WO2020107560A1 - 基于滚环扩增和DNA折纸术检测microRNAs的方法 - Google Patents

基于滚环扩增和DNA折纸术检测microRNAs的方法 Download PDF

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WO2020107560A1
WO2020107560A1 PCT/CN2018/121971 CN2018121971W WO2020107560A1 WO 2020107560 A1 WO2020107560 A1 WO 2020107560A1 CN 2018121971 W CN2018121971 W CN 2018121971W WO 2020107560 A1 WO2020107560 A1 WO 2020107560A1
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rolling circle
circle amplification
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何丹农
徐艳
陈玮嘉
张兆坤
王萍
金彩虹
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上海纳米技术及应用国家工程研究中心有限公司
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  • the invention relates to a method for detecting microRNAs based on rolling circle amplification and DNA origami, in particular to a method for amplifying target signals by rolling circle amplification, and simultaneously introducing and orderly arranging detection units by using DNA origami to realize nucleic acid molecules Of detection.
  • the invention belongs to the field of nano biological sensing.
  • MicroRNAs are a group of endogenous single-stranded small RNA molecules containing 19 to 25 bases that do not translate any protein or polysaccharide. Studies have shown that miRNAs can also activate the expression of target genes. Regulating the expression of genes related to individual growth, development, disease and other physiological processes plays an important role in life processes, such as hematopoiesis, early development of individuals, cell proliferation and apoptosis, cell differentiation, cell death, and hormone and fat metabolism. In recent years, the close relationship between miRNAs and tumors has attracted researchers' attention. More than 50% of miRNAs are located in unstable regions of DNA, and these regions are closely related to the shearing or amplification of tumor cell chromosomes. miRNAs play the role of tumor suppressor genes or oncogenes when cancer occurs, and miRNAs are abnormal in many cancer tumors, indicating that the expression level of miRNAs is of great significance for tumor diagnosis and prognosis.
  • Rolling circle amplification uses circular DNA molecules as templates and short DNA or RNA complementary to or partially complementary to the circular DNA as primers.
  • a synthesis with a large number Nucleic acid amplification method of very long DNA single strand of repeated sequence at the same time it is also a constant temperature nucleic acid amplification method, which does not require high temperature denaturation process.
  • the synthesis method of cyclic DNA is the conventional synthesis method in the art, or the selected method refers to the issue published in Angewandte Chemie Vol. 127, No. 8 on February 19, 2015.
  • the title is Growth and Origami Folding of DNA Nanoparticles for High- efficiency The method described in the Supporting Online Information page 1 of the paper "Transport in In Cellular" Imaging and Drug Delivery.
  • DNA origami can use hundreds of complementary short ss-DNA (staple chains) to fold a 7308 base long ss-DNA (scaffold chain) into any pattern under high cation concentration.
  • the long single-stranded DNA obtained by rolling circle amplification is a good template for DNA origami. Since the long chains obtained by rolling circle amplification carry a large number of repetitive sequences, the use of staple chains can be further reduced.
  • the present invention aims to provide a method for detecting miRNAs based on rolling circle amplification and DNA origami.
  • the object of the present invention is achieved by the following scheme: a method for detecting miRNAs based on rolling circle amplification and DNA origami, using rolling circle amplification to amplify the detected nucleic acid, and then using DNA origami to amplify the long chain Perform orderly folding and introduce the detection sequence through the staple chain to achieve the determination of the content of miRNAs, including the following steps:
  • Rolling circle amplification design a pair of hairpin structures composed of two single strands as recognition units, one of which contains a recognition sequence complementary to the target miRNA, at least DNA3, Seq.No of Seq.No.3 .6 DNA6, Seq.No.8 DNA8 or Seq.No.10 DNA10 any one of the DNA; the other is Seq.No.4 DNA as the initiator chain, take 100 ⁇ M recognition sequence and 100 ⁇ M initiator chain, React at a concentration ratio of 1:1 at 95°C for 5 min and slowly cool down to obtain a recognition unit; take 5 ⁇ L of 10 ⁇ M recognition unit and 5 ⁇ L of the target miRNA with a concentration of 0.1 nM-1 ⁇ M to mix evenly.
