CN114921533A - Methods and adaptors for characterising a target polynucleotide - Google Patents

Abstract

Translated fromChinese

本发明提供了用于表征目标多核苷酸的方法及衔接体。本发明提供了一种表征目标多核苷酸的方法，包括：(a)使目标多核苷酸穿过纳米孔移动，其中，所述目标多核苷酸的测序终端包含修饰部分，所述修饰部分使纳米孔堵塞；(b)随着所述目标多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值。本发明还提供了用于表征目标多核苷酸的衔接体，其中，所述衔接体包含修饰部分，所述修饰部分结合到所述目标多核苷酸的测序终端，所述修饰部分使纳米孔堵塞。本发明提供的方法避免了测序完成的文库大量富集在纳米孔测序装置的反式(Trans)端，并进一步提高了反式(Trans)端改造的可变性。

The present invention provides methods and adapters for characterizing polynucleotides of interest. The present invention provides a method of characterizing a target polynucleotide, comprising: (a) moving the target polynucleotide through a nanopore, wherein the sequencing terminus of the target polynucleotide comprises a modified portion that enables Nanopore plugging; (b) taking one or more electrical and/or optical measurements as the target polynucleotide moves relative to the pore. The present invention also provides an adaptor for characterizing a polynucleotide of interest, wherein the adaptor comprises a modified moiety that binds to a sequencing terminus of the polynucleotide of interest, the modified moiety clogging the nanopore . The method provided by the present invention avoids a large amount of enrichment of the sequenced library at the trans (Trans) end of the nanopore sequencing device, and further improves the variability of the trans (Trans) end modification.

Description

Translated fromChinese

用于表征目标多核苷酸的方法及衔接体Methods and adapters for characterizing target polynucleotides

技术领域technical field

本发明属于基因测序领域，涉及一种表征多核苷酸的方法，本发明还涉及所述方法中使用的衔接体。The present invention belongs to the field of gene sequencing, relates to a method for characterizing polynucleotides, and also relates to an adaptor used in the method.

背景技术Background technique

纳米孔测序技术具有长读长、直接读取修饰信息和实时数据生产并行分析的特点，在长片段核酸检测变异(包括但不仅限于点突变、插入缺失、倒位易位、基因融合、RNA异常剪切、RNA编辑等多种核酸相关变异)和修饰信息(包括但不仅限于甲基化、乙酰化等)检测方面比二代测序或其他测序平台有更明显优势。该平台支持数据生产和分析并行的特点实现了实时变异/修饰检出和诊断，加上便携式的设计，使其具有广泛的应用前景。Nanopore sequencing technology has the characteristics of long read length, direct read modification information and parallel analysis of real-time data production. It has obvious advantages over next-generation sequencing or other sequencing platforms in the detection of various nucleic acid-related variants such as splicing, RNA editing) and modification information (including but not limited to methylation, acetylation, etc.). The platform supports data production and analysis in parallel, realizes real-time variant/modification detection and diagnosis, and the portable design makes it have broad application prospects.

对纳米孔两侧施加电压后，当分析物(例如多核苷酸、多肽、多糖和脂质)通过纳米孔时造成电流下降，不同结构的分析物所引起的电流阻断程度不同。当分析物在纳米孔的桶(barrel)中暂时停留一段时间时，电流会发生变化。纳米孔检测核苷酸给出已知特征和持续时间的电流变化。When a voltage is applied to both sides of the nanopore, the current decreases when analytes (such as polynucleotides, polypeptides, polysaccharides and lipids) pass through the nanopore, and analytes with different structures cause different degrees of current blocking. The current changes when the analyte stays temporarily in the barrel of the nanopore for a period of time. Nanopores detect nucleotides given current changes of known characteristics and duration.

在目前的进行纳米孔测序中，测序起始时所有的待测分析物均被加载到纳米孔测序装置的顺式(Cis)端。而在测序过程中，待测分析物会不断过孔，并大量聚集到纳米孔测序仪的反式(Trans)端。In current nanopore sequencing, all analytes to be tested are loaded into the cis (Cis) end of the nanopore sequencing device at the start of sequencing. During the sequencing process, the analyte to be tested will continuously pass through the pore and accumulate in a large amount on the trans (Trans) end of the nanopore sequencer.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种表征目标多核苷酸的方法，本发明还提供了所述方法中使用的衔接体。本发明的衔接体可用于避免测序文库富集到测序装置的反式(Trans)端，从而避免该富集可能的干扰，并进一步提高了反式(Trans)端改造的可变性。The purpose of the present invention is to provide a method for characterizing a target polynucleotide, and the present invention also provides an adaptor used in the method. The adaptor of the present invention can be used to avoid the enrichment of the sequencing library to the trans (Trans) end of the sequencing device, thereby avoiding possible interference of the enrichment, and further improving the variability of the trans (Trans) end modification.

本发明的目的是通过以下技术方案实现的：The purpose of this invention is to realize through the following technical solutions:

第一方面，本发明提供了一种表征目标多核苷酸的方法，包括：In a first aspect, the present invention provides a method for characterizing a target polynucleotide, comprising:

(a)使目标多核苷酸穿过纳米孔移动，(a) moving the target polynucleotide through the nanopore,

其中，所述目标多核苷酸的测序终端包含修饰部分，所述修饰部分使纳米孔堵塞；Wherein, the sequencing terminal of the target polynucleotide comprises a modified part, and the modified part blocks the nanopore;

(b)随着所述目标多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。(b) obtaining one or more electrical and/or optical measurements as the target polynucleotide moves relative to the pore, wherein the measurements represent one or more characteristics of the target polynucleotide, and thereby characterize the target polynucleotide.

根据本发明所述的方法，还包括：The method according to the present invention further comprises:

(c)使所述目标多核苷酸相对于所述纳米孔反向移动，回到所述纳米孔的起始侧。(c) moving the target polynucleotide in the opposite direction relative to the nanopore, back to the start side of the nanopore.

根据本发明所述的方法，其中，According to the method of the present invention, wherein,

所述纳米孔包括固态纳米孔和/或生物纳米孔；所述生物纳米孔包括跨膜孔；所述跨膜孔包括跨膜蛋白孔。The nanopores include solid state nanopores and/or biological nanopores; the biological nanopores include transmembrane pores; the transmembrane pores include transmembrane protein pores.

根据本发明所述的方法，其中，步骤(c)不包括：According to the method of the present invention, wherein, step (c) does not include:

随着所述多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。As the polynucleotide moves relative to the pore, one or more electrical and/or optical measurements are taken, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the the target polynucleotide.

根据本发明所述的方法，其中，步骤(c)包括：According to the method of the present invention, wherein, step (c) comprises:

随着所述多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。As the polynucleotide moves relative to the pore, one or more electrical and/or optical measurements are taken, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the the target polynucleotide.

根据本发明所述的方法，其中，步骤(c)中，所述使所述目标多核苷酸相对于所述孔反向移动通过包括如下至少一种方式实现：施加反向电压。According to the method of the present invention, wherein, in step (c), the reverse movement of the target polynucleotide relative to the pore is achieved by at least one of the following methods: applying a reverse voltage.

所述目标多核苷酸相对于所述孔反向移动的方式还可以包括：AFM(原子力显微镜)牵引，或使所述目标多核苷酸相对于所述纳米孔反向移动的反向酶的牵引，如使所述目标多核苷酸朝向所述纳米孔移动的解旋酶为5’-3’方向的解旋酶，则反向酶可以为3’-5’方向的解旋酶。The manner in which the target polynucleotide moves in the opposite direction relative to the pore may also include: AFM (atomic force microscopy) traction, or the pulling of a reverse enzyme that makes the target polynucleotide move in the opposite direction relative to the nanopore , if the helicase that moves the target polynucleotide toward the nanopore is a helicase in the 5'-3' direction, the reverse enzyme can be a helicase in the 3'-5' direction.

根据本发明所述的方法，其中，步骤(a)包括：According to the method of the present invention, wherein, step (a) comprises:

将包含修饰部分的衔接体附接到所述目标多核苷酸，以使所述目标多核苷酸的测序终端包含修饰部分。An adaptor comprising a modified moiety is attached to the target polynucleotide such that the sequencing terminus of the target polynucleotide comprises the modified moiety.

优选地，所述修饰部分包含侧链不带电荷或侧链带正电荷的修饰部分；和/或Preferably, the modified moiety comprises a modified moiety with an uncharged side chain or a positively charged side chain; and/or

所述侧链不带电荷的修饰部分包括PNA、多肽和磷酸骨架烷基化修饰的核苷酸中任一种或任两种以上的组合；和/或The modified moieties with uncharged side chains include any one or a combination of any two or more of PNAs, polypeptides, and phosphate backbone alkylation-modified nucleotides; and/or

所述侧链带正电荷的修饰部分包括磷酸骨架阳离子寡聚物修饰的核苷酸；和/或The side chain positively charged modified moiety comprises a phosphate backbone cationic oligomer modified nucleotide; and/or

所述阳离子寡聚物包括精胺、亚精胺、腐胺任一种或任两种以上的组合；和/或The cationic oligomer includes spermine, spermidine, putrescine, or any combination of any two or more; and/or

所述修饰部分包含相互结合的配基和配体，所述配基和配体包括链霉亲和素和生物素、抗原和抗体。The modified moiety comprises ligands and ligands that bind to each other, including streptavidin and biotin, antigens and antibodies.

根据本发明所述的方法，其中所述衔接体为包含双链区和至少一个单链区的Y型衔接体，或包含双链区并且不包含单链区的E型衔接体。According to the method of the present invention, wherein the adapter is a Y-type adapter comprising a double-stranded region and at least one single-stranded region, or an E-type adapter comprising a double-stranded region and no single-stranded region.

其中所述E型衔接体为常规测序用衔接体，并且适用于本申请的E型衔接体包含修饰部分。Wherein the E-type adapter is a conventional sequencing adapter, and the E-type adapter suitable for the present application comprises a modified part.

根据本发明所述的方法，其中，所述衔接体为Y型衔接体，所述Y型衔接体的修饰部分位于所述Y型衔接体的悬突部分或所述Y型衔接体的修饰部分形成所述Y型衔接体的悬突部分；和/或The method according to the present invention, wherein the adaptor is a Y-shaped adaptor, and the modified part of the Y-shaped adaptor is located in the overhang part of the Y-shaped adaptor or the modified part of the Y-shaped adaptor forming an overhang portion of the Y-shaped adaptor; and/or

所述修饰部分包含侧链不带电荷的修饰部分，更优选地，所述侧链不带电荷的修饰部分为PNA或多肽。The modified moiety comprises a modified moiety with an uncharged side chain, more preferably, the modified moiety with an uncharged side chain is a PNA or a polypeptide.

和/或and / or

所述修饰部分共价地连接到所述Y型衔接体或所述修饰通过点击化学反应连接到所述Y型衔接体。The modified moiety is covalently attached to the Y-type adaptor or the modification is attached to the Y-type adaptor through a click chemistry reaction.

根据本发明所述的方法，其中，所述衔接体为E型衔接体，所述修饰部分位于没有附接至所述目标多核苷酸的一端；和/或The method according to the present invention, wherein the adaptor is an E-type adaptor, and the modified moiety is located at the end not attached to the target polynucleotide; and/or

所述修饰部分包含亲和物质，优选地，所述亲和物质为链霉亲和素和生物素；或The modified part comprises an affinity substance, preferably, the affinity substance is streptavidin and biotin; or

所述修饰部分包含胆固醇。The modified moiety includes cholesterol.

