CN116479089A - 一种纳米核酸探针及其制备方法和应用 - Google Patents
一种纳米核酸探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了本发明提供了一种可有效、快速、实时进行多种肿瘤标志物筛查的纳米核酸探针,由DNA四面体、检测单链DNA和辅助单链DNA组成;所述DNA四面体由4条单链DNA经碱基互补配对形成,检测单链DNA带有荧光基团和荧光淬灭基团,且检测单链DNA和辅助单链DNA分别与DNA四面体的两条不同单链通过碱基互补配对连接。本发明纳米核酸探针检测灵敏度高,生物安全性好,可自身自转运进入细胞,无需其他载体,适用于对活体细胞胞内靶点进行检测,可用于动物活体成像,实现对肿瘤的实时荧光标记。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种纳米核酸探针及其制备方法和应用。
背景技术
诊断肿瘤的传统方法有病理组织活检,核磁共振成像,电子计算机断层扫描等技术,但是这些技术都很难在肿瘤早期对其进行有效的检测与筛查。随着对肿瘤疾病研究的不断深入,肿瘤标志物检测在肿瘤早期检测和筛查中展现出重要意义。肿瘤标志物主要包括蛋白质类、糖类、酶类、激素类物质和一些分化紊乱的基因片段等,是由肿瘤细胞本身合成、释放,或是机体对肿瘤细胞反应而产生或升高的一类物质,存在于血液、细胞、组织或体液中,反映肿瘤的存在和生长,通过化学、免疫学以及基因组学等方法测定肿瘤标志物,对肿瘤的诊断、疗效和复发的监测、预后的判断具有一定的价值。
核仁素是一种多功能蛋白,主要功能是参与核糖体的生物形成,同时可参与rRNA的加工和mRNA的稳定等多种过程。近年研究发现其在肿瘤发生发展中也发挥着重要作用。核仁素在多种增生活跃的癌症细胞膜上以及细胞质中过表达,恶性肿瘤中高表达的核仁素直接或间接参与信号转导,机制各异,影响肿瘤细胞的存活、增殖、转移并促进肿瘤的进展,可作为肿瘤的生物学标志物。研究发现,核仁素适配子AS1411对核仁素有特异性亲和的能力,AS1441结合核仁素并抑制核仁素表达,诱导肿瘤细胞凋亡。因此,以anti-AS1411为靶DNA进行核仁素检测是肿瘤筛查的一种可行策略。
现有的肿瘤标志物检测手段主要有定量荧光聚合酶链式反应技术、酶联免疫技术(ELISA)等,但这些手段所针对的检测目标单一且检测时间长,前者仅用于核酸检测,用时需2小时以上,后者仅用于抗原抗体肿瘤标志物检测,用时超过4小时,且无法用于活细胞、活体组织中实现实时直观的肿瘤检测和标记。
因此,为了提高肿瘤检测筛查的效率和准确率,提供一种新的检测手段,实现肿瘤的实时检测标记,为临床决策提供可靠的依据,具有非常重要的意义。
发明内容
本发明的目的在于提供一种可有效、快速、实时进行多种肿瘤标志物筛查的纳米核酸探针。
本发明提供了一种纳米核酸探针,它由如下组分组成:
DNA四面体、检测单链DNA、辅助单链DNA;
所述DNA四面体由4条单链DNA经碱基互补配对形成;
所述检测单链DNA带有荧光基团和荧光淬灭基团;
所述检测单链DNA和辅助单链DNA分别与DNA四面体的两条不同单链通过碱基互补配对连接;
进一步地,上述荧光基团为FAM、Cy5、Cy3、ROX、TAMRA中的至少一种,所述淬灭基团为BHQ1、BHQ2、Dabcyl中的至少一种;优选地,所述荧光基团为Cy5,所述淬灭基团为BHQ2。
进一步地,上述检测单链DNA形成发卡结构,辅助单链DNA形成发卡结构。
更进一步地,上述检测单链DNA的荧光基团位于从5’端起第11位和第12位核苷酸之间,所述荧光淬灭基团位于从5’端起第35位和第36位核苷酸之间;
所述辅助单链DNA从5’端起第1~11位核苷酸与检测单链DNA从5’端起第1~11位核苷酸反向互补。
更进一步地,上述检测单链DNA从3’端起第1~11位核苷酸与所述DNA四面体的一条单链从3’端起第1~11位核苷酸反向互补配对连接;所述辅助单链DNA从3’端起第1~11位核苷酸与所述DNA四面体的另一条单链从3’端起第1~11位核苷酸反向互补配对连接。
更进一步地,上述检测单链DNA从5’端至3’端的序列如SEQ ID NO.1所示;所述辅助单链DNA从5’端至3’端的序列如SEQ ID NO.2所示。