  • the target miRNAs are Seq.No. 5 miRNA5, Seq.No.7 miRNA7, Seq.No.9 miRNA9 or Seq.No.11 miRNA11 any one of the RNA, incubated at 37 °C for 30min, recorded as mixture I; take 10 ⁇ L of mixture I , 3 ⁇ L 4 ⁇ M cyclic DNA, 3 ⁇ L phi29 DNA polymerase, 1mM deoxyribonucleoside triphosphate (dNTP) in RCA reaction buffer (33mM Tris, 10mM magnesium acetate, 66mM potassium acetate, 0.1% by volume Tween 20, 1mM dithiosu Sugar alcohol) mixed evenly, and reacted at 30°C for a certain period of time; the reaction solution was reacted at 65°C for 10min to inactivate the DNA polymerase, and the single-stranded DNA produced by the reaction was precipitated with ethanol, which was recorded as the scaffold chain used for the origami reaction;
  • the present invention first designs a pair of recognition units with a hairpin structure.
  • the primer chain is released to initiate rolling circle amplification.
  • a long single strand of DNA with a repeating sequence is obtained to achieve the target signal Amplification; using DNA origami technology to fold the amplified products in an orderly manner while introducing a sequence rich in guanine (G), which can form a G-quadruplex in the presence of K +
  • G guanine
  • the DNAzyme formed has high peroxidase activity (HRP DNAzyme for short), and the DNAzyme has the ability to catalytically reduce H 2 O 2 and can oxidize luminol Chemiluminescence can also oxidize ABTS to generate blue-green free radical cations (ABTS .+ ) to achieve the output of the detection signal.
  • 100 ⁇ M and 100 ⁇ M priming strands of the recognition sequence DNA are taken and reacted at a temperature ratio of 1:1 at 95° C. for 5 min, and the temperature is slowly lowered to obtain a recognition unit.
  • the RCA reaction buffer is: 33 mM Tris, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% by volume of Tween 20, 1 mM dithiothreitol.
  • the present invention first uses rolling circle amplification to amplify the detected nucleic acid, and then uses DNA origami to fold the amplified long chain in an orderly manner, and introduces the detection sequence through the staple chain to achieve the content of miRNAs Perform the measurement.
  • the miRNAs may be conventional miRNAs in the art, preferably Let-7a, has-miR-21, miR-1, has-miR-144.
  • the method for synthesizing cyclic DNA is a conventional synthesis method in the art.
  • For the preferred method refer to Volume 127, Issue 8 published in Angewandte Chemie on February 19, 2015.
  • the title is Growth and Origami Folding of DNA Nanoparticles for High- efficiency The method described in the Supporting Online Information page 1 of the paper "Transport in In Cellular” Imaging and Drug Delivery.
  • the rolling circle amplification time is a conventional reaction time in the art.
  • the preferred time range is 5-30 min, and the preferred reaction time is 10 min.
  • the mixing ratio of the scaffold chain and the staple chain is a conventional ratio in the art, preferably the reaction molar ratio is 1:10-1:100, and the preferred reaction molar ratio is 1:20.
  • the present invention uses rolling circle amplification for signal amplification, does not require a high-temperature denaturation process, reacts quickly, and improves detection sensitivity.
  • the present invention uses the technique of DNA origami to fold the amplified product, so that the introduced detection sequence can be arranged in an orderly manner, and avoids signal interference caused by the detection sequence directly surrounding.
  • the detection sequence is introduced into the nail chain to determine the content of miRNAs, including the following steps:
  • the resulting circular DNA was mixed with T4 polymerase and the reaction mixture, and then reacted at 37°C for 16 hr to remove the linked template DNA. Then, the reaction solution was reacted at 85°C for 10 min to inactivate the enzyme. The reaction mixture was ultrafiltered with 3000kD ultrafiltration tube at 3000g for 10min, and the pure solution at the bottom of the tube was collected to obtain circular DNA.