根据本发明所述的方法，其中，所述衔接体包含阻断链，所述阻断链具有与所述多核苷酸不同的结构，用于阻滞马达蛋白；The method according to the present invention, wherein the adaptor comprises a blocking strand having a different structure from the polynucleotide for blocking motor proteins;

优选地，所述阻断链包含一个或多个硝基吲哚、一个或多个肌苷、一个或多个吖啶、一个或多个2-氨基嘌呤、一个或多个2-6-二氨基嘌呤、一个或多个5-溴-脱氧尿嘧啶、一个或多个反向胸苷、一个或多个反向二脱氧胸苷、一个或多个二脱氧胞苷、一个或多个5-甲基胞苷酸、一个或多个5-羟甲基胞苷、一个或多个2’烷氧基修饰的核糖核苷酸优选2’甲氧基修饰的核糖核苷酸、一个或多个异脱氧胞苷、一个或多个异脱氧鸟苷、一个或多个C3基团、一个或多个光裂解的基团、一个或多个己二醇、一个或多个iSp9基团、一个或多个iSp18基团、聚合物或一个或多个硫醇连接。Preferably, the blocking chain comprises one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-di aminopurine, one or more 5-bromo-deoxyuracil, one or more reverse thymidine, one or more reverse dideoxythymidine, one or more dideoxycytidine, one or more 5- Methyl cytidine, one or more 5-hydroxymethyl cytidine, one or more 2' alkoxy modified ribonucleotides, preferably 2' methoxy modified ribonucleotides, one or more Isodeoxycytidine, one or more isodeoxyguanosine, one or more C3 groups, one or more photocleavable groups, one or more hexanediol, one or more iSp9 groups, one or more Multiple iSp18 groups, polymers or one or more thiol linkages.

第二方面，本发明提供了用于表征目标多核苷酸的衔接体，其中，所述衔接体包含修饰部分，所述修饰部分用于结合到所述目标多核苷酸的测序终端，所述修饰部分能够使纳米孔堵塞。In a second aspect, the present invention provides an adaptor for characterizing a polynucleotide of interest, wherein the adaptor comprises a modified moiety for binding to a sequencing terminus of the polynucleotide of interest, the modification Partially able to block nanopores.

第三方面，本发明提供了用于表征目标多核苷酸的构建体，所述构建体包含衔接体，和目标多核苷酸；In a third aspect, the present invention provides a construct for characterizing a polynucleotide of interest, the construct comprising an adaptor, and a polynucleotide of interest;

所述衔接体包含修饰部分，所述修饰部分结合到所述目标多核苷酸的测序终端，所述修饰部分使纳米孔堵塞；the adaptor comprises a modified moiety that binds to the sequencing terminus of the target polynucleotide, the modified moiety plugs the nanopore;

优选地，所述目标多核苷酸为双链多核苷酸。Preferably, the target polynucleotide is a double-stranded polynucleotide.

第四方面，本发明提供了用于表征目标多核苷酸的复合物，所述复合物包含构建体和马达蛋白，其中，In a fourth aspect, the present invention provides a complex for characterizing a polynucleotide of interest, the complex comprising a construct and a motor protein, wherein,

所述构建体包含衔接体和目标多核苷酸，所述衔接体包含修饰部分，所述修饰部分结合到所述目标多核苷酸的测序终端，所述修饰部分使纳米孔堵塞；the construct comprises an adaptor and a target polynucleotide, the adaptor comprising a modified moiety that binds to a sequencing terminus of the target polynucleotide, the modified moiety clogging the nanopore;

所述马达蛋白为能够结合到所述目标多核苷酸并且控制其移动穿过孔的蛋白；the motor protein is a protein capable of binding to the target polynucleotide and controlling its movement through the pore;

优选地，所述马达蛋白选自聚合酶、核酸外切酶、解旋酶和拓扑异构酶中的一种或多种；Preferably, the motor protein is selected from one or more of polymerase, exonuclease, helicase and topoisomerase;

更优选地，所述解旋酶选自Hel308解旋酶、RecD解旋酶、Tral解旋酶、TrwC解旋酶、XPD解旋酶和DDA解旋酶中的一种或多种。More preferably, the helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase and DDA helicase.

第五方面，本发明提供了用于纳米孔表征多核苷酸的试剂盒，所述试剂盒的组成包含：In a fifth aspect, the present invention provides a kit for nanopore characterization of polynucleotides, the kit comprising:

1)衔接体，所述衔接体包含修饰部分，所述修饰部分结合到所述目标多核苷酸的测序终端，所述修饰部分使纳米孔堵塞；和1) an adaptor comprising a modified moiety that binds to the sequencing terminus of the polynucleotide of interest, the modified moiety clogging the nanopore; and

2)马达蛋白。2) Motor proteins.

在纳米孔测序中，测序起始时所有的文库均被加载到纳米孔测序装置的顺式(Cis)端。而在测序过程中，随着待测序文库不断过孔，核酸文库会大量聚集到纳米孔测序仪的反式(Trans)端，由于核酸携带电荷，该富集会对测序结果造成可能的干扰。本发明的发明人提供一种方法，可以避免该富集。本发明的技术构思结合图1和图2描述如下，图1为使用本发明的衔接体从而避免测序文库在反式端(Trans)大量富集的原理示意图；图2为包含本发明的衔接体、目标多核苷酸和解旋酶的复合物的示意图；其中，图1中，所述衔接体连接到待表征的目标多核苷酸双链的两端，在解旋酶的作用下，所述双链边解旋边相对于孔移动，通过孔的电流变化来确定所述目标多核苷酸的序列，即为链测序。由于本发明的衔接体中修饰部分存在于所述测序链的测序终端，因此在目标多核苷酸链的测序终端，该修饰未能穿越纳米孔从而造成堵孔，导致完成测序的目标多核苷酸链被从孔中踢出回到顺式(Cis)端。简言之，测序过程中，加入所述解旋酶为5’-3’解旋酶，在复合物的引导下，文库的5’端进孔，解旋酶沿着5‘-3’移位并进行测序；当运行到3’末端时，因为不能跨过末端的能量障碍，会出现堵孔现象，进而使系统施加反向电压踢孔，将测序完成的单链文库从反式(Trans)端踢回到顺式(Cis)端。In nanopore sequencing, all libraries are loaded into the cis (Cis) end of the nanopore sequencing device at the start of sequencing. During the sequencing process, as the library to be sequenced continues to pass through the pores, a large number of nucleic acid libraries will accumulate on the trans (Trans) end of the nanopore sequencer. Since the nucleic acids carry charges, this enrichment may cause possible interference to the sequencing results. The inventors of the present invention provide a method whereby this enrichment can be avoided. The technical concept of the present invention is described below in conjunction with FIG. 1 and FIG. 2 . FIG. 1 is a schematic diagram of the principle of using the adaptor of the present invention to avoid a large amount of enrichment of the sequencing library at the trans end (Trans); FIG. 2 is a schematic diagram that includes the adaptor of the present invention. , a schematic diagram of a complex of a target polynucleotide and a helicase; wherein, in Figure 1, the adapter is connected to both ends of the double-stranded target polynucleotide to be characterized, and under the action of the helicase, the double-stranded The unwinding side of the strand moves relative to the pore, and the sequence of the target polynucleotide is determined by the current change in the pore, which is strand sequencing. Since the modified part of the adaptor of the present invention exists at the sequencing terminal of the sequencing strand, at the sequencing terminal of the target polynucleotide chain, the modification fails to pass through the nanopore, thereby causing blocking of the pore, resulting in the completion of the sequencing target polynucleotide The chain is kicked out of the hole back to the cis (Cis) end. In short, during the sequencing process, the helicase is added as a 5'-3' helicase. Under the guidance of the complex, the 5' end of the library enters the hole, and the helicase moves along the 5'-3'. When running to the 3' end, the hole blocking phenomenon will occur because the energy barrier at the end cannot be crossed, and the system will then apply a reverse voltage to kick the hole, and the sequenced single-stranded library will be converted from trans (Trans ) end kicks back to the cis (Cis) end.

其中，在图2中，所述复合物包含所述衔接体，待表征的目标多核苷酸双链和马达蛋白，所述Y1链包含阻断链S和与阻断链S连接的多核苷酸链D'，以及停滞在所述阻断链S上的马达蛋白，所述Y2链与Y1链的互补区域为多核苷酸链L的双链部分；所述YB-Dn链包含修饰部分，该修饰部分在一个具体的实施例中包含PNA，PNA由于不带电荷会导致堵孔，从而最终被踢出。所述YB-Up链与Y1链的互补区域为双链多核苷酸D；其中所述YB-Dn链和YB-Up链通过点击化学连接，具体的实施例中为DBCO和N3。Wherein, in Figure 2, the complex comprises the adaptor, the target polynucleotide duplex to be characterized and a motor protein, the Y1 strand comprises a blocking strand S and a polynucleotide linked to the blocking strand S chain D', and the motor protein stagnated on the blocking chain S, the complementary region of the Y2 chain and the Y1 chain is the double-stranded part of the polynucleotide chain L; the YB-Dn chain contains a modified part, the The modified moiety in a specific embodiment comprises PNA, which, due to its lack of charge, can block the pores and be eventually kicked out. The complementary region of the YB-Up chain and the Y1 chain is a double-stranded polynucleotide D; wherein the YB-Dn chain and the YB-Up chain are connected by click chemistry, in a specific embodiment, DBCO and N3.

与现有技术相比，本发明的技术方案具备以下优点：Compared with the prior art, the technical solution of the present invention has the following advantages:

本发明提供的方法避免了测序完成的文库大量富集在纳米孔测序装置的反式(Trans)端，相较于现有的测序技术，对于反式(Trans)端的因大量富集带电荷核酸可能造成的干扰会大大降低，并进一步提高了反式(Trans)端改造的可变性。The method provided by the present invention avoids that the sequenced library is enriched in the trans (Trans) end of the nanopore sequencing device. The potential interference is greatly reduced and further increases the variability of trans (Trans) end remodeling.

另外，与使用如链霉亲和素和生物素结合产生堵孔的方法相比，使用侧链不带电荷或带正电荷的修饰产生堵孔的方法，在测序时用一种衔接体如Y衔接体即可，制备和操作更简便，功能也更多。In addition, compared with methods such as streptavidin and biotin combined to produce pore blocking methods, using uncharged or positively charged side chain modifications to produce pore blocking methods, use an adaptor such as Y during sequencing The adaptor is enough, which is easier to prepare and operate, and has more functions.