更进一步地,上述DNA四面体是4条单链DNA的序列分别一对一地选自SEQ ID NO.3~6的所述序列。
更进一步地,上述的纳米核酸探针由如下方法制备而成:
分别将检测单链DNA、辅助单链DNA、DNA四面体的四条单链DNA的混合物置于足以使DNA变性的温度下维持3~8min,再将温度降低到2~8℃维持20min以上,然后混合,在室温孵育30min以上。
更进一步地,它由如下方法制备而成:
分别将检测单链DNA、辅助单链DNA、DNA四面体的四条单链DNA的混合物置于95℃下维持10min,再将温度降低到4℃维持20min以上,然后混合,在室温孵育30min以上。
本发明还提供了上述纳米核酸探针的制备方法,包括如下步骤:
(1)将DNA四面体由4条单链DNA等量溶于TM缓冲液中得溶液a,同时将检测单链DNA溶于TM缓冲液中得溶液b,将辅助单链DNA溶于TM溶液中得溶液c;
(2)溶液a、溶液b、溶液c加热到足以使DNA变性的温度维持3~8min,然后降温到2~8℃维持20min以上;
(3)将溶液a、溶液b、溶液c混合,在室温孵育30min以上。
本发明还提供了上述的纳米核酸探针在基因和/或蛋白的实时荧光检测试剂中的应用。
进一步地,上述试剂是对肿瘤标志物进行检测和/或标记的试剂。
本发明还提供了上述的纳米核酸探针在活体成像试剂中的应用。
进一步地,上述试剂是在活细胞或活体组织中成像的试剂。
本发明还提供了一种核酸和/或蛋白的检测或对活细胞中的核酸和/或蛋白靶点进行标记方法,其特征在于,包括将上述的纳米核酸探针与样本在室温下共同孵育5~30min,检测荧光信号强度的步骤。
进一步地,上述样本为待测核酸和/或蛋白,或为活细胞或活体组织。
本发明的有益效果:本发明纳米核酸探针可对多种物质进行检测,包括肿瘤特征蛋白,肿瘤特征基因等;检测时间显著缩短,在数分钟内即可对检测目标进行检出,且可对活细胞和活体组织进行实时直观的荧光检测。
本发明纳米核酸探针与检测目标在室温下进行孵育时,可自发形成杂交链式反应,形成级联扩增,放大检测信号,检测浓度可低至0.1nM,灵敏度高。
本发明探针可自身自转运进入细胞,无需其他载体,且生物安全性好;因此,适用于对活体细胞胞内靶点进行检测,可用于动物活体成像,实现对肿瘤的实时荧光标记。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明纳米核酸探针结构和检测过程示意图。
图2为T-probe检测探针灵敏性结果验证。
图3为T-probe检测探针特异性结果验证。
图4为T-probe检测探针反应动力学结果验证。
图5为T-probe检测探针细胞标记结果。
图6为T-probe经肿瘤周围组织注射在小鼠体内标记结果。
图7为T-probe经尾静脉注射在小鼠体内标记结果。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
本发明实施例以靶向核仁素的检测探针为例,但需要特别说明的是,基于本发明的设计理念,可以针对不同的靶点设计不同的检测单链DNA和辅助单链DNA序列,与TDN结合,均可实现快速、灵敏度高的检测。因此,本领域技术人员在本发明基本技术设计思想的基础上,设计的其他检测探针也属于本发明的范围。
实施例1、本发明纳米核酸探针的制备
本实施例设计了一种靶向核仁素的纳米核酸探针,靶点DNA为anti-AS1411,序列为CCACCACCACCACAACCACCACCACC(SEQ ID NO.7)。
首先,将等量(1μM,1μL)的四条单链DNA(S1-a*、S2-b*、S3、S4)混合在96μLTM缓冲液中得到DNA四面体(TDN)溶液,同时,将两种发卡核酸单链H1(检测单链DNA)、H2(辅助单链DNA)分别在TM缓冲液中稀释至10μM。之后将TDN溶液和两种发卡核酸单链DNA溶液均加热至95℃,10分钟并快速淬火至4℃,20分钟。获得的TDN溶液及卡核酸单链DNA溶液在25℃下混合孵育30min后成功合成本发明纳米核酸探针(T-probe)溶液。制备获得的T-probe溶液在避光条件下于4℃保存以供后续使用。
TDN的四条单链DNA:S1-a*、S2-b*、S3、S4以及检测单链DNA:H1和辅助单链DNA:H2的序列如下:
H1(5’→3’):
GGTGGTGGTGGTGTGGTGGTGGTGGACGTACCCACCACCACCACA ATTTTTTTTTTTTTCCTCTACCACCTACATCAC(SEQ ID NO.1;荧光基团Cy5位于下划线标记的两个核苷酸之间,荧光淬灭基团位于波浪线标记的两个核苷酸之间;具体为:
GGTGGTGGTGG-Cy5-TGTGGTGGTGGTGGACGTACCCACCACCAC CACAA-BHQ2-TTTTTTTTTTTTTCCTCTACCACCTACATCAC);
H2(5’→3’):
CCACCACCACCACAACCACCACCACCGTTGTGGTGGTGGTGGGTA CGTTTTTTTTTTTCTTCACACCACACTCCATCTA(SEQ ID NO.2);
S1-a*(5’→3’):
ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGA ACATTCCTAAGTCTGAATTGTGATGTAGGTGGTAGAGGAA(SEQ ID NO.3);
S2-b*(5’→3’):
ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTC AGACTTAGGAATGTTCGTTTAGATGGAGTGTGGTGTGAAG(SEQ ID NO.4);
S3(5’→3’):
ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGG GAAGAGCATGCCCATCC(SEQID NO.5);
S4(5’→3’):
ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGA TGGGCATGCTCTTCCCG(SEQID NO.6)。
以下通过实验例证明本发明的有益效果。
实验例1、检测效能验证试验
1、灵敏性实验
将实施例1的T-probe与不同浓度的靶点DNA进行混合孵育(T-probe最终浓度为200nM,靶点DNA最终浓度为0,0.1nM,0.5nM,1nM,10nM,20nM,40nM,60nM,80nM,100nM),在25℃环境下孵育1小时,检测其荧光信号强度,判断探针的检测灵敏度。(靶点DNA为anti-AS1411,下同) 。
结果如图2所示,T-probe荧光信号强度随靶点DNA浓度呈对数型上升,并且可对最低浓度为0.1nM的靶DNA进行有效检出。
2、特异性实验
在原T-probe结构基础上,对检测序列结构进行调整,构建non-T-probe,使得该T-probe与原靶点DNA不再存在碱基配对关系。同时设计一段随机核酸序列,作为阴性靶点DNA,与原靶点进行对照。将探针与靶点DNA按照以下分组混合孵育:T-probe与靶点DNA;T-probe与随机序列;non-T-probe与靶点DNA;单独的裸T-probe。在25℃环境下孵育1小时,检测其荧光信号强度,判断探针的检测特异性。
结果如图3所示,对于无特异性碱基配对的T-probe及DNA序列,T-probe无法表现出荧光响应信号,说明本发明探针检测特异性高。
实验例2、反应动力学探究
将实施例1的T-probe与靶点DNA混合孵育(T-probe最终浓度为200nM,靶点DNA最终浓度为100nM)。每隔10分钟对样本荧光信号强度进行检测,观察T-probe的时间梯度荧光强度变化。同时,未经DNA四面体修饰的发卡结构也与响应靶点DNA共同孵育作为对照。
如图4所示,T-probe在前30分钟的荧光反应速度约为裸发卡结构的2-3倍左右,最终荧光强度约为裸发卡结构的2倍;说明本发明修饰了DNA四面体结构的探针检测速度快,检测信号强。
实验例3、细胞标记实验
如图5所示,采用实施例1的探针T-probe,可实现三种靶肿瘤细胞表面荧光信号标记,且标记范围与胞膜核仁素所在位置大致相同。而对于胞膜核仁素表达阴性的L929细胞则无明显荧光信号显示。
说明本发明探针能够有效实现细胞标记。
实验例4、动物体内标记效果
采用BALB/c裸鼠构筑小鼠荷瘤模型,并通过肿瘤周围原位注射以及经尾静脉注射两种给药途径注射实施例1的T-probe探针溶液,并观察小鼠体内荧光标记效果。
首先采用肿瘤周围原位注射方式进行给药,如图7所示,荷瘤小鼠肿瘤周围快速出现荧光信号响应。而对照组中无明显荧光信号显示,表现了T-probe良好的特异性。
之后采用经尾静脉注射方式进行给药,实验组小鼠肿瘤区域有显著荧光信号显示,但相较于肿瘤周围原位给药,经尾静脉注射给药方式荧光信号响应较慢。同样,对照组中无明显荧光信号显示,表现本发明T-probe具有较强的特异性及生物稳定性。
综上,本发明提供了一种可有效、快速、实时进行多种肿瘤标志物筛查的纳米核酸探针,检测灵敏度高,生物安全性好,可自身自转运进入细胞,无需其他载体,适用于对活体细胞胞内靶点进行检测,可用于动物活体成像,实现对肿瘤的实时荧光标记。
Claims (10)
1.一种纳米核酸探针,其特征在于,它由DNA四面体、检测单链DNA和辅助单链DNA组成;
所述DNA四面体由4条单链DNA经碱基互补配对形成;
所述检测单链DNA带有荧光基团和荧光淬灭基团;
所述检测单链DNA和辅助单链DNA分别与DNA四面体的两条不同单链通过碱基互补配对连接。
2.如权利要求1所述的纳米核酸探针,其特征在于,所述荧光基团为FAM、Cy5、Cy3、ROX、TAMRA中的至少一种,所述淬灭基团为BHQ1、BHQ2、Dabcyl中的至少一种;优选地,所述荧光基团为Cy5,所述淬灭基团为BHQ2。
3.如权利要求1所述的纳米核酸探针,其特征在于,所述检测单链DNA形成发卡结构,所述辅助单链DNA形成发卡结构;
优选地,所述检测单链DNA的荧光基团位于从5’端起第11位和第12位核苷酸之间,所述荧光淬灭基团位于从5’端起第35位和第36位核苷酸之间;
所述辅助单链DNA从5’端起第1~11位核苷酸与检测单链DNA从5’端起第1~11位核苷酸反向互补。
4.如权利要求3所述的纳米核酸探针,其特征在于,所述检测单链DNA从3’端起第1~11位核苷酸与所述DNA四面体的一条单链从3’端起第1~11位核苷酸反向互补配对连接;所述辅助单链DNA从3’端起第1~11位核苷酸与所述DNA四面体的另一条单链从3’端起第1~11位核苷酸反向互补配对连接;
优选地,所述检测单链DNA从5’端至3’端的序列如SEQ ID NO.1所示;所述辅助单链DNA从5’端至3’端的序列如SEQ ID NO.2所示。
5.如权利要求1~4任一项所述的纳米核酸探针,其特征在于,所述DNA四面体是4条单链DNA的序列分别一对一地选自SEQ ID NO.3~6的所述序列。
6.如权利要求1~4任一项所述的纳米核酸探针,其特征在于,它由如下方法制备而成:
分别将检测单链DNA、辅助单链DNA、DNA四面体的四条单链DNA的混合物置于足以使DNA变性的温度下维持3~8min,再将温度降低到2~8℃维持20min以上,然后混合,在室温孵育30min以上;
优选地,它由如下方法制备而成:
分别将检测单链DNA、辅助单链DNA、DNA四面体的四条单链DNA的混合物置于95℃下维持10min,再将温度降低到4℃维持20min以上,然后混合,在室温孵育30min以上。
7.权利要求1~6任一项所述纳米核酸探针的制备方法,其特征在于,包括如下步骤:
(1)将DNA四面体由4条单链DNA等量溶于TM缓冲液中得溶液a,同时将检测单链DNA溶于TM缓冲液中得溶液b,将辅助单链DNA溶于TM溶液中得溶液c;
(2)溶液a、溶液b、溶液c加热到足以使DNA变性的温度维持3~8min,然后降温到2~8℃维持20min以上;
(3)将溶液a、溶液b、溶液c混合,在室温孵育30min以上。
8.权利要求1~6任一项所述的纳米核酸探针在基因和/或蛋白的实时荧光检测试剂中的应用;
优选地,所述试剂是对肿瘤标志物进行检测和/或标记的试剂;更优选地,所述肿瘤标志物是核仁素。
9.权利要求1~6任一项所述的纳米核酸探针在活体成像试剂中的应用;
优选地,所述试剂是在活细胞或活体组织中成像的试剂。
10.一种核酸和/或蛋白的检测或对活细胞中的核酸和/或蛋白靶点进行标记方法,其特征在于,包括将权利要求1~6任一项所述的纳米核酸探针与样本在室温下共同孵育5~30min,检测荧光信号强度的步骤;
优选地,所述样本为待测核酸和/或蛋白,或为活细胞或活体组织。
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