  • the reaction solution was reacted at a temperature of 65°C for 10 minutes to inactivate phi29 DNA polymerase.
  • the single-stranded DNA produced by the reaction is precipitated with ethanol and is referred to as a scaffold chain to be used in the origami reaction.
  • Synthetic loop DNA The synthesis method of loop DNA refers to Volume 127, Issue 8, published in Angewandte Chemie on February 19, 2015. The title is Growth and Origami Folding of DNA Nanoparticles for High-efficiency Molecular Molecular Transport Cellular Imaging The method described in the Supporting Online Information page 1 of Drug Delivery paper. After 100 ⁇ M phosphate-modified DNA 1 and 100 ⁇ M ligated template DNA were mixed well, react at 90°C for 1min. After the solution was cooled to room temperature, T4 ligase and T4 ligase reaction buffer were added and reacted at 25°C for 16hr. The enzyme was inactivated by reacting at 65°C for 10 min.
  • the resulting circular DNA was mixed with T4 polymerase and the reaction mixture, and then reacted at 37°C for 16 hr to remove the linked template DNA. Then, the reaction solution was reacted at 85°C for 10 min to inactivate the enzyme. The reaction mixture was ultrafiltered with 3000kD ultrafiltration tube at 3000g for 10min, and the pure solution at the bottom of the tube was collected to obtain circular DNA.
  • the resulting circular DNA was mixed with T4 polymerase and the reaction mixture, and then reacted at 37°C for 16 hr to remove the linked template DNA. Then, the reaction solution was reacted at 85°C for 10 min to inactivate the enzyme. The reaction mixture was ultrafiltered with 3000kD ultrafiltration tube at 3000g for 10min, and the pure solution at the bottom of the tube was collected to obtain circular DNA.
  • the resulting circular DNA was mixed with T4 polymerase and the reaction mixture, and then reacted at 37°C for 16 hr to remove the linked template DNA. Then, the reaction solution was reacted at 85°C for 10 min to inactivate the enzyme. The reaction mixture was ultrafiltered with 3000kD ultrafiltration tube at 3000g for 10min, and the pure solution at the bottom of the tube was collected to obtain circular DNA.

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Abstract

一种基于滚环扩增和DNA折纸术检测microRNAs的方法，具体涉及一种以滚环扩增放大靶标信号，同时利用DNA折纸术引入并有序排列检测单元，从而实现对核酸分子的检测。优点在于：（1）以滚环扩增进行信号放大，不需要高温变性过程，反应迅速，提高检测灵敏度。（2）利用DNA折纸的技术对扩增产物进行折叠，使引入的检测序列可以有序的排布，避免了检测序列直接环绕造成的信号干扰。

Description

基于滚环扩增和DNA折纸术检测microRNAs的方法 技术领域
本发明涉及一种基于滚环扩增和DNA折纸术检测microRNAs的方法，具体涉及一种以滚环扩增放大靶标信号，同时利用DNA折纸术引入并有序排列检测单元，从而实现对核酸分子的检测。本发明属于纳米生物传感领域。
背景技术
MicroRNAs(miRNAs)是一组内源性的含有19～25个碱基的不翻译任何蛋白或多糖的单链小RNA分子。研究表明，miRNAs也可以激活靶标基因的表达。调节个体生长、发育、疾病等生理过程相关基因的表达，在生命过程中其中起重要作用的，如造血、个体早期发育、细胞增殖和凋亡、细胞分化、细胞死亡以及激素和脂肪代谢。近几年来miRNAs与肿瘤之间的密切关系引起研究人员的关注。超过50％的miRNAs位于DNA的不稳定区域，而这些区域与肿瘤细胞染色体的剪切或扩增息息相关。miRNAs在癌症发生时起着抑癌基因或致癌基因的功能，并且miRNAs在许多癌症肿瘤中不正常表，这表明miRNAs的表达水平对肿瘤诊断和预后具有十分重要的意义。
滚环扩增(rolling circle amplification，RCA)是以环状DNA分子为模板，以短的与环DNA互补或部分互补的DNA或RNA为引物，在phi29DNA聚合酶的催化下，合成一条带有大量重复序列的很长的DNA单链的核酸扩增方法，同时它也是一种恒温的核酸扩增方法，不需要高温变性的过程。其中，环DNA的合成方法为本领域常规的合成方法，或选择的方法参考2015年2月19日发表在Angewandte Chemie中第127卷第8期题目为Growth and Origami Folding of DNA on Nanoparticles for High-efficiency Molecular Transport in Cellular Imaging and Drug Delivery的论文的Supporting Online Information第1页中所记载的方法。
DNA折纸术可以利用数百条互补的短ss-DNA(订书钉链)，在高阳离子浓度下，将一条7308个碱基的长ss-DNA(脚手架链)折叠为任意图案。而滚环扩增所得到的长的单链DNA是很好的进行DNA折纸的模板。由于滚环扩增所得长链携带大量重复序列，因此又可以进一步减少订书钉链的使用。
发明内容
针对现有技术的不足，本发明目的在于提供一种基于滚环扩增和DNA折纸术检测miRNAs的方法。
本发明目的通过下述方案实现：一种基于滚环扩增和DNA折纸术检测miRNAs的方法，利用滚环扩增对所检测核酸进行信号放大，随后利用DNA折纸术对扩增后的长链进行有序的折叠，通过订书钉链引入检测序列，从而实现对miRNAs的含量进行测定，包括如下步骤：
(1)滚环扩增：设计一对由两条单链构成的发夹结构作为识别单元，其中一条链含有与靶标miRNA互补的识别序列，至少为Seq.No.3的DNA3、Seq.No.6的DNA6、Seq.No.8的DNA8或Seq.No.10的DNA10中的任一种DNA；另一条为Seq.No.4的DNA作为引发链，取100μM识别序列与100μM引发链，以浓度比1:1在95℃温度下反应5min，缓慢降温，得到识别单元；取5μL 10μM识别单元与5μL浓度在0.1nM-1μM的靶标miRNA混合均匀，所述的靶标miRNAs为Seq.No.5的miRNA 5、Seq.No.7的miRNA7、Seq.No.9的miRNA 9或Seq.No.11的miRNA 11中的任一RNA，37℃孵育30min，记为混合物I；取10μL混合物I，3μL4μM环DNA，3μL phi29DNA聚合酶，1mM脱氧核糖核苷三磷酸(dNTP)在RCA反应缓冲液(33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％体积百分比的吐温20，1mM二硫苏糖醇)混合均匀，30℃反应一定时间；反应液在65℃温度下反应10min，使DNA聚合酶失活，反应产生的单链DNA用乙醇沉淀，记为脚手架链被用于进行折纸反应；
(2)DNA折纸：各取浓度为100uM 5uL Seq.No.12的DNA12、Seq.No.13的DNA13和Seq.No.14的DNA 14富含鸟嘌呤的订书钉链混合均匀，记为混合物III，脚手架单链DNA和混合物III以一定的摩尔比混合均匀，在含有K +的1×TAE-Mg 2+(50mM K +，40mM Tris(pH＝7.4)，20mM醋酸，2mM EDTA，12.5mM醋酸镁)缓冲溶液中，与PCR仪中从95℃退火至4℃，速率为1℃/min，反应物记为混合物II；
(3)检测：取10uL混合物II，加入1μl 20μM氯高铁血红素(hemin)、1μl 40mM H 2O 2以及50μl 2mM ABTS溶液混合均匀后，监测其在410nm处吸光值的变化。
本发明首先设计一对发夹结构的识别单元，通过与靶标miRNAs结合，释放引物链，引发滚环扩增，通过控制扩增条件，得到一条长的具有重复序列的DNA单链，实现靶标信号放大；利用DNA折纸技术，将扩增产物有序折叠，同时引入富含鸟嘌呤(G)的序列，在有K +存在的条件下，该序列能形成G-四联体(G-quadruplex)，G-四联体与氯高铁血红素(hemin)孵育后，形成的DNAzyme 具有很高过氧化物酶活性(简称HRP DNAzyme)，DNAzyme具有催化还原H 2O 2的能力，可氧化鲁米诺化学发光，也可氧化ABTS生成蓝绿色的自由基阳离子(ABTS .+)从而实现检测信号的输出。
进一步的，取识别序列DNA100μM与100μM引发链，以浓度比1:1在95℃温度下反应5min，缓慢降温，得到识别单元。
所述的RCA反应缓冲液为：33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％体积百分比的吐温20，1mM二硫苏糖醇。
本发明首先利用了滚环扩增对所检测核酸进行信号放大，随后利用DNA折纸术对扩增后的长链进行有序的折叠，通过订书钉链引入检测序列，从而实现对miRNAs的含量进行测定。
所述的miRNAs可以为本领域常规的miRNAs，优选的为Let-7a，has-miR-21，miR-1，has-miR-144。
所用的反应缓冲溶液均采用焦碳酸二乙酯(DEPC)处理后的milliQ水中进行配置。
所述的环DNA的合成方法为本领域常规的合成方法，优选的方法参考2015年2月19日发表在Angewandte Chemie中第127卷第8期题目为Growth and Origami Folding of DNA on Nanoparticles for High-efficiency Molecular Transport in Cellular Imaging and Drug Delivery的论文的Supporting Online Information第1页中所记载的方法。
所述的滚环扩增的时间为本领域常规的反应时间，较佳的时间范围是5-30min，优选的反应时间为10min。
所述的脚手架链与订书钉链的混合比例为本领域常规的比例，较佳的为反应摩尔比为1:10-1:100，优选的反应摩尔比为1:20。
本发明的优点在于：
(1)本发明以滚环扩增进行信号放大，不需要高温变性过程，反应迅速，提高检测灵敏度。
(2)本发明利用DNA折纸的技术对扩增产物进行折叠，使引入的检测序列可以有序的排布，避免了检测序列直接环绕造成的信号干扰。
具体实施方式
以下通过具体的实施例对本发明的技术方案作进一步描述。以下的实施例 是对本发明的进一步说明，而不限制本发明的范围。
实施例1
一种基于滚环扩增和DNA折纸术检测miRNAs的方法，利用滚环扩增对所检测核酸进行信号放大，随后利用DNA折纸术对扩增后的长链进行有序的折叠，通过订书钉链引入检测序列，从而实现对miRNAs的含量进行测定，包括如下步骤：
1、滚环扩增：
(1)合成环DNA：环DNA的合成方法参考2015年2月19日发表在Angewandte Chemie中第127卷第8期题目为Growth and Origami Folding of DNA on Nanoparticles for High-efficiency Molecular Transport in Cellular Imaging and Drug Delivery的论文的Supporting Online Information第1页中所记载的方法。100μM磷酸修饰DNA 1与100μM连接模板DNA 2混合均匀后，与90℃反应1min。待溶液降温至室温后，加入T4连接酶及T4连接酶反应缓冲液，于25℃反应16hr，随后反应液于65℃反应10min使酶失活。所得的环DNA与T4聚合酶及反应混合液混合均匀后，于37℃反应16hr，以去除连接模板DNA 2，随后反应液于85℃反应10min使酶失活。反应混合液采用30kD超滤管于3000g超滤10min，收集管底纯净的溶液，即可得环DNA。
(2)滚环扩增：2.5μL 100μM识别序列DNA 3与2.5μL 100μM引发链DNA 4，以浓度比1:1混合均匀后在95℃温度下反应5min，缓慢降温，得到识别单元；取5μL 10μM识别单元与5μL 1nM靶标miRNA 5混合均匀，37℃孵育30min，记为混合物I；取10μL混合物I、3μL 4μM环DNA，3μL phi29DNA聚合酶，1mM dNTP在RCA反应缓冲液(33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％(体积/体积)吐温20，1mM二硫苏糖醇)混合均匀，30℃反应10min。反应液在65℃温度下反应10min，使phi29DNA聚合酶失活。反应产生的单链DNA用乙醇沉淀，记为脚手架链被用于进行折纸反应。
2、DNA折纸：
各取5uL富含鸟嘌呤的订书钉链DNA 12-14(100uM)混合均匀，记为混合物III，脚手架单链DNA和混合物III以1:20的摩尔比混合均匀，在含有K +的1×TAE-Mg 2+(50mM K +，40mM Tris(pH＝7.4)，20mM醋酸，2mM EDTA，12.5mM醋酸镁)缓冲溶液中，与PCR仪中从95℃退火至4℃，速率为1℃/min，反应 后记为混合物II。
3、检测：
取10uL混合物II，加入1μl 20μM hemin、1μl 40mM H 2O 2以及50μl 2mM ABTS溶液混合均匀后，监测其在410nm处吸光值的变化，从而实现对miRNAs的含量进行测定。
实施例2
与实施例1步骤近似，包括：
1、滚环扩增：
(1)合成环DNA：环DNA的合成方法参考2015年2月19日发表在Angewandte Chemie中第127卷第8期题目为Growth and Origami Folding of DNA on Nanoparticles for High-efficiency Molecular Transport in Cellular Imaging and Drug Delivery的论文的Supporting Online Information第1页中所记载的方法。100μM磷酸修饰DNA 1与100μM连接模板DNA 2混合均匀后，于90℃反应1min，待溶液降温至室温后，加入T4连接酶及T4连接酶反应缓冲液，于25℃反应16hr，随后反应液于65℃反应10min使酶失活。所得的环DNA与T4聚合酶及反应混合液混合均匀后，于37℃反应16hr，以去除连接模板DNA 2，随后反应液于85℃反应10min使酶失活。反应混合液采用30kD超滤管于3000g超滤10min，收集管底纯净的溶液，即可得环DNA。
(2)滚环扩增：2.5μL 100μM识别序列DNA 6与2.5μL 100μM引发链DNA 4，以浓度比1:1混合均匀后在95℃温度下反应5min，缓慢降温，得到识别单元。取5μL 10μM识别单元与5μL 1nM靶标miRNA 7混合均匀，37℃孵育30min，记为混合物I。取10μL混合物I、3μL 4μM环DNA，3μL phi29DNA聚合酶，1mM dNTP在RCA反应缓冲液(33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％(体积/体积)吐温20，1mM二硫苏糖醇)混合均匀，30℃反应10min。反应液在65℃温度下反应10min，使phi29DNA聚合酶失活。反应产生的单链DNA用乙醇沉淀，记为脚手架链被用于进行折纸反应。
2、DNA折纸：
各取5uL富含鸟嘌呤的订书钉链DNA 12-14(100uM)混合均匀，记为混合物III，脚手架单链DNA和混合物III以1:20的摩尔比混合均匀，在含有K +的1×TAE-Mg 2+(50mM K +，40mM Tris(pH＝7.4)，20mM醋酸，2mM EDTA，12.5 mM醋酸镁)缓冲溶液中，与PCR仪中从95℃退火至4℃，速率为1℃/min，反应后记为混合物II。
3、检测：
取10uL混合物II，加入1μl 20μM hemin、1μl 40mM H 2O 2以及50μl 2mM ABTS溶液混合均匀后，监测其在410nm处吸光值的变化。
实施例3
与实施例1步骤近似，包括：
1、滚环扩增：
(1)合成环DNA：环DNA的合成方法参考2015年2月19日发表在Angewandte Chemie中第127卷第8期题目为Growth and Origami Folding of DNA on Nanoparticles for High-efficiency Molecular Transport in Cellular Imaging and Drug Delivery的论文的Supporting Online Information第1页中所记载的方法。100μM磷酸修饰DNA 1与100μM连接模板DNA 2混合均匀后，于90℃反应1min。待溶液降温至室温后，加入T4连接酶及T4连接酶反应缓冲液，于25℃反应16hr，随后反应液于65℃反应10min使酶失活。所得的环DNA与T4聚合酶及反应混合液混合均匀后，于37℃反应16hr，以去除连接模板DNA 2，随后反应液于85℃反应10min使酶失活。反应混合液采用30kD超滤管于3000g超滤10min，收集管底纯净的溶液，即可得环DNA。
(2)滚环扩增：2.5μL 100μM识别序列DNA 8与2.5μL 100μM引发链DNA 4，以浓度比1:1混合均匀后在95℃温度下反应5min，缓慢降温，得到识别单元。取5μL 10μM识别单元与5μL 1nM靶标miRNA 9混合均匀，37℃孵育30min，记为混合物I。取10μL混合物I、3μL 4μM环DNA，3μL phi29DNA聚合酶，1mM dNTP在RCA反应缓冲液(33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％(体积/体积)吐温20，1mM二硫苏糖醇)混合均匀，30℃反应10min。反应液在65℃温度下反应10min，使phi29DNA聚合酶失活。反应产生的单链DNA用乙醇沉淀，记为脚手架链被用于进行折纸反应。
2、DNA折纸：
各取5uL富含鸟嘌呤的订书钉链DNA 12-14(100uM)混合均匀，记为混合物III，脚手架单链DNA和混合物III以1:20的摩尔比混合均匀，在含有K +的1×TAE-Mg 2+(50mM K +，40mM Tris(pH＝7.4)，20mM醋酸，2mM EDTA，12.5 mM醋酸镁)缓冲溶液中，与PCR仪中从95℃退火至4℃，速率为1℃/min，反应后记为混合物II。
3、检测：
取10uL混合物II，加入1μl 20μM hemin、1μl 40mM H 2O 2以及50μl 2mM ABTS溶液混合均匀后，监测其在410nm处吸光值的变化。
实施例4
与实施例1步骤近似，包括：
1、滚环扩增：
(1)合成环DNA：环DNA的合成方法参考2015年2月19日发表在Angewandte Chemie中第127卷第8期题目为Growth and Origami Folding of DNA on Nanoparticles for High-efficiency Molecular Transport in Cellular Imaging and Drug Delivery的论文的Supporting Online Information第1页中所记载的方法。100μM磷酸修饰DNA 1与100μM连接模板DNA 2混合均匀后，与90℃反应1min。待溶液降温至室温后，加入T4连接酶及T4连接酶反应缓冲液，于25℃反应16hr，随后反应液于65℃反应10min使酶失活。所得的环DNA与T4聚合酶及反应混合液混合均匀后，于37℃反应16hr，以去除连接模板DNA 2，随后反应液于85℃反应10min使酶失活。反应混合液采用30kD超滤管于3000g超滤10min，收集管底纯净的溶液，即可得环DNA。
(2)滚环扩增：2.5μL 100μM识别序列DNA 10与2.5μL 100μM引发链DNA 4，以浓度比1:1混合均匀后在95℃温度下反应5min，缓慢降温，得到识别单元。取5μL 10μM识别单元与5μL 1nM靶标miRNA 11混合均匀，37℃孵育30min，记为混合物I。取10μL混合物I、3μL 4μM环DNA，3μL phi29DNA聚合酶，1mM dNTP在RCA反应缓冲液(33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％(体积/体积)吐温20，1mM二硫苏糖醇)混合均匀，30℃反应10min。反应液在65℃温度下反应10min，使phi29DNA聚合酶失活。反应产生的单链DNA用乙醇沉淀，记为脚手架链被用于进行折纸反应。
2、DNA折纸：
各取5uL富含鸟嘌呤的订书钉链DNA 12-14(100uM)混合均匀，记为混合物III，脚手架单链DNA和混合物III以1:20的摩尔比混合均匀，在含有K +的1×TAE-Mg 2+(50mM K +，40mM Tris(pH＝7.4)，20mM醋酸，2mM EDTA，12.5 mM醋酸镁)缓冲溶液中，与PCR仪中从95℃退火至4℃，速率为1℃/min，反应后记为混合物II。
3、检测：
取10uL混合物II，加入1μl 20μM hemin、1μl 40mM H 2O 2以及50μl 2mM ABTS溶液混合均匀后，监测其在410nm处吸光值的变化。
Figure PCTCN2018121971-appb-000001
Figure PCTCN2018121971-appb-000002
Figure PCTCN2018121971-appb-000003

Claims (7)

  1. 一种基于滚环扩增和DNA折纸术检测miRNAs的方法，其特征在于，利用滚环扩增对所检测核酸进行信号放大，随后利用DNA折纸术对扩增后的长链进行有序的折叠，通过订书钉链引入检测序列，从而实现对miRNAs的含量进行测定，包括如下步骤：
    (1)滚环扩增：设计一对由两条单链构成的发夹结构作为识别单元，其中一条链含有与靶标miRNA互补的识别序列，至少为Seq.No.3、Seq.No.6、Seq.No.8或Seq.No.10中的任一种DNA；另一条为Seq.No.4的DNA4作为引发链，识别序列的DNA与引发链的浓度比为1:1，在95℃温度下反应5min，缓慢降温，得到识别单元；取5μL 10μM识别单元与5μL浓度在0.1nM-1μM的靶标miRNA混合均匀，所述的靶标miRNAs为Seq.No.5、Seq.No.7、Seq.No.9或Seq.No.11的任一miRNA，37℃孵育30min，记为混合物I；取10μL混合物I，3μL 4μM环DNA，3μL phi29 DNA聚合酶，1mM脱氧核糖核苷三磷酸(dNTP)在RCA反应缓冲液(33mM Tris，10mM醋酸镁，66mM醋酸钾，0.1％体积百分比的吐温20，1mM二硫苏糖醇)混合均匀，30℃反应一定时间；反应液在65℃温度下反应10min，使DNA聚合酶失活，反应产生的单链DNA用乙醇沉淀，记为脚手架链被用于进行折纸反应；
    (2)DNA折纸：各取5uL Seq.No.12、Seq.No.13和Seq.No.14富含鸟嘌呤的订书钉链DNA 12-14(100uM)混合均匀，记为混合物III，脚手架单链DNA和混合物III以一定的摩尔比混合均匀，在含有K +的1×TAE-Mg 2+(50mM K +，40mM Tris(pH＝7.4)，20mM醋酸，2mM EDTA，12.5mM醋酸镁)缓冲溶液中，与PCR仪中从95℃退火至4℃，速率为1℃/min，反应后记为混合物II；
    (3)检测：取10uL混合物II，加入1μl 20μM氯高铁血红素(hemin)、1μl 40mM H 2O 2以及50μl 2mM ABTS溶液混合均匀后，监测其在410nm处吸光值的变化。
  2. 根据权利要求1所述基于滚环扩增和DNA折纸术检测miRNAs的方法，其特征在于，取任一识别序列DNA100μM与100μM引发链，以浓度比1:1在95℃温度下反应5min，缓慢降温，得到识别单元。
  3. 根据权利要求1所述基于滚环扩增和DNA折纸术检测miRNAs的方法，其特征在于所述的miRNAs优选的为Let-7a，has-miR-21，miR-1，has-miR-144。
  4. 根据权利要求1所述基于滚环扩增和DNA折纸术检测miRNAs的方法， 其特征在于，所用的反应缓冲溶液均采用焦碳酸二乙酯(DEPC)处理后的milliQ水中进行配置。
  5. 根据权利要求1所述基于滚环扩增和DNA折纸术检测miRNAs的方法，其特征在于，所述的环DNA由Seq.No.1 DNA 1和Seq.No.2 DNA 2合成得到。
  6. 根据权利要求1所述基于滚环扩增和DNA折纸术检测miRNAs的方法，其特征在于所述的滚环扩增的时间5-30min，优选的反应时间为10min。
  7. 根据权利要求1所述基于滚环扩增和DNA折纸术检测miRNAs的方法，其特征在于所述的脚手架链与订书钉链的混合比例为本领域常规的比例，较佳的为反应摩尔比为1:10-1:100，优选的反应摩尔比为1:20。
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