附图说明Description of drawings

为了更清楚地说明本发明实施例的技术方案，下面将对本发明实施例中所需要使用的附图作简单地介绍，显而易见地，下面所描述的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings that need to be used in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

图1示出为使用本发明的衔接体从而避免测序文库在反式端(Trans)大量富集的原理示意图；Figure 1 shows a schematic diagram of the principle of using the adaptor of the present invention to avoid a large enrichment of the sequencing library at the trans end (Trans);

图2示出为一个具体实施例中包含本发明的衔接体、目标多核苷酸和解旋酶的复合物的示意图；Figure 2 shows a schematic diagram of a complex comprising an adaptor of the present invention, a target polynucleotide and a helicase in one embodiment;

其中，所述Y1链包含阻断链S和与阻断链S连接的多核苷酸链D'，以及停滞在所述阻断链S上的马达蛋白，所述Y2链与Y1链的互补区域为多核苷酸链L的双链部分，所述YB-Dn链包含修饰部分，所述YB-Up链与Y1链的互补区域为双链多核苷酸D；其中所述YB-Dn链和YB-Up链通过点击化学连接；Wherein, the Y1 chain comprises a blocking chain S and a polynucleotide chain D' connected to the blocking chain S, and a motor protein stagnated on the blocking chain S, the Y2 chain and the complementary region of the Y1 chain is the double-stranded part of the polynucleotide chain L, the YB-Dn chain includes a modified part, and the complementary region of the YB-Up chain and the Y1 chain is the double-stranded polynucleotide D; wherein the YB-Dn chain and the YB -Up chains are linked by click chemistry;

图3示出为另一个具体实施例中，包含本发明的衔接体、目标多核苷酸和解旋酶的复合物的示意图；其中，将目标多核苷酸的测序终端引入生物素与链霉亲和素复合物，测序进行到末端时电场力不足以将其撕扯来，造成堵孔，从而使得测序链被踢出。3 is a schematic diagram of a complex comprising an adaptor of the present invention, a target polynucleotide and a helicase in another specific embodiment; wherein, the sequencing terminal of the target polynucleotide is introduced into biotin and streptavidin When the sequencing reaches the end, the electric field force is not enough to tear it apart, causing the hole to be blocked, so that the sequencing chain is kicked out.

图4示出为使用本发明的实施例1的衔接体进行测序时的测序信号图；FIG. 4 shows a graph of sequencing signals when sequencing is performed using the adaptor of Example 1 of the present invention;

图5示出为使用本发明的实施例2的衔接体进行测序时的测序信号图；FIG. 5 shows a graph of sequencing signals when sequencing is performed using the adaptor of Example 2 of the present invention;

图6示出为发明的实施例3的衔接体以及使用本发明的实施例3的衔接体进行测序时的测序信号图。FIG. 6 shows a graph of the sequencing signal when the adapter of Example 3 of the present invention is used and the adapter of Example 3 of the present invention is used for sequencing.

具体实施方式Detailed ways

应理解，公开的产品和方法的不同应用可以根据本领域的具体需求而调整。可以理解本文中使用的术语仅是为了描述本发明的具体实施方式的目的，而不意为对本发明的限制。It will be appreciated that different applications of the disclosed products and methods can be tailored to the specific needs of the art. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments of the present invention only, and is not intended to limit the present invention.

另外，除非本文另有明确规定，否则本说明书和随附的权利要求中所使用的单数形式的“一”、“一个”和“所述”包括复数指代。因此，例如，涉及“多核苷酸”时包括两个或多核苷酸，涉及“多核苷酸结合蛋白质包括两个或多个这样的蛋白，涉及“解旋酶”时包括两个或多个解旋酶，涉及“单体”指的是两个或多个单体，涉及“孔”时包括两个或多个孔，等。Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "polynucleotide" includes two or more nucleotides, reference to "polynucleotide-binding protein" includes two or more such proteins, and reference to "helicase" includes two or more helicases Gyrase, reference to "monomer" refers to two or more monomers, reference to "pore" includes two or more pores, etc.

本文所引用的所有公开物、专利和专利申请，无论在前文或在后文，均以引用的方式全文引入。All publications, patents, and patent applications cited herein, whether supra or hereinafter, are incorporated by reference in their entirety.

方法method

本发明提供了一种表征目标多核苷酸的方法，包括：The present invention provides a method for characterizing a target polynucleotide, comprising:

(a)使目标多核苷酸穿过纳米孔移动，(a) moving the target polynucleotide through the nanopore,

其中，所述目标多核苷酸的测序终端包含修饰部分，所述修饰部分使纳米孔堵塞；Wherein, the sequencing terminal of the target polynucleotide comprises a modified part, and the modified part blocks the nanopore;

(b)随着所述多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。(b) taking one or more electrical and/or optical measurements as the polynucleotide moves relative to the pore, wherein the measurements represent one or more characteristics of the polynucleotide and are determined by This characterizes the target polynucleotide.

根据本发明所述的方法，还包括：The method according to the present invention further comprises:

(c)任选地施加反向电压，使所述目标多核苷酸相对于所述纳米孔反向移动，回到所述纳米孔的起始侧。(c) optionally applying a reverse voltage to move the target polynucleotide in the opposite direction relative to the nanopore, back to the start side of the nanopore.

根据本发明所述的方法，其中，步骤(c)不包括：According to the method of the present invention, wherein, step (c) does not include:

随着所述多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。As the polynucleotide moves relative to the pore, one or more electrical and/or optical measurements are taken, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the the target polynucleotide.

根据本发明所述的方法，其中，步骤(c)可以包括：According to the method of the present invention, wherein, step (c) can comprise:

随着所述多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。As the polynucleotide moves relative to the pore, one or more electrical and/or optical measurements are taken, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the the target polynucleotide.

根据本发明所述的方法，其中，步骤(c)中，所述使所述目标多核苷酸相对于所述孔反向移动通过包括如下至少一种方式实现：施加反向电压。According to the method of the present invention, wherein, in step (c), the reverse movement of the target polynucleotide relative to the pore is achieved by at least one of the following methods: applying a reverse voltage.

本发明的方法包括测量所述目标多核苷酸的一个或多个特征。该方法可以包括测量2个，3个，4个，5个或更多个目标多核苷酸的特征。所述一个或多个特征，优选选自(i)目标多核苷酸的长度，(ii)所述目标多核苷酸的同一性，(iii)所述目标多核苷酸的序列，(iv)所述目标多核苷酸的二级结构；以及(v)目标多核苷酸是否是经修饰的。(i)至(v)的任何组合可以根据本发明进行测量。The methods of the present invention comprise measuring one or more characteristics of the target polynucleotide. The method can include measuring the characteristics of 2, 3, 4, 5 or more target polynucleotides. The one or more features are preferably selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the the secondary structure of the target polynucleotide; and (v) whether the target polynucleotide is modified. Any combination of (i) to (v) can be measured according to the present invention.

对于(i)，多核苷酸的长度例如可以通过确定所述目标多核苷酸和所述孔的相互作用数量，以及目标多核苷酸与所述孔相互作用之间的持续时间来测定。For (i), the length of a polynucleotide can be determined, for example, by determining the number of interactions between the target polynucleotide and the pore, and the duration between interactions between the target polynucleotide and the pore.

对于(ii)，所述多核苷酸的同一性可以通过多种方式测定。多核苷酸的同一性可以联合目标多核苷酸的序列的测定，或不联合目标多核苷酸的序列的测定进行测定。前者是直接的；对所述多核苷酸进行测序，并由此进行鉴定。后者可以以几种方式来完成。例如，可以测定多核苷酸中特定模序的存在(而无需测定该多核苷酸的其余序列)。或者，方法中测定的特定的电和/或光信号可鉴定来自特定来源的目标多核苷酸。For (ii), the identity of the polynucleotides can be determined in a variety of ways. The identity of a polynucleotide can be determined in conjunction with the determination of the sequence of the target polynucleotide, or not in conjunction with the determination of the sequence of the target polynucleotide. The former is straightforward; the polynucleotide is sequenced and identified therefrom. The latter can be done in several ways. For example, the presence of a particular motif in a polynucleotide can be determined (without determining the remaining sequence of the polynucleotide). Alternatively, a specific electrical and/or optical signal measured in the method can identify a polynucleotide of interest from a specific source.

对于(iii)，多核苷酸的序列可以如前所述确定。合适的测序方法，特别是那些使用电测量的方法，描述于Stoddart D et al.,Proc Natl Acad Sci,12；106(19):7702-7,Lieberman KR et al,J Am Chem Soc.2010；132(50):17961-72,和国际申请WO 2000/28312中。For (iii), the sequence of the polynucleotide can be determined as previously described. Suitable sequencing methods, especially those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50):17961-72, and in International Application WO 2000/28312.

对于(iv)，所述二级结构可以以多种方式测量。例如，如果该方法包含电测量，二级结构可以利用穿过孔的停留时间的改变或电流变化进行测量。这使得单链和双链多核苷酸的区域能够得到识别。For (iv), the secondary structure can be measured in a variety of ways. For example, if the method includes electrical measurements, secondary structure can be measured using changes in residence time or changes in current through the pores. This allows regions of single- and double-stranded polynucleotides to be identified.

对于(v)，可以测定任何存在或不存在修饰。该方法优选包括确定所述目标多核苷酸是否通过甲基化，氧化，损伤，用一个或多个蛋白质或一个或多个标记物，标签或阻断链进行了修饰。特异性修饰将导致与孔的特异性相互作用，其可以使用下面描述的方法来测定。例如，可以基于孔与每个核苷酸的相互作用过程中穿过孔的电流，识别胞嘧啶与甲基化的胞嘧啶。For (v), the presence or absence of any modification can be determined. The method preferably includes determining whether the target polynucleotide has been modified by methylation, oxidation, damage, with one or more proteins or one or more labels, tags or blocking strands. Specific modifications will result in specific interactions with the pore, which can be determined using the methods described below. For example, cytosines and methylated cytosines can be identified based on the current flowing through the pore during its interaction with each nucleotide.

该方法通常在缓冲剂存在下实施。在上面讨论的示例性设备中，缓冲剂存在所述室的水溶液中。本发明的方法可使用任何缓冲剂。通常地，缓冲剂是磷酸盐缓冲液。其他合适的缓冲剂是HEPES和Tris-HCl缓冲剂。该方法通常在pH值为4.0至12.0，4.5至10.0，5.0至9.0，5.5至8.8，6.0至8.7，7.0至8.8，或7.5至8.5下实施。所使用的pH优选约为7.5。The method is usually carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution of the chamber. Any buffer may be used in the methods of the present invention. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The method is typically carried out at pH values of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

该方法可在0至100℃，15℃至95℃，16℃至90℃，17℃至85℃，18℃至80℃，19℃至70℃，或20℃至60℃实施。该方法通常在室温下进行。该方法可选地在支持解旋酶功能的温度下，例如约37℃实施。The method can be carried out at 0 to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C to 80°C, 19°C to 70°C, or 20°C to 60°C. The method is usually carried out at room temperature. The method is optionally carried out at a temperature that supports helicase function, eg, about 37°C.

该方法可以在游离核苷酸或游离核苷酸类似物和/或辅助解旋酶功能发挥的辅助因子存在下实施。该方法也可以在游离核苷酸或游离核苷酸类似物不存在且解旋酶的辅助因子不存在下实施。所述游离核苷酸可以为如上面讨论的单个核苷酸的任意一个或多个。游离核苷酸包括，但不限于，单磷酸腺苷(AMP)，二磷酸腺苷(ADP)，三磷酸腺苷(ATP)，单磷酸鸟苷(GMP)，二磷酸鸟苷(GDP)，三磷酸鸟苷(GTP)，单磷酸胸苷(TMP)，二磷酸胸苷(TDP)，三磷酸胸苷(TTP)，单磷酸尿苷(UMP)，二磷酸尿苷(UDP)，三磷酸尿苷(UTP)，单磷酸胞苷(CMP)，二磷酸胞苷(CDP)，三磷酸胞苷(CTP)，环磷酸腺苷(cAMP)，环单磷酸鸟苷(cGMP)，脱氧单磷酸腺苷(dAMP)，脱氧二磷酸腺苷(DADP)，脱氧三磷酸腺苷(dATP)，脱氧单磷酸鸟苷(dGMP)，脱氧二磷酸鸟苷(dGDP)，脱氧三磷酸鸟苷(dGTP)，脱氧单磷酸胸苷(dTMP)，脱氧二磷酸胸苷(dTDP)，脱氧三磷酸胸苷(dTTP)，脱氧二磷酸尿苷(dUMP)，脱氧二磷酸尿苷(dUDP)，脱氧三磷酸尿苷(dUTP)，脱氧单磷酸胞苷(dCMP)，脱氧二磷酸胞苷(dCDP)和脱氧三磷酸胞苷(dCTP)。该游离核苷酸优选选自AMP，TMP，GMP，CMP，UMP，dAMP，dTMP，dGMP或dCMP。该游离核苷酸优选三磷酸腺苷(ATP)。解旋酶辅助因子是使解旋酶或构建体发挥功能的因子。解旋酶辅助因子优选为二价金属阳离子。所述二价金属阳离子优选为Mg²⁺，Mn²⁺，Ca²⁺或Co²⁺。解旋酶辅助因子最优选Mg²⁺。The method can be carried out in the presence of free nucleotides or analogs of free nucleotides and/or cofactors that aid in the functioning of the helicase. The method can also be carried out in the absence of free nucleotides or free nucleotide analogs and in the absence of cofactors for helicases. The free nucleotides can be any one or more of the single nucleotides discussed above. Free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate glycoside (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate ( UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate ( dAMP), deoxyadenosine diphosphate (DADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), Deoxythymidine Diphosphate (dTDP), Deoxythymidine Triphosphate (dTTP), Deoxyuridine Diphosphate (dUMP), Deoxyuridine Diphosphate (dUDP), Deoxyuridine Triphosphate (dUTP), Deoxyuridine Diphosphate (dTMP) Cytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). A helicase cofactor is a factor that enables a helicase or construct to function. The helicase cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg²⁺ , Mn²⁺ , Ca²⁺ or Co²⁺ . The helicase cofactor is most preferably Mg²⁺ .

衔接体adaptor

本发明的衔接体包含修饰部分，修饰部分结合到所述目标多核苷酸的测序终端，以使纳米孔堵塞。The adaptor of the present invention comprises a modified moiety that binds to the sequencing terminus of the target polynucleotide to block the nanopore.

其中，所述修饰部分包含侧链不带电荷或侧链带正电荷的修饰部分。可选地，所述侧链不带电荷的修饰部分包括PNA、多肽、和磷酸骨架烷基化修饰的核苷酸中任一种或任两种以上的组合。可选地，所述侧链带正电荷的修饰部分包括磷酸骨架阳离子寡聚物修饰的核苷酸，详见描述于CN101370817A的那些，通过引用将其全部内容并入本文。Wherein, the modified moiety comprises a modified moiety with an uncharged side chain or a positively charged side chain. Optionally, the uncharged modified moiety of the side chain includes any one or a combination of any two or more of PNA, polypeptide, and nucleotides modified by alkylation of the phosphate backbone. Alternatively, the side chain positively charged modified moiety comprises phosphate backbone cationic oligomer modified nucleotides, as described in detail in CN101370817A, which is incorporated herein by reference in its entirety.

具体地，所述阳离子寡核苷酸A_iB_jH具有寡核苷酸部分A_i和寡阳离子部分B_j，其中，A_i是i聚体的寡核苷酸残基，i＝5-50，具有天然或非天然核碱基和/或戊呋喃糖基和/或天然磷酸二酯键。B_j是j聚体的有机寡阳离子部分，j＝1-50，其中B选自包括以下基团的组：Specifically, the cationic oligonucleotide A_i B_j H has an oligonucleotide moiety A_i and an oligocationic moiety B_j , wherein A_i is an i-mer oligonucleotide residue, i=5- 50, with natural or unnatural nucleobases and/or pentofuranosyl and/or natural phosphodiester linkages. B_j is the organo-oligocationic moiety of the j-mer, j = 1-50, where B is selected from the group consisting of:

-HPO₃-R¹-(X-R²_n)_n1-X-R³-O-，其中R¹、R²_n和R³是相同或不同的低级亚烷基，X是NH或NC(NH₂)₂，n1＝2-20，-HPO₃ -R¹ -(XR²_n )_n1 -XR³ -O-, wherein R¹ , R²_n and R³ are the same or different lower alkylene groups, and X is NH or NC(NH₂ )₂ , n1=2-20,

-HPO₃-R⁴-CH(R⁵X¹)-R⁶-O-，其中R⁴是低级亚烷基，R⁵和R⁶是相同或不同的低级亚烷基，X¹为腐胺、亚精胺或精胺残基，-HPO₃ -R⁴ -CH(R⁵ X¹ )-R⁶ -O-, wherein R⁴ is lower alkylene, R⁵ and R⁶ are the same or different lower alkylene, X¹ is putrescine , spermidine or spermine residues,

-HPO₃-R⁷-(aa)_n2-R⁸-O-，其中R⁷是低级亚烷基，R⁸是低级亚烷基、丝氨酸、通过还原天然氨基酸获得的氨基醇，(aa)_n2是含有带阳离子侧链的天然氨基酸如精氨酸、赖氨酸、鸟氨酸、组氨酸、二氨基丙酸的肽，n2＝2-20。-HPO₃ -R⁷ -(aa)_n2 -R⁸ -O-, wherein R⁷ is lower alkylene, R⁸ is lower alkylene, serine, amino alcohol obtained by reducing natural amino acids, (aa)_n2 It is a peptide containing natural amino acids with cationic side chains such as arginine, lysine, ornithine, histidine, and diaminopropionic acid, n2=2-20.

本说明书和权利要求书所使用的“低级烷基”和“低级亚烷基”优选地分别系指任选取代的C₁-C₅直链或支链烷基或亚烷基。As used herein and in the claims, "lower alkyl" and "lower alkylene" preferably refer to optionally substituted_C1 -_C5 straight or branched chain alkyl or alkylene groups, respectively.

例如A选自包括脱氧核糖核苷酸、核糖核苷酸、锁核苷酸(LNA)以及它们的化学修饰或取代如硫代磷酸酯(phosphorothioate)(也被称为硫代磷酸酯(thiophosphate))，2′-氟基，2′-O-烷基或标记基团如荧光剂的组。优选地，所述阳离子寡聚物包括精胺、亚精胺、腐胺中任一种或任两种以上的组合。For example A is selected from the group consisting of deoxyribonucleotides, ribonucleotides, locked nucleotides (LNA) and their chemical modifications or substitutions such as phosphorothioate (also known as thiophosphate) ), 2'-fluoro, 2'-O-alkyl or a group of labelling groups such as fluorescent agents. Preferably, the cationic oligomer includes any one of spermine, spermidine, and putrescine, or a combination of any two or more.

可选地，所述修饰部分包含相互结合的配基和配体，所述配基和配体包括链霉亲和素和生物素、和/或抗原和抗体。Optionally, the modified moiety comprises ligands and ligands that bind to each other, including streptavidin and biotin, and/or antigens and antibodies.

所述修饰部分的长度依据修饰部分化学结构的不同而改变。在一个具体的实施方案中，所述修饰部分为PNA，修饰的多核苷酸可为3-100个多核苷酸，优选5-50个多核苷酸，更优选10-30个多核苷酸，进一步优选13-20个多核苷酸。The length of the modified moiety varies depending on the chemical structure of the modified moiety. In a specific embodiment, the modified part is PNA, and the modified polynucleotide can be 3-100 polynucleotides, preferably 5-50 polynucleotides, more preferably 10-30 polynucleotides, further 13-20 polynucleotides are preferred.

所述衔接体连接到待表征的目标多核苷酸双链的两端，在解旋酶的作用下，所述双链边解旋边相对于孔移动，通过孔的电流变化来确定所述目标多核苷酸的序列，即为链测序。由于本发明的衔接体中修饰部分存在于所述测序链的测序终端，因此在目标多核苷酸链的测序终端，该修饰未能穿越纳米孔从而造成堵孔，导致完成测序的目标多核苷酸链被从孔中踢出回到顺式(Cis)端。The adaptor is connected to both ends of the target polynucleotide duplex to be characterized. Under the action of the helicase, the duplex moves relative to the pore while unwinding, and the target is determined by the current change in the pore. The sequence of a polynucleotide is called strand sequencing. Since the modified part of the adaptor of the present invention exists at the sequencing terminal of the sequencing strand, at the sequencing terminal of the target polynucleotide chain, the modification fails to pass through the nanopore, thereby causing blocking of the pore, resulting in the completion of the sequencing target polynucleotide The chain is kicked out of the hole back to the cis (Cis) end.

其中所述衔接体可以为包含双链区和至少一个单链区的Y型衔接体，或包含双链区并且不包含单链区的E型衔接体。Wherein the adapter can be a Y-type adapter comprising a double-stranded region and at least one single-stranded region, or an E-type adapter comprising a double-stranded region and no single-stranded region.

在一个具体的实施方案中，所述衔接体为Y型衔接体，所述Y型衔接体的修饰部分位于所述Y型衔接体的悬突部分或所述Y型衔接体的修饰部分形成所述Y型衔接体的悬突部分；和/或In a specific embodiment, the adaptor is a Y-shaped adaptor, and the modified part of the Y-shaped adaptor is located where the overhang part of the Y-shaped adaptor or the modified part of the Y-shaped adaptor is formed. the overhanging portion of the Y adaptor; and/or

所述修饰部分包含侧链不带电荷的修饰部分，更优选地，所述侧链不带电荷的修饰部分为PNA或多肽。The modified moiety comprises a modified moiety with an uncharged side chain, more preferably, the modified moiety with an uncharged side chain is a PNA or a polypeptide.

和/或and / or

所述修饰部分共价地连接到所述Y型衔接体或所述修饰通过点击化学反应连接到所述Y型衔接体。The modified moiety is covalently attached to the Y-type adaptor or the modification is attached to the Y-type adaptor through a click chemistry reaction.

具体地，所述Y型衔接体在5'到3'端方向包含{S-D}_n或{D-S}_n，其中，D为包含修饰部分的双链多核苷酸，S为阻断链，n为正整数；Specifically, the Y-shaped adaptor comprises {SD}_n or {DS}_n in the direction from the 5' to 3' end, wherein D is a double-stranded polynucleotide comprising a modified moiety, S is a blocking strand, and n is positive integer;

并且，所述D双链包含与S连接的多核苷酸链D'及所述D'的互补链D”，其中马达蛋白在表征过程中沿S→D'的方向移动，所述修饰部分位于互补链D”。And, the D duplex comprises a polynucleotide chain D' linked to S and a complementary strand D' of the D', wherein the motor protein moves in the direction of S→D' during the characterization process, and the modified part is located in Complementary strand D".

例如，在一个具体的实施方案中，所述解旋酶为5’-3’解旋酶，在复合物的引导下，文库的5’端进孔，解旋酶沿着5‘-3’移位并进行测序；当运行到3’末端时，因为不能跨过末端的能量障碍，会出现堵孔现象，进而使系统施加反向电压踢孔，将测序完成的单链文库从反式(Trans)端踢回到顺式(Cis)端。For example, in a specific embodiment, the helicase is a 5'-3' helicase, and under the guidance of the complex, the 5' end of the library enters the hole, and the helicase is along the 5'-3' Shift and sequence; when running to the 3' end, because the energy barrier at the end cannot be crossed, the hole blocking phenomenon will occur, which in turn causes the system to apply a reverse voltage to kick the hole, and the sequenced single-stranded library will be changed from trans ( The Trans) end kicks back to the cis (Cis) end.

根据本发明所述的衔接体，其中所述衔接体在5'到3'端方向包含{L-S-D}_n或{D-S-L}_n；其中，所述L为多核苷酸链；优选地，所述L的至少部分是双链的；和/或所述L的至少部分是单链的；和/或所述L包含一个或多个阻断分子；和/或所述L包含优先地旋入孔中的先导序列。The adaptor according to the present invention, wherein the adaptor comprises {LSD}_n or {DSL}_n in the 5' to 3' end direction; wherein, the L is a polynucleotide chain; preferably, the L and/or at least a portion of said L is single-stranded; and/or said L comprises one or more blocking molecules; and/or said L comprises preferentially threaded into pores the leading sequence.

可以理解的是，所述L部分为首先接触测序孔的部分。It can be understood that the L part is the part that first contacts the sequencing well.

在一个具体的实施方案中，所述目标多核苷酸被修饰为包括含有先导序列的Y型衔接体和E型衔接体。其中，含有先导序列的Y型衔接体连接到多核苷酸的一端，而E型衔接体连接到另一端，本发明所述的修饰部分位于E型衔接体没有附接至所述目标多核苷酸的一端；所述修饰部分包含亲和物质，所述亲和物质为链霉亲和素和生物素；或所述修饰部分包含胆固醇。In a specific embodiment, the polynucleotide of interest is modified to include a Y-type adaptor and an E-type adaptor containing a leader sequence. Wherein, the Y-type adapter containing the leader sequence is connected to one end of the polynucleotide, and the E-type adapter is connected to the other end, and the modified part of the present invention is located in the E-type adapter that is not attached to the target polynucleotide one end of ; the modified part contains an affinity substance, and the affinity substance is streptavidin and biotin; or the modified part contains cholesterol.

先导序列优先进入纳米孔，且不包含单链区的双链衔接体由于包含修饰部分使纳米孔堵塞，从而无法穿过所述孔，进而使系统施加反向电压踢孔，将测序完成的单链文库从反式(Trans)端踢回到顺式(Cis)端。The leader sequence preferentially enters the nanopore, and the double-stranded adaptor that does not contain the single-stranded region blocks the nanopore due to the modified part, so that it cannot pass through the pore, so that the system applies a reverse voltage to kick the pore, and the sequenced single The strand library is kicked from the trans (Trans) end back to the cis (Cis) end.

在本发明中，关于所述顺式(Cis)端和反式(Trans)端具有以下理解：In the present invention, the cis (Cis) end and the trans (Trans) end have the following understandings:

纳米孔通常具有两个开口：第一开口和第二开口。这样的开口通常被称为纳米孔的顺式开口和反式开口。通常第一开口是顺式开口而第二开口是反式开口。纳米孔中的符号“顺式”和“反式”开口在本领域中是常规的。例如，纳米孔的顺式开口通常面向纳米孔的顺式端，并且反式开口通常面向反式端。可以理解的是，所述顺式端为目标多核苷酸移入纳米孔的端，所述反式端为目标多核苷酸移出纳米孔的端。Nanopores typically have two openings: a first opening and a second opening. Such openings are commonly referred to as cis- and trans-openings of nanopores. Typically the first opening is a cis opening and the second opening is a trans opening. The notation "cis" and "trans" openings in nanopores are conventional in the art. For example, the cis opening of the nanopore typically faces the cis end of the nanopore, and the trans opening typically faces the trans end. It can be understood that the cis end is the end where the target polynucleotide moves into the nanopore, and the trans end is the end where the target polynucleotide moves out of the nanopore.

阻断链block chain

所述一个或多个阻断链包括在目标多核苷酸中。一个或多个阻断链优选是目标多核苷酸的一部分，例如它/它们中断多核苷酸序列。一个或多个阻断链优选不为一个或多个嵌段分子的一部分，该嵌段分子如与目标多核苷酸杂交的减速带。The one or more blocking strands are included in the target polynucleotide. The one or more blocking strands are preferably part of the target polynucleotide, eg it/they interrupt the polynucleotide sequence. The one or more blocking strands are preferably not part of one or more block molecules, such as speed bumps that hybridize to the target polynucleotide.

在目标多核苷酸中具有任意数量的阻断链，如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个或更多个阻断链。优选在目标多核苷酸中具有2个，4个或6个阻断链。目标多核苷酸的不同区域中可具有阻断链，例如前导序列中的阻断链和发卡环中的阻断链。Have any number of blocking strands in the target polynucleotide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more blocks chain. Preferably there are 2, 4 or 6 blocking strands in the target polynucleotide. The target polynucleotide may have blocking strands in different regions, eg, blocking strands in the leader sequence and blocking strands in the hairpin loop.

一个或多个阻断链各提供了能量障碍，所述一个或多个解旋酶甚至在活动模式也不能克服该能量障碍。所述一个或多个阻断链可通过减少解旋酶的牵拉(例如通过除去目标多核苷酸中的核苷酸的碱基)或物理性地阻断一个或多个解旋酶的移动(例如利用庞大的化学基团)来停滞一个或多个解旋酶。The one or more blocking chains each provide an energy barrier that the one or more helicases cannot overcome even in active mode. The one or more blocking strands can block the movement of the one or more helicases by reducing the pull of the helicase (eg, by removing bases of nucleotides in the target polynucleotide) or physically (eg using bulky chemical groups) to arrest one or more helicases.

所述一个或多个阻断链可包括停滞一个或多个解旋酶的任意分子或任意分子的组合。所述一个或多个阻断链可以包括阻止所述一个或多个解旋酶沿目标多核苷酸移动的任意分子或任意分子的组合。其直接地确定在缺少纳米孔和施加的电势的条件下，一个或多个解旋酶是否停留在一个或多个阻断链处。例如，这可如实施例中所示进行测试，例如解旋酶穿过阻断链且置换DNA的互补链的能力可以通过PAGE进行测量。The one or more blocking chains may comprise any molecule or combination of any molecules that arrest one or more helicases. The one or more blocking strands may comprise any molecule or combination of any molecules that prevents the one or more helicases from moving along the target polynucleotide. It directly determines whether one or more helicases stay at one or more blocking strands in the absence of nanopore and applied potential. For example, this can be tested as shown in the Examples, eg the ability of the helicase to cross the blocking strand and displace the complementary strand of DNA can be measured by PAGE.

一个或多个阻断链通常包括直链分子如聚合物。所述一个或多个阻断链通常具有与目标多核苷酸不同的结构。例如，如果所述目标多核苷酸是DNA，一个或多个阻断链通常不是脱氧核糖核酸。特别是，如果目标多核苷酸是脱氧核糖核酸(DNA)或核糖核酸(RNA)，所述一个或多个阻断链优选包括肽核酸(PNA)，甘油核酸(GNA)，苏糖核酸(TNA)，锁核酸(LNA)或具有核苷酸侧链的合成聚合物。The one or more blocking chains typically comprise linear molecules such as polymers. The one or more blocking strands typically have a different structure than the target polynucleotide. For example, if the target polynucleotide is DNA, one or more blocking strands are typically not deoxyribonucleic acid. In particular, if the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the one or more blocking strands preferably comprise peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA) ), locked nucleic acids (LNA) or synthetic polymers with nucleotide side chains.

一个或多个阻断链优选包括一个或多个硝基吲哚，例如一个或多个5-硝基吲哚，一个或多个肌苷，一个或多个吖啶，一个或多个2-氨基嘌呤，一个或多个2-6-二氨基嘌呤，一个或多个5-溴-脱氧尿嘧啶，一个或多个反向胸苷(反向dTs)，一个或多个反向脱氧胸苷(ddTs)，一个或多个二脱氧胞苷(ddCs)，一个或多个5-甲基胞苷，一个或多个5-羟甲基胞苷，一个或多个2’烷氧基修饰的核糖核苷酸(优选2’甲氧基修饰的核糖核苷酸)，一个或多个异脱氧胞苷(异-dCs)，一个或多个异脱氧鸟苷(异dGs)，一个或多个iSpC3基团(即缺少糖和碱基的核苷酸)，一个或多个光裂解(PC)基团，一个或多个己二醇基团，一个或多个阻断链9(iSp9)基团，一个或多个阻断链18(iSp18)基团，聚合物或一个或多个硫醇连接。所述一个或多个阻断链可包括这些基团的任意组合。许多这些基团可以购自(Integrated DNA)。The one or more blocking chains preferably comprise one or more nitroindoles, such as one or more 5-nitroindole, one or more inosine, one or more acridine, one or more 2- Aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuracils, one or more reverse thymidines (reverse dTs), one or more reverse deoxythymidines (ddTs), one or more dideoxycytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'alkoxy modified Ribonucleotides (preferably 2' methoxy-modified ribonucleotides), one or more isodeoxycytidines (iso-dCs), one or more isodeoxyguanosines (iso-dGs), one or more iSpC3 groups (i.e. nucleotides lacking sugars and bases), one or more photocleavable (PC) groups, one or more hexanediol groups, one or more blocking strand 9 (iSp9) groups group, one or more blocking chain 18 (iSp18) groups, polymer or one or more thiol linkages. The one or more blocking chains can include any combination of these groups. Many of these groups are commercially available from (Integrated DNA).

所述一个或多个阻断链可包含任何数量的这些基团。例如，对于2-氨基嘌呤，2-6-二氨基嘌呤，5-溴脱氧尿苷，反向dTs，ddTs，ddCs，5-甲基胞苷，5-羟甲基胞苷，2’烷氧基修饰的核糖核苷酸(优选2’甲氧基修饰的核糖核苷酸)，异dCs，异dGs，iSpC3基团，PC基团，己二醇基团和硫醇连接，一个或多个阻断链优选包含2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多。一个或多个阻断链优选包含2个、3个、4个、5个、6个、7个、8个或更多iSp9基团。一个或多个阻断链优选包含2个、3个、4个、5个或6个或更多iSp18基团。最优选的阻断链基团是4个iSp18基团。The one or more blocking chains may contain any number of these groups. For example, for 2-aminopurine, 2-6-diaminopurine, 5-bromodeoxyuridine, reverse dTs, ddTs, ddCs, 5-methylcytidine, 5-hydroxymethylcytidine, 2'alkoxy base-modified ribonucleotides (preferably 2' methoxy-modified ribonucleotides), iso-dCs, iso-dGs, iSpC3 groups, PC groups, hexanediol groups and thiol linkages, one or more Blocking chains preferably comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. The one or more blocking chains preferably comprise 2, 3, 4, 5, 6, 7, 8 or more iSp9 groups. The one or more blocking chains preferably comprise 2, 3, 4, 5 or 6 or more iSp18 groups. The most preferred chain blocking groups are 4 iSp18 groups.

聚合物优选为多肽或聚乙二醇(PEG)。所述多肽优选地包含2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多个氨基酸。所述PEG优选包含2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多单体单元。The polymer is preferably a polypeptide or polyethylene glycol (PEG). The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomer units.

一个或多个阻断链优选包括一个或多个无碱基核苷酸(即缺乏核碱基的核苷酸)，例如2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多个无碱基的核苷酸。核碱基可以被无碱基核苷酸中的-H(idSp)或-OH置换。无碱基的阻断链可以通过从一个或多个相邻的核苷酸中除去核碱基而被插入到目标多核苷酸中。The one or more blocking strands preferably comprise one or more abasic nucleotides (ie nucleotides lacking nucleobases), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. Nucleobases can be replaced by -H(idSp) or -OH in abasic nucleotides. An abasic blocker strand can be inserted into a target polynucleotide by removing nucleobases from one or more adjacent nucleotides.

一个或多个阻断链优选包含一个或多个物理上导致一个或多个解旋酶停滞的化学基团。所述一个或多个化学基团优选为一个或多个侧挂的化学基团。所述一个或多个化学基团可以连接到目标多核苷酸中的一个或更多个核碱基。所述一个或多个化学基团可以连接到目标多核苷酸的骨架。可存在任何数量，如2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多个这些化学基团。合适的基团包括但不限于，荧光团，链霉亲和素和/或生物素，胆固醇，亚甲基蓝，二硝基苯酚(DNPs)，洋地黄毒苷和/或抗洋地黄毒苷和二苯基环辛炔基团。The one or more blocking strands preferably comprise one or more chemical groups that physically cause the stalling of one or more helicases. The one or more chemical groups are preferably one or more pendant chemical groups. The one or more chemical groups can be attached to one or more nucleobases in the target polynucleotide. The one or more chemical groups can be attached to the backbone of the polynucleotide of interest. Any number, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of these chemical groups may be present. Suitable groups include, but are not limited to, fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin and diphenyl cyclooctyne group.

目标多核苷酸中的不同阻断链可以包含不同停滞分子。例如，一个阻断链可以包括如上讨论的一个线性分子，另一个阻断链可以包括一个或多个物理上导致一个或多个解旋酶停滞的化学基团。阻断链可包括如上讨论的任何线性分子和一个或多个物理上导致一个或多个解旋酶停滞的化学基团，例如一个或多个无碱基和荧光团。Different blocking strands in the target polynucleotide may contain different arresting molecules. For example, one blocking chain can include a linear molecule as discussed above, and the other blocking chain can include one or more chemical groups that physically cause one or more helicases to stall. Blocking strands can include any linear molecule as discussed above and one or more chemical groups that physically cause one or more helicases to stall, such as one or more abasic and fluorophores.

复合物Complex

本发明提供了一种复合物，所述复合物包含本发明所述的衔接体和马达蛋白，其中所述马达蛋白位于阻断链；The present invention provides a complex comprising the adaptor of the present invention and a motor protein, wherein the motor protein is located in the blocking chain;

优选地，所述马达蛋白为能够结合到多核苷酸并且控制其移动穿过孔的蛋白；优选为酶。例如，所述酶选自聚合酶、核酸外切酶、解旋酶和拓扑异构酶中的一种或多种。例如，所述解旋酶选自Hel308解旋酶、RecD解旋酶、Tral解旋酶、TrwC解旋酶、XPD解旋酶和DDA解旋酶中的一种或多种。Preferably, the motor protein is a protein capable of binding to a polynucleotide and controlling its movement through a pore; preferably an enzyme. For example, the enzyme is selected from one or more of polymerases, exonucleases, helicases, and topoisomerases. For example, the helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase, and DDA helicase.

多核苷酸polynucleotide

多核苷酸如核酸是含有两个或更多个核苷酸的大分子。多核苷酸或核酸可包括任何核苷酸的任意组合。核苷酸可以是天然存在的或人工合成的。多核苷酸中的一个或多个核苷酸可以被氧化或甲基化。多核苷酸中的一个或多个核苷酸的可被损坏。例如，多核苷酸可包含嘧啶二聚体。此类二聚体通常与紫外线导致的损坏相关联，且是皮肤黑素瘤的首要原因。多核苷酸中的一个或多个核苷酸可被修饰，例如用标记物或标签。合适的标记物如下所述。Polynucleotides such as nucleic acids are macromolecules containing two or more nucleotides. A polynucleotide or nucleic acid can include any combination of any nucleotides. Nucleotides can be naturally occurring or artificially synthesized. One or more nucleotides in a polynucleotide can be oxidized or methylated. One or more nucleotides in a polynucleotide can be damaged. For example, a polynucleotide can comprise a pyrimidine dimer. Such dimers are often associated with UV-induced damage and are the leading cause of cutaneous melanoma. One or more nucleotides in a polynucleotide can be modified, eg, with a label or tag. Suitable labels are described below.

多核苷酸中的核苷酸通常为核糖核苷酸或脱氧核糖核苷酸。所述多核苷酸可包含以下核苷：腺苷，尿苷，鸟苷和胞苷。所述核苷酸优选脱氧核糖核苷酸。所述多核苷酸优选包括下列核苷：脱氧腺苷(dA)，脱氧尿苷(dU)和/或胸苷(dT)，脱氧鸟苷(dG)和脱氧胞苷(dC)。The nucleotides in a polynucleotide are typically ribonucleotides or deoxyribonucleotides. The polynucleotide may comprise the following nucleosides: adenosine, uridine, guanosine and cytidine. The nucleotides are preferably deoxyribonucleotides. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

核苷酸通常含有单磷酸、二磷酸或三磷酸。磷酸盐可连接在核苷酸的5”或3”侧上。Nucleotides typically contain monophosphates, diphosphates, or triphosphates. Phosphates can be attached on the 5" or 3" side of the nucleotide.

合适的核苷酸包括但不限于，单磷酸腺苷(AMP)、单磷酸鸟苷(GMP)、单磷酸胸苷(TMP)、单磷酸尿苷(UMP)、单磷酸胞苷(CMP)，环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)、脱氧单磷酸腺苷(dAMP)，脱氧单磷酸鸟苷(dGMP)、脱氧单磷酸胸苷(dTMP)、脱氧单磷酸尿苷(dUMP)和脱氧单磷酸胞苷(dCMP)。所述核苷酸优选选自AMP、TMP、GMP、CMP、UMP、dAMP、dTMP、dGMP、dCMP和dUMP。所述核苷酸最优选自dAMP、dTMP、dGMP、dCMP和dUMP。所述多核苷酸优选包括以下核苷酸：dAMP、dUMP和/或dTMP和dCMP。Suitable nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), Cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) ) and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from the group consisting of AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP. The nucleotides are optimally selected from dAMP, dTMP, dGMP, dCMP and dUMP. The polynucleotide preferably comprises the following nucleotides: dAMP, dUMP and/or dTMP and dCMP.

所述多核苷酸中的核苷酸可以任何方式彼此连接。核苷酸通常被它们的糖和的磷酸基连接，如在核酸中。所述核苷酸可通过它们的核碱基的连接，如嘧啶二聚体中。The nucleotides in the polynucleotide can be linked to each other in any manner. Nucleotides are usually linked by their sugars and phosphate groups, as in nucleic acids. The nucleotides may be linked by their nucleobases, as in pyrimidine dimers.

所述多核苷酸可以是核酸。所述多核苷酸可以是任何本领域已知的合成的核酸，例如肽核酸(PNA)、甘油核酸(GNA)、苏糖核酸(TNA)、锁核酸(LNA)，或其它具有核苷酸侧链的合成聚合物。该PNA骨架由通过肽键连接的重复的N-(2-氨基乙基)-甘氨酸单元组成。该GNA骨架由通过磷酸二酯键连接的重复的乙二醇单元组成。该TNA骨架由通过磷酸二酯键连接在一起的重复的苏糖组成。该LNA是由如上所讨论的具有在核糖中连接2′'氧和4”碳的额外的桥的核苷酸形成。The polynucleotide may be a nucleic acid. The polynucleotide can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or others with nucleotide side Chain of synthetic polymers. The PNA backbone consists of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone consists of repeating ethylene glycol units linked by phosphodiester bonds. The TNA backbone consists of repeating threose sugars linked together by phosphodiester bonds. The LNA is formed from nucleotides with an additional bridge linking the 2'' oxygen and the 4' carbon in the ribose as discussed above.

该多核苷酸是最优选核糖核酸(RNA)或脱氧核糖核酸(DNA)。The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

所述多核苷酸可以为任何长度。例如，多核苷酸可以为至少10个，至少50个，至少100，至少150，至少200，至少250，至少300，至少400或至少500个核苷酸长度。所述多核苷酸可以为1000或更多个核苷酸，5000或更多个核苷酸长度或100000或更多个核苷酸长度。The polynucleotides can be of any length. For example, a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides in length. The polynucleotide may be 1000 or more nucleotides, 5000 or more nucleotides in length, or 100,000 or more nucleotides in length.

解旋酶可沿本发明的方法中的全部或仅部分目标多核苷酸移动。全部或部分目标多核苷酸可以使用本发明的方法进行表征。The helicase can move along all or only a portion of the target polynucleotide in the methods of the invention. All or a portion of a polynucleotide of interest can be characterized using the methods of the present invention.

目标多核苷酸可以是单链的。所述目标多核苷酸的至少一部分优选为双链。解旋酶通常结合至单链多核苷酸。如果目标多核苷酸的至少一部分是双链的，所述目标多核苷酸优选地包括单链区域或非杂交区域。所述一个或多个解旋酶能够结合到单链区域或非杂交区域的一条链上。所述目标多核苷酸优选地包括一个或多个单链区域或一个或多个非杂交的区域。The target polynucleotide can be single-stranded. At least a portion of the target polynucleotide is preferably double-stranded. Helicases typically bind to single-stranded polynucleotides. If at least a portion of the target polynucleotide is double-stranded, the target polynucleotide preferably includes a single-stranded region or a non-hybridizing region. The one or more helicases are capable of binding to one strand of the single-stranded region or the non-hybridizing region. The target polynucleotide preferably includes one or more single-stranded regions or one or more non-hybridizing regions.

样品sample

目标多核苷酸存在于任何合适的样品中。本发明通常在已知含有或怀疑含有目标多核苷酸的样品上实施。替换的，可以对样本实施本发明，以确认所识别的一个或多个目标多核苷酸，所述目标多核苷酸已知或预期存在于所述样本中。The polynucleotide of interest is present in any suitable sample. The present invention is generally practiced on samples known to contain or suspected of containing the polynucleotide of interest. Alternatively, the present invention can be performed on a sample to identify one or more target polynucleotides that are known or expected to be present in the sample.

所述样品可以是生物样品。本发明可以针对从任何生物体或微生物中获得或提取的样品在体外实施。所述生物体或微生物通常是古核的(archaean)，原核的或真核的，并且通常属于以下五界中的一个：植物界，动物界，真菌，原核生物和原生生物。本发明针对从任何病毒中获得或提取的样品在体外实施。所述样品优选是液体样品。样品通常包括患者的体液。所述样品可以是尿液，淋巴液，唾液，粘液或羊水，但优选血液，血浆或血清。通常，所述样品是来源于人的，但可替代地可以是来自其他哺乳动物的，如商业上养殖的动物，如马，牛，绵羊或猪，或者可以是宠物如猫或狗。或者，植物来源的样品通常从商业作物获得，如谷类，豆类，水果或蔬菜，例如小麦，藜，大麦，燕麦，芸苔，玉米，大豆，水稻，香蕉，苹果，西红柿，土豆，葡萄，烟草，菜豆，小扁豆，甘蔗，可可，棉花。The sample may be a biological sample. The present invention can be practiced in vitro on samples obtained or extracted from any organism or microorganism. The organism or microorganism is usually archaean, prokaryotic or eukaryotic, and usually belongs to one of five kingdoms: plant, animal, fungus, prokaryotes and protists. The present invention is practiced in vitro for samples obtained or extracted from any virus. The sample is preferably a liquid sample. The sample typically includes the patient's body fluids. The sample may be urine, lymph, saliva, mucus or amniotic fluid, but preferably blood, plasma or serum. Typically, the sample is of human origin, but may alternatively be from other mammals, such as commercially farmed animals such as horses, cattle, sheep or pigs, or may be pets such as cats or dogs. Alternatively, samples of plant origin are typically obtained from commercial crops, such as cereals, pulses, fruits or vegetables, such as wheat, quinoa, barley, oats, canola, corn, soybeans, rice, bananas, apples, tomatoes, potatoes, grapes, Tobacco, kidney beans, lentils, sugar cane, cocoa, cotton.

所述样品可以是非生物样品。非生物样品优选为液体样品。非生物样品的示例包括外科手术液体，水如饮用水，海水或河水，以及用于实验室测试的试剂。The sample may be a non-biological sample. The non-biological sample is preferably a liquid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, seawater or river water, and reagents for laboratory testing.

样品通常在测试前被处理，例如通过离心或通过膜滤除不需要的分子或细胞，例如红血细胞。可以在获取所述样本后立即进行检测。通常也可以在分析前储存所述样本，优选低于-70℃。Samples are typically processed prior to testing, such as by centrifugation or filtering through membranes to remove unwanted molecules or cells, such as red blood cells. Detection can be performed immediately after taking the sample. The sample can also generally be stored prior to analysis, preferably below -70°C.

点击化学click chemistry

本申请的多核苷酸之间可共价地连接。例如，可使用游离铜点击化学或铜催化的点击化学。由于点击化学令人满意的性质和其对于在多种构建块(building blocks)之间生成共价连接的范围，使得在这些应用中使用点击化学。例如，它是快速的，清洁的并且无毒的，只产生无害的副产物。点击化学是由Kolb等在2001首次介绍的术语，为描述更广泛的一系列强大，有选择性的和模块化的构建块，所述构建块可靠地用于小规模和大规模应用(Kolb HC Finn,MG,Sharp less KB,click chemistry:diverse chemical function froma few good reactions,Angew.Chem.Int.Ed.40(2001)2004-2021)。他们定义了如下一系列严格标准用于点击化学：“反应必须是模块化的，宽的范围，给出非常高的产量，只产生无害的可通过非色谱法去除的副产物，并且是立体定向的(但不必然是对映选择性)。所要求的方法特征包括简单的反应条件(理想地所述方法应对氧气和水不敏感)，容易获得的起始物质和试剂，无溶剂或溶剂的使用，所述溶剂是温和的(例如水)或容易去除的，和简单的产物分离。纯化如果需要必须是通过非色谱法，例如结晶或蒸馏，并且所述产物在生理状态下必须是稳定的”。The polynucleotides of the present application can be covalently linked. For example, free copper click chemistry or copper catalyzed click chemistry can be used. Click chemistry is used in these applications due to its desirable properties and its scope for generating covalent linkages between various building blocks. For example, it is fast, clean and non-toxic, producing only harmless by-products. Click chemistry is a term first introduced by Kolb et al. in 2001 to describe a broader set of robust, selective and modular building blocks that are reliable for small-scale and large-scale applications (Kolb HC Finn, MG, Sharp less KB, click chemistry: reverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021). They define the following set of stringent criteria for click chemistry: "The reaction must be modular, broad in scope, give very high yields, produce only harmless non-chromatographically removable by-products, and be steric Directed (but not necessarily enantioselective). Desired process characteristics include simple reaction conditions (ideally the process should be insensitive to oxygen and water), readily available starting materials and reagents, no solvent or solvents For use, the solvent is mild (e.g. water) or easily removable, and simple product isolation. Purification must be by non-chromatographic methods, such as crystallization or distillation, if desired, and the product must be stable under physiological conditions of".

点击化学的合适例子包括但不限于以下：Suitable examples of click chemistry include, but are not limited to, the following:

(a)游离铜的变体的1,3偶级环加成反应，其中叠氮化物与炔烃在应力例如在环辛烷环中反应；(a) 1,3 bi-cycloaddition reactions of variants of free copper in which azides react with alkynes under stress such as in a cyclooctane ring;

(b)在一个连接体上的氧亲核试剂与在其他连接体上的环氧化合物或氮杂环丙烷反应性部分的反应；和(b) the reaction of an oxygen nucleophile on one linker with an epoxy or aziridine reactive moiety on the other linker; and

(c)施陶丁格连接，其中炔烃部分可被芳基膦取代，导致和叠氮化物的特异性反应，从而得到酰胺键。(c) Staudinger linkages, in which the alkyne moiety can be substituted with an arylphosphine, resulting in a specific reaction with azides, resulting in an amide bond.

优选所述点击化学反应是在炔烃和叠氮化物之间Cu(I)催化的1,3偶极环加成反应。在优选实施例中，第一基团是叠氮化物基团，第二基团是炔烃基团。核酸碱基已被合成，在优选位置中插入叠氮化物和炔烃基团(例如Kocalka P,El-Sagheer AH,Brown T,Rapidand efficient DNA strand cross-linking by click chemistry,Chembiochem.2008.9(8):1280-5)。炔烃基团是从Berry Associates(Michigan,USA)商业可获得的，并且叠氮化物基团是通过ATDBio或IDT bio合成的。Preferably the click chemistry reaction is a Cu(I) catalyzed 1,3 dipolar cycloaddition reaction between an alkyne and an azide. In a preferred embodiment, the first group is an azide group and the second group is an alkyne group. Nucleic acid bases have been synthesized with azide and alkyne groups inserted in preferred positions (eg Kocalka P, El-Sagheer AH, Brown T, Rapid and efficient DNA strand cross-linking by click chemistry, Chembiochem. 2008.9(8): 1280-5). Alkyne groups were commercially available from Berry Associates (Michigan, USA) and azide groups were synthesized by ATDBio or IDT bio.

在本申请的一个具体的实施方案中，优选地反应基团是叠氮化物和己基基团，例如叠氮化物N3和DBCO。In a specific embodiment of the present application, the preferred reactive groups are azide and hexyl groups, such as azide N3 and DBCO.

试剂盒Reagent test kit

再一方面，本发明还提供了一种用于表征多核苷酸的试剂盒，所述试剂盒包含所述的衔接体或所述的复合物。In yet another aspect, the present invention also provides a kit for characterizing a polynucleotide, the kit comprising the adaptor or the complex.

所述试剂盒包括(a)一个或多个衔接体，(b)一个或多个解旋酶。所述试剂盒可包括上面讨论的任何解旋酶和孔。The kit includes (a) one or more adaptors, (b) one or more helicases. The kit can include any of the helicases and wells discussed above.

所述试剂盒还可以包括膜的成分，如形成两性分子层需要的磷脂如脂质双分子层。The kit may also include components of the membrane, such as phospholipids such as lipid bilayers, required to form an amphiphilic layer.

本发明的试剂盒可以另外包含使得上述提及的任何实施例能够实施的一个或多个其它试剂或仪器。这类试剂或仪器包括以下试剂或仪器中的一个或多个：合适的缓冲液(水溶液)，从受体(subject)中得到样品的工具(例如包含针的容器或仪器)，扩增和/或表达多核苷酸的工具，上文定义的膜或压力钳或膜片钳装置。试剂在试剂盒中可以干燥状态存在，使得流体样品重新悬浮试剂。所述试剂盒还可，可选地，包括使本发明的方法中如何使用试剂盒的说明书，或者该方法可用于何种患者的详细信息。所述试剂盒可选地包括促进解旋酶移动的必要的组分(例如ATP和Mg²⁺)。The kits of the present invention may additionally comprise one or more other reagents or instruments that enable any of the above-mentioned embodiments to be practiced. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), means for obtaining a sample from a subject (eg, a container or instrument containing a needle), amplification and/or Or means for expressing a polynucleotide, a membrane or pressure clamp or patch clamp device as defined above. The reagents may be present in the kit in a dry state, allowing fluid samples to resuspend the reagents. The kit may also, optionally, include instructions on how to use the kit in the method of the invention, or details of which patients the method may be used for. The kit optionally includes the necessary components (eg, ATP and^Mg2+ ) to facilitate the movement of the helicase.

实施例1：本发明的Y衔接体-酶复合物的制备及测序Example 1: Preparation and sequencing of the Y adapter-enzyme complex of the present invention

SEQ ID NO:1GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTTSEQ ID NO: 1GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT

SEQ ID NO:2ACTGCTCATTCGGTCCTGCTGACTSEQ ID NO: 2ACTGCTCATTCGGTCCTGCTGACT

SEQ ID NO:3CGACTTCTACCGTTTGACTCCGCSEQ ID NO: 3CGACTTCTACCGTTTGACTCCGC

SEQ ID NO:4GTCAGCAGGACCGAATGAGCAGTSEQ ID NO: 4GTCAGCAGGACCGAATGAGCAGT

SEQ ID NO:5AGTCCAGCACCGACC，其中SEQ ID NO:5由PNA组成。SEQ ID NO: 5AGTCCAGCACCGACC, wherein SEQ ID NO: 5 consists of PNA.

复合物是由4个不同链杂交在一起构成的；The complex is made up of 4 different strands hybridized together;

第一链(Y1)，依次包含前导序列，即iSpC3阻断链，表示为3，其连接到SEQ ID NO:1的5′端，SEQ ID NO:1的3′端依次连接到阻断链iSpC18(表示为8888)和SEQ ID NO:2的5′端。The first strand (Y1), which in turn comprises the leader sequence, i.e. the iSpC3 blocking chain, is denoted as 3, which is connected to the 5' end of SEQ ID NO: 1, and the 3' end of SEQ ID NO: 1 is connected to the blocking chain in turn iSpC18 (denoted 8888) and the 5' end of SEQ ID NO:2.

第二链(Y2)，如SEQ ID NO:3所示。The second strand (Y2), as shown in SEQ ID NO:3.

第三链(YB-Up)，SEQ ID NO:4的5′端包含P，用于连接待表征的多核苷酸，SEQ IDNO:4的3′端包含点击化学基团DBCO；The third strand (YB-Up), the 5' end of SEQ ID NO:4 contains P, used to connect the polynucleotide to be characterized, and the 3' end of SEQ ID NO:4 contains a click chemistry group DBCO;

第四链(YB-Dn)，N-Lys(azide)-OO-AGTCCAGCACCGACC-RR-C，其中O为O-liker(也称为AEEA或eg1)，R为Lys，两者均用于增加溶解性。Fourth strand (YB-Dn), N-Lys(azide)-OO-AGTCCAGCACCGACC-RR-C, where O is O-liker (also known as AEEA or eg1) and R is Lys, both used to increase dissolution sex.

Y1：5’-333333333333333333333333333333GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCT GACT-3’Y1: 5’-33333333333333333333333333333GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCT GACT-3’

Y2：5'-CGACTTCTACCGTTTGACTCCGC-3’Y2: 5'-CGACTTCTACCGTTTGACTCCGC-3'

YB-Up：5'-P-GTCAGCAGGACCGAATGAGCAGT-DBCO-3’YB-Up: 5'-P-GTCAGCAGGACCGAATGAGCAGT-DBCO-3'

YB-Dn：N-Lys(azide)-OO-AGTCCAGCACCGACC-RR-CYB-Dn: N-Lys(azide)-OO-AGTCCAGCACCGACC-RR-C

将等物质的量的YB-Up和YB-Dn混合均匀(浓度从10μM-100μM均可)；将其放置在50℃，反应4h，之后通过Urea-PAGE胶进行连接产物的分离以及纯化，制备得到YB-PNA链。Mix equal amounts of YB-Up and YB-Dn evenly (concentrations can range from 10 μM to 100 μM); place them at 50°C, react for 4 hours, and then separate and purify the ligation products by Urea-PAGE gel to prepare A YB-PNA chain was obtained.

修饰Y型衔接体复合物的制备：将Y1；Y2；YB-PNA三条合成单链以1：1.1：1.1的比例进行退火(从95℃缓慢降温到25℃，降温幅度不超过0.1℃/s)。退火终体系包括160mMHEPES 7.0；200mM NaCl,Y1的终浓度为4-8μM，最终形成Y型衔接体。将Y型衔接体(500nM)与6倍物质的量的T4 Dda-M1G/E94C/C109A/C136A/A360C(3μM)(SEQ ID NO:6所示)在缓冲液(100mM NaAc(pH 7)；1.5mM TMAD)中混合并室温孵育30分钟。得到样品1。Preparation of modified Y-type adaptor complexes: anneal the three synthetic single strands of Y1; Y2; YB-PNA at a ratio of 1:1.1:1.1 (slowly cool down from 95°C to 25°C, and the cooling range does not exceed 0.1°C/s ). The final annealing system includes 160 mM HEPES 7.0; 200 mM NaCl, the final concentration of Y1 is 4-8 μM, and finally the Y-shaped adaptor is formed. The Y-type adaptor (500 nM) was combined with 6 times the amount of T4 Dda-M1G/E94C/C109A/C136A/A360C (3 μM) (SEQ ID NO: 6) in buffer (100 mM NaAc (pH 7); 1.5mM TMAD) and incubated for 30 minutes at room temperature.Sample 1 was obtained.

SEQ ID NO:6SEQ ID NO: 6

GTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYECNVLFEQKEVPDLAKARVLICDEVSMYDRKLFKILLSTIPPWATIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLGKTCETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYVGTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYECNVLFEQKEVPDLAKARVLICDEVSMYDRKLFKILLSTIPPWATIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLGKTCETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV

其中，如图2示出为该复合物的示意图；其中，所述Y1链包含阻断链S和与阻断链S连接的多核苷酸链D'，以及停滞在所述阻断链S上的马达蛋白，所述Y2链与Y1链的互补区域为多核苷酸链L的双链部分，所述YB-Dn链包含修饰部分，所述YB-Up链与Y1链的互补区域为双链多核苷酸D；其中所述YB-Dn链和YB-Up链通过点击化学连接。2 shows a schematic diagram of the complex; wherein, the Y1 chain comprises a blocking chain S and a polynucleotide chain D' connected to the blocking chain S, and is stagnant on the blocking chain S The motor protein, the complementary region of the Y2 chain and the Y1 chain is the double-stranded part of the polynucleotide chain L, the YB-Dn chain contains a modified part, and the complementary region of the YB-Up chain and the Y1 chain is double-stranded Polynucleotide D; wherein the YB-Dn chain and the YB-Up chain are linked by click chemistry.

然后将样品1使用DNAPac PA200柱，使用下列洗脱缓冲液(缓冲液A:20mM Na-CHES,250mM NaCl,4％(W/V)甘油，pH 8.6,缓冲液B:20mM Na-CHES,1M NaCl,4％(W/V)甘油，pH 8.6)进行纯化，将样品1装在所述柱子上，并且用缓冲液A将没有结合到DNA上的酶从柱上洗脱掉。然后用10倍柱体积的0-100％缓冲液B将结合了酶的Y型衔接体复合物进行洗脱。然后汇集主洗脱峰，测量其浓度，即得到本发明的衔接体复合物。Sample 1 was then applied to a DNAPac PA200 column using the following elution buffers (buffer A: 20 mM Na-CHES, 250 mM NaCl, 4% (w/v) glycerol, pH 8.6, buffer B: 20 mM Na-CHES, 1 M NaCl, 4% (w/v) glycerol, pH 8.6) for purification,sample 1 was loaded on the column and the enzyme not bound to DNA was eluted from the column with buffer A. The enzyme-bound Y-adapter complex was then eluted with 10 column volumes of 0-100% buffer B. The main elution peaks are then pooled and their concentrations measured to obtain the adaptor complex of the present invention.

用T4连接酶将2.7Kb的多核苷酸文库(即待测分析物)两端与该衔接体复合物连接起来，然后使用成都齐碳科技有限公司纳米孔测序仪QNome-9604进行测序，测序缓冲液：终浓度10mM HEPEs，100mM MgCl₂，375mM KCl，ATP 100mM，pH 7.1，测序温度：30-40℃。测序结果如图4所示。测序过程中，文库的5’端进孔，解旋酶沿着5‘-3’移位并进行测序；待测物穿过纳米孔会引起电流变化，当运行到3’末端时(即YB-Dn链区域)，由于此区域是PNA，PNA不带电，过孔时不会引起电流变化，系统认为出现了堵孔现象，进而系统会施加反向电压踢孔，将测序完成的单链文库从反式(Trans)端踢回到顺式(Cis)端。Use T4 ligase to connect the two ends of the 2.7Kb polynucleotide library (that is, the analyte to be tested) with the adaptor complex, and then use the nanopore sequencer QNome-9604 of Chengdu Qi Carbon Technology Co., Ltd. for sequencing, sequencing buffer Solution: final concentration of 10 mM HEPEs, 100 mM MgCl₂ , 375 mM KCl,ATP 100 mM, pH 7.1, sequencing temperature: 30-40°C. The sequencing results are shown in Figure 4. During the sequencing process, the 5' end of the library enters the pore, and the helicase is shifted along the 5'-3' and sequenced; the passage of the analyte through the nanopore will cause a current change, and when running to the 3' end (i.e. YB -Dn chain region), since this region is PNA, PNA is not charged, and will not cause current changes when passing through the hole. The system thinks that there is a hole blocking phenomenon, and then the system will apply a reverse voltage to kick the hole, and the sequenced single-stranded library will be completed. Kick back from the trans (Trans) end to the cis (Cis) end.

实施例2：本发明另一种E型衔接体-酶复合物的制备及测序Example 2: Preparation and sequencing of another E-type adapter-enzyme complex of the present invention

SEQ ID NO:7GGTAGTCAGCAGGACCGAATGAGCAGTTTSEQ ID NO: 7GGTAGTCAGCAGGACCGAATGAGCAGTTT

SEQ ID NO:8ACTGCTCATTCGGTCCTGCTGACSEQ ID NO: 8ACTGCTCATTCGGTCCTGCTGAC

E型衔接体序列E-type adaptor sequence

EA-1:5’-P-GGTAGTCAGCAGGACCGAATGAGCAGTTT-biotin-3’EA-1: 5'-P-GGTAGTCAGCAGGACCGAATGAGCAGT TT-biotin-3'

EA-2:5’-ACTGCTCATTCGGTCCTGCTGAC-3’EA-2:5'-ACTGCTCATTCGGTCCTGCTGAC -3'

其中，EA-1的5′端包含P，用于连接待表征的多核苷酸，末端使用生物素(biotin)标记。Among them, the 5' end of EA-1 contains P, which is used to connect the polynucleotide to be characterized, and the end is labeled with biotin.

修饰E型衔接体复合物的制备：将EA-1:EA-2两条合成单链以1：1.1的摩尔比进行退火(从95℃缓慢降温到25℃，降温幅度不超过0.1℃/s)。退火终体系包括160mM HEPES7.0；200mM NaCl，产物终浓度为4-8μM。之后按照1：1的摩尔比加入链霉亲和素，在30℃孵育10min，链霉亲和素与EA-1末端的生物素连接，即获得E型衔接体。图3示出为所述复合物的示意图；其中，将目标多核苷酸的测序终端引入生物素与链霉亲和素复合物，测序进行到末端时电场力不足以将其撕扯来，造成堵孔，从而使得测序链被踢出。Preparation of the modified E-type adaptor complex: anneal the two synthetic single strands of EA-1:EA-2 at a molar ratio of 1:1.1 (slowly cool down from 95°C to 25°C, and the cooling range does not exceed 0.1°C/s ). The final annealing system includes 160 mM HEPES 7.0; 200 mM NaCl, with a final product concentration of 4-8 μM. Then, streptavidin was added at a molar ratio of 1:1, incubated at 30°C for 10 min, and streptavidin was linked to the biotin at the end of EA-1 to obtain an E-type adaptor. Figure 3 is a schematic diagram of the complex; wherein, the sequencing terminal of the target polynucleotide is introduced into the biotin and streptavidin complex, and the electric field force is not enough to tear it off when the sequencing is performed to the terminal, causing blockage holes, thereby allowing the sequencing strand to be kicked out.

用单酶切方法制备非对称的2.7Kb的多核苷酸(即待测分析物)文库，分别加入实施例1制备的Y型衔接体复合物以及本实施例制备的E型衔接体进行连接并且制备纯化，使用成都齐碳科技有限公司纳米孔测序仪QNome-9604进行测序，测序缓冲液：终浓度10mMHEPEs，100mM MgCl₂，375mM KCl，ATP 100mM，pH 7.1，测序温度：30-40℃。测序结果如图5所示。开始测序时，在复合物的引导下，待测物的5’端进孔，解旋酶沿着5‘-3’移位并进行测序；当运行到3’末端时，由于电场力的作用不足以使3‘末端的生物素与链霉亲和素跨过纳米孔，会出现堵孔现象，进而使系统施加反向电压踢孔，将测序完成的单链文库从反式(Trans)端踢回到顺式(Cis)端。An asymmetric 2.7Kb polynucleotide (that is, the analyte to be tested) library was prepared by a single enzyme digestion method, and the Y-type adaptor complex prepared in Example 1 and the E-type adaptor prepared in this example were respectively added for connection and Preparation and purification, using the nanopore sequencer QNome-9604 of Chengdu Qitan Technology Co., Ltd. for sequencing, sequencing buffer: final concentration of 10mM HEPEs, 100mM MgCl₂ , 375mM KCl, ATP 100mM, pH 7.1, sequencing temperature: 30-40 ℃. The sequencing results are shown in Figure 5. At the beginning of sequencing, under the guidance of the complex, the 5' end of the analyte enters the hole, and the helicase is displaced along the 5'-3' and sequenced; when it runs to the 3' end, due to the effect of the electric field force It is not enough for the biotin and streptavidin at the 3' end to cross the nanopore, and the hole blocking phenomenon will occur, and then the system will apply a reverse voltage to kick the hole, and the sequenced single-stranded library will be removed from the trans (Trans) end. Kick back to the cis (Cis) end.

实施例3：本发明的另一种Y衔接体的制备及测序Example 3:Preparation and sequencing of another Y adapter of the present invention

按照实施例1的方法进行，不同之处在于：SEQ ID NO:5的多核苷酸5’端的磷酸进行精胺修饰。According to the method of Example 1, the difference is that: the phosphoric acid at the 5' end of the polynucleotide of SEQ ID NO: 5 is modified with spermine.

复合物是由3个不同链杂交在一起构成的；The complex is formed by the hybridization of 3 different strands;

第一链(Y-Top-1-NS)，依次包含前导序列，即iSpC3阻断链，表示为3，其连接到SEQID NO:1的5′端，SEQ ID NO:1的3′端依次连接到阻断链iSpC18(表示为8888)和SEQ ID NO:2的5′端。The first strand (Y-Top-1-NS), which in turn comprises the leader sequence, i.e. the iSpC3 blocking chain, is denoted as 3, which is connected to the 5' end of SEQ ID NO: 1, and the 3' end of SEQ ID NO: 1 is in turn Ligated to the 5' end of the blocking chain iSpC18 (denoted 8888) and SEQ ID NO:2.

第二链(Y-Top-2-NS)，如SEQ ID NO:3所示。The second strand (Y-Top-2-NS) is shown in SEQ ID NO:3.

第三链(Y-Bottom-S)，SEQ ID NO:4的5′端包含P，用于连接待表征的多核苷酸，SEQ ