
7.1靶标DNA的准备
靶标DNA的准备与实施例6一致。
7.2硫修饰向导DNA以及扩增引物的设计
硫修饰向导DNA设计与实施例6一致。
根据靶标SAP2序列,设计RPA扩增引物,序列如下:
RF1:ATCAACATCAGCTAAAACTTGATTTTTAAT(SEQ ID NO:37)
RR1:GATCCAAGTGGTTCATCAGCTTCACAAGAT(SEQ ID NO:38)
7.3检测方法
首先,使用RPA试剂盒(君诺德)在37℃通过不对称RPA扩增30min放大靶标DNA含量,加入10pmol的gDNA-4和gDNA-5,进行退火。退火后在体系中加入终浓度为100nM的SBDgsu-FlucN和SBDgsu-FlucC融合蛋白,室温下放置10min,然后加入萤光素酶底物。检测实时荧光。
7.4检测结果
如图12所示,与RPA结合,使用SBDgsu-FlucN、SBDgsu-FlucC检测靶标DNA的检测极限为1000拷贝/微升。
实施例8
RPA扩增技术联合SBDgsu-Fluc检测系统用于检测白色念珠菌基因组DNA
8.1白色念珠菌基因组DNA的准备
白色念珠菌基因组DNA的准备与实施例4一致。利用类似的方法制备的幽门螺杆菌基因组DNA为阴性对照组。
8.2硫修饰向导DNA以及扩增引物的设计
硫修饰向导DNA设计与实施例6一致。
扩增引物的设计与实施例7一致。
8.3检测方法
首先,使用RPA试剂盒(君诺德)在37℃扩增30min放大白色念珠菌基因组DNA含量,加入10pmol的gDNA-4和gDNA-5,进行退火。退火后在体系中加入终浓度为100nM的SBDgsu-FlucN和SBDgsu-FlucC融合蛋白,室温下放置10min,然后加入萤光素酶底物。检测实时荧光。
8.4检测结果
如图13所示,RPA扩增技术联合SBDgsu-Fluc检测系统可以特异性检测白色念珠菌基因组DNA。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 上海交通大学
杭州比格飞序生物科技有限公司
<120> 基于硫修饰核酸及硫修饰核酸识别蛋白的核酸检测方法
<130> P2022-0414
<160> 38
<170> PatentIn version 3.5
<210> 1
<211> 867
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> SBDgsu-BclI融合蛋白核苷酸序列
<400> 1
ggtaccatgc agccgaaccc gaaatttatt aacaaaagca gcgcgttttg ggcgtatgcg 60
aaactgctga gcgaacagct gggctatagc aaagatggcg tggtgattag ctatagcgaa 120
gcgcaagcgc gcgcgaaact gaaaaaactg ggcattaacg tgaaagaagg catttttaaa 180
gatgtgctgc gctatctgaa atatcgcgcg gaactgctga acaaacacaa agattatctg 240
atggatgtgg aagaagcgcg caaatatttt caagtggcgc tgaaacagca tcagcagaac 300
aactacacct gcaaactgcc gttaaacaag caaaaaaatg aaaaaaaaga ttatgcgtat 360
tttacctgca ttattaacat tattgcggaa accgaactgc gctattttgc gaacaataac 420
ggcctggtgt atggcaaaga tatttatttt gatgataacc cgatgaacct gagctatatt 480
ctgaacttta accgcgaact ggaaggcatt atgagccgcc gctttgatgg cgcgtttccg 540
agcaccgtga acccgattct gatttgggaa attaaagaat attactatac cacgaccttt 600
ggcagccgca ttgcggatgg cgtgtatgaa acgcagctgg atggctatga aattaaaacc 660
attcgcgaag aaaccaacaa aaacattcag catatttatt ttattgatga ttataacacc 720
tggtggaaca tgggcaaaag ctatctgtgc cgcattattg atatgctgca tatgggcctg 780
gtggatgaag tgattatggg caaagaagtg tttgaacgct ggccgcagat tctgcgcgcg 840
gtgctgaatc agtattataa agaattc 867
<210> 2
<211> 72
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 2
ctggaagaac aagcccatct gggcggcggc ggcagcggcg gcggcggcag catgcagccg 60
aacccgaaat tt 72
<210> 3
<211> 46
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 3
cagtggtggt ggtggtggtg ctcgagtcat ttataatact gattca 46
<210> 4
<211> 46
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 4
agcaaatggg tcgcggatcc gaattcatga ccagtctgac cccaag 46
<210> 5
<211> 72
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 5
aaatttcggg ttcggctgca tgctgccgcc gccgccgctg ccgccgccgc ccagatgggc 60
ttgttcttcc ag 72
<210> 6
<211> 517
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> SBDgsu-BclI融合蛋白氨基酸序列
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Met Thr Ser Leu Thr Pro Ser Leu Lys Leu Phe Ser
35 40 45
Ser Leu Ser Arg Ala Pro Gly Ala Val Trp Thr Glu Ala Thr Arg Arg
50 55 60
Lys Ala Pro His Lys Pro Leu Leu Leu Leu Ala Val Leu Asp Leu Val
65 70 75 80
His Arg Gly Val Ile Thr Ser Pro Phe Ile Ala Val Ser Gly Asp Leu
85 90 95
Val Glu Leu Asn Glu Leu Phe Asn Leu Tyr Trp Arg Arg Val Val Pro
100 105 110
Leu Gly Leu Thr Ser Ser Ile Ala Phe Pro Phe Ser Arg Leu Ser Arg
115 120 125
Glu Pro Phe Trp Glu Leu Val Pro Gln Pro Gly Ile Ala Ile Thr Asp
130 135 140
Ala Val Ile Asn Asn Thr Ser Ser Val Ser Tyr Leu Arg Lys Tyr Ala
145 150 155 160
Leu Gly Ala Lys Leu Asp Asp Gly Leu Phe Arg Val Met Gln Ser Gly
165 170 175
Glu Gly Arg Glu Ala Leu Arg Glu Ala Leu Leu Leu Ser Cys Phe Ser
180 185 190
Ala Asp Ala Ala Ala Leu Leu Arg Glu Gln Ser Val Ile Asn Arg Glu
195 200 205
Ala Phe Asp Tyr Ser Arg Val Leu Glu Glu Gln Ala His Leu Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Met Gln Pro Asn Pro Lys Phe Ile
225 230 235 240
Asn Lys Ser Ser Ala Phe Trp Ala Tyr Ala Lys Leu Leu Ser Glu Gln
245 250 255
Leu Gly Tyr Ser Lys Asp Gly Val Val Ile Ser Tyr Ser Glu Ala Gln
260 265 270
Ala Arg Ala Lys Leu Lys Lys Leu Gly Ile Asn Val Lys Glu Gly Ile
275 280 285
Phe Lys Asp Val Leu Arg Tyr Leu Lys Tyr Arg Ala Glu Leu Leu Asn
290 295 300
Lys His Lys Asp Tyr Leu Met Asp Val Glu Glu Ala Arg Lys Tyr Phe
305 310 315 320
Gln Val Ala Leu Lys Gln His Gln Gln Asn Asn Tyr Thr Cys Lys Leu
325 330 335
Pro Leu Asn Lys Gln Lys Asn Glu Lys Lys Asp Tyr Ala Tyr Phe Thr
340 345 350
Cys Ile Ile Asn Ile Ile Ala Glu Thr Glu Leu Arg Tyr Phe Ala Asn
355 360 365
Asn Asn Gly Leu Val Tyr Gly Lys Asp Ile Tyr Phe Asp Asp Asn Pro
370 375 380
Met Asn Leu Ser Tyr Ile Leu Asn Phe Asn Arg Glu Leu Glu Gly Ile
385 390 395 400
Met Ser Arg Arg Phe Asp Gly Ala Phe Pro Ser Thr Val Asn Pro Ile
405 410 415
Leu Ile Trp Glu Ile Lys Glu Tyr Tyr Tyr Thr Thr Thr Phe Gly Ser
420 425 430
Arg Ile Ala Asp Gly Val Tyr Glu Thr Gln Leu Asp Gly Tyr Glu Ile
435 440 445
Lys Thr Ile Arg Glu Glu Thr Asn Lys Asn Ile Gln His Ile Tyr Phe
450 455 460
Ile Asp Asp Tyr Asn Thr Trp Trp Asn Met Gly Lys Ser Tyr Leu Cys
465 470 475 480
Arg Ile Ile Asp Met Leu His Met Gly Leu Val Asp Glu Val Ile Met
485 490 495
Gly Lys Glu Val Phe Glu Arg Trp Pro Gln Ile Leu Arg Ala Val Leu
500 505 510
Asn Gln Tyr Tyr Lys
515
<210> 7
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 单链DNA序列T1
<400> 7
agagtaagta gttcgccagt taatagtttg 30
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 单链DNA序列T2
<400> 8
actgagatac ctacagcgtg agctatgaga 30
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> gDNA-1
<400> 9
caaactatta actggcgaac tacttactct 30
<210> 10
<211> 10
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> Signal DNA-F
<400> 10
gctgatcatg 10
<210> 11
<211> 10
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> Signal DNA-R
<400> 11
catgatcagc 10
<210> 12
<211> 90
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 靶标DNA F1
<400> 12
atccgcctcc atccagtgag tccggcgttg ccgggaagct agagtaagta gaacgccagt 60
taatagtttg cgcaacgttg ttgccattgc 90
<210> 13
<211> 90
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 靶标DNA R1
<400> 13
gcaatggcaa caacgttgcg caaactatta actggcgttc tacttactct agcttcccgg 60
caacgccgga ctcactggat ggaggcggat 90
<210> 14
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> gDNA-2
<400> 14
caaactatta actggcgttc tacttactct 30
<210> 15
<211> 33
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 15
gcttcttact gagtccggcg ttgccgggaa gct 33
<210> 16
<211> 34
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 16
gccgtgtata gagtccgtac aaactattaa ctgg 34
<210> 17
<211> 13
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 17
tccgcctcca tcc 13
<210> 18
<211> 12
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 18
gcaatggcaa ca 12
<210> 19
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> gDNA-3
<400> 19
cagcatctgg agaattaaga taaagtgaat 30
<210> 20
<211> 31
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 20
aaaaaacaag gagtcattgc taagaatgct t 31
<210> 21
<211> 35
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 21
gccgtgtata gagtccgtag tggcagcatc tggag 35
<210> 22
<211> 13
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 22
gtccctgtca ctt 13
<210> 23
<211> 15
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 23
ccgaaaatta tttgt 15
<210> 24
<211> 72
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 24
aaaggtggca agatcgcggt gggcggcggc ggcagcggag gaggaggaag taccagtctg 60
accccaagtc tg 72
<210> 25
<211> 42
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 25
tgtattttca gggcgaattc accagtctga ccccaagtct ga 42
<210> 26
<211> 45
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 26
acggagctcg aattcggatc ctcacagatg ggcttgttct tccag 45
<210> 27
<211> 72
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 27
cttaatgttt ttcgcatctt cacttcctcc tcctccgctg ccgccgccgc ccagatgggc 60
ttgttcttcc ag 72
<210> 28
<211> 72
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 28
ctggaagaac aagcccatct gggcggcggc ggcagcggag gaggaggaag tgaagatgcg 60
aaaaacatta ag 72
<210> 29
<211> 45
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 29
cctgcaggtc gactctagag gatccaccgt ctttatcaat cagcg 45
<210> 30
<211> 40
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 30
tgccacgagg aagccatatg agcggttacg tgaacaaccc 40
<210> 31
<211> 72
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 31
cagacttggg gtcagactgg tacttcctcc tcctccgctg ccgccgccgc ccaccgcgat 60
cttgccacct tt 72
<210> 32
<211> 1025
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> SBDgsu-FlucN氨基酸序列
<400> 32
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn
355 360 365
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile
370 375 380
Glu Gly Arg Ile Ser Glu Asn Leu Tyr Phe Gln Gly Glu Phe Thr Ser
385 390 395 400
Leu Thr Pro Ser Leu Lys Leu Phe Ser Ser Leu Ser Arg Ala Pro Gly
405 410 415
Ala Val Trp Thr Glu Ala Thr Arg Arg Lys Ala Pro His Lys Pro Leu
420 425 430
Leu Leu Leu Ala Val Leu Asp Leu Val His Arg Gly Val Ile Thr Ser
435 440 445
Pro Phe Ile Ala Val Ser Gly Asp Leu Val Glu Leu Asn Glu Leu Phe
450 455 460
Asn Leu Tyr Trp Arg Arg Val Val Pro Leu Gly Leu Thr Ser Ser Ile
465 470 475 480
Ala Phe Pro Phe Ser Arg Leu Ser Arg Glu Pro Phe Trp Glu Leu Val
485 490 495
Pro Gln Pro Gly Ile Ala Ile Thr Asp Ala Val Ile Asn Asn Thr Ser
500 505 510
Ser Val Ser Tyr Leu Arg Lys Tyr Ala Leu Gly Ala Lys Leu Asp Asp
515 520 525
Gly Leu Phe Arg Val Met Gln Ser Gly Glu Gly Arg Glu Ala Leu Arg
530 535 540
Glu Ala Leu Leu Leu Ser Cys Phe Ser Ala Asp Ala Ala Ala Leu Leu
545 550 555 560
Arg Glu Gln Ser Val Ile Asn Arg Glu Ala Phe Asp Tyr Ser Arg Val
565 570 575
Leu Glu Glu Gln Ala His Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly
580 585 590
Ser Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro
595 600 605
Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg
610 615 620
Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu
625 630 635 640
Val Asp Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala
645 650 655
Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val
660 665 670
Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu
675 680 685
Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg
690 695 700
Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val
705 710 715 720
Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro
725 730 735
Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly
740 745 750
Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe
755 760 765
Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile
770 775 780
Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val
785 790 795 800
Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp
805 810 815
Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val
820 825 830
Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu
835 840 845
Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu
850 855 860
Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val
865 870 875 880
Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr
885 890 895
Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser
900 905 910
Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile
915 920 925
Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr
930 935 940
Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe
945 950 955 960
Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val
965 970 975
Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly
980 985 990
Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly
995 1000 1005
Gly Ser Ser Arg Val Asp Leu Gln Ala Lys Leu His His His His
1010 1015 1020
His His
1025
<210> 33
<211> 368
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> SBDgsu-FlucC氨基酸序列
<400> 33
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Gly Tyr Val Asn Asn Pro Glu Ala Thr Asn
20 25 30
Ala Leu Ile Asp Lys Asp Gly Trp Leu His Ser Gly Asp Ile Ala Tyr
35 40 45
Trp Asp Glu Asp Glu His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu
50 55 60
Ile Lys Tyr Lys Gly Tyr Gln Val Ala Pro Ala Glu Leu Glu Ser Ile
65 70 75 80
Leu Leu Gln His Pro Asn Ile Phe Asp Ala Gly Val Ala Gly Leu Pro
85 90 95
Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala Val Val Val Leu Glu His
100 105 110
Gly Lys Thr Met Thr Glu Lys Glu Ile Val Asp Tyr Val Ala Ser Gln
115 120 125
Val Thr Thr Ala Lys Lys Leu Arg Gly Gly Val Val Phe Val Asp Glu
130 135 140
Val Pro Lys Gly Leu Thr Gly Lys Leu Asp Ala Arg Lys Ile Arg Glu
145 150 155 160
Ile Leu Ile Lys Ala Lys Lys Gly Gly Lys Ile Ala Val Gly Gly Gly
165 170 175
Gly Ser Gly Gly Gly Gly Ser Thr Ser Leu Thr Pro Ser Leu Lys Leu
180 185 190
Phe Ser Ser Leu Ser Arg Ala Pro Gly Ala Val Trp Thr Glu Ala Thr
195 200 205
Arg Arg Lys Ala Pro His Lys Pro Leu Leu Leu Leu Ala Val Leu Asp
210 215 220
Leu Val His Arg Gly Val Ile Thr Ser Pro Phe Ile Ala Val Ser Gly
225 230 235 240
Asp Leu Val Glu Leu Asn Glu Leu Phe Asn Leu Tyr Trp Arg Arg Val
245 250 255
Val Pro Leu Gly Leu Thr Ser Ser Ile Ala Phe Pro Phe Ser Arg Leu
260 265 270
Ser Arg Glu Pro Phe Trp Glu Leu Val Pro Gln Pro Gly Ile Ala Ile
275 280 285
Thr Asp Ala Val Ile Asn Asn Thr Ser Ser Val Ser Tyr Leu Arg Lys
290 295 300
Tyr Ala Leu Gly Ala Lys Leu Asp Asp Gly Leu Phe Arg Val Met Gln
305 310 315 320
Ser Gly Glu Gly Arg Glu Ala Leu Arg Glu Ala Leu Leu Leu Ser Cys
325 330 335
Phe Ser Ala Asp Ala Ala Ala Leu Leu Arg Glu Gln Ser Val Ile Asn
340 345 350
Arg Glu Ala Phe Asp Tyr Ser Arg Val Leu Glu Glu Gln Ala His Leu
355 360 365
<210> 34
<211> 50
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 靶标单链DNA序列
<400> 34
cttatataaa gtaccttgag atgaagatcc atcaccataa ccaattttga 50
<210> 35
<211> 15
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> gDNA-4
<400> 35
attggttatg gtgat 15
<210> 36
<211> 15
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> gDNA-5
<400> 36
tcaaggtact ttata 15
<210> 37
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 37
atcaacatca gctaaaactt gatttttaat 30
<210> 38
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<220>
<223> 引物
<400> 38
gatccaagtg gttcatcagc ttcacaagat 30
Claims (10)
1.一种核酸检测效应器,其特征在于,所述核酸检测效应器包括靶向元件和报告元件,其中所述靶向元件与所述报告元件形成一融合蛋白,其中所述靶向元件包含硫修饰核酸识别蛋白。
2.如权利要求1所述的核酸检测效应器,其特征在于,所述核酸检测效应器在硫修饰向导核酸的引导下,可结合于待检测核酸的预定区域,并通过报告元件与分子信标接触从而产生报告信号。
3.如权利要求2所述的核酸检测效应器,其特征在于,所述的硫修饰向导核酸包含磷硫酰化修饰的gDNA或gRNA。
4.如权利要求1所述的核酸检测效应器,其特征在于,所述硫修饰核酸识别蛋白为硫结合结构域蛋白(SBD),其特异性识别被硫修饰的核酸;
所述的硫修饰的核酸指核酸中磷酸基团中的一个OH(O或O-)被SH(或S-)所取代和或一个=0被=S替换。
5.如权利要求4所述的核酸检测效应器,其特征在于,所述硫结合结构域蛋白(SBD)包括:SBDgsu、SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu或SBD同源蛋白。
6.如权利要求1所述的核酸检测效应器,其特征在于,所述报告元件选自下组:限制性核酸内切酶、荧光双分子互补报告系统、或其组合。
7.如权利要求2所述的核酸检测效应器,其特征在于,所述分子信标选自下组:双链DNA、荧光素酶、或其组合。
8.如权利要求1所述的核酸检测效应器,其特征在于,所述融合蛋白的结构如下式I或I'或I”所示:
A-L-B(I)
B-L-A(I')
A-L-B-L-A(I”)
式中,
A为靶向元件SBD;
B为报告元件;
L各自独立地为无或连接肽;
各“-”独立地为化学键。
9.一种核酸检测的方法,其特征在于,包含步骤:
(i)在硫修饰向导核酸的存在下,使得如权利要求1所述的核酸检测效应器与待检测的核酸形成“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物;和
(ii)使分子信标与所述“核酸检测效应器-硫修饰向导核酸-待检测核酸”复合物接触并检测报告信号。
10.一种用于核酸检测的反应体系,其特征在于,所述的反应体系包括:
(a)如权利要求1所述的核酸检测效应器;
(b)硫修饰向导核酸,所述硫修饰向导核酸引导所述核酸检测效应器特异性结合于待检测核酸的预定区域;和
(c)分子信标,所述分子信标与所述核酸检测效应器接触后从而产生报告信号。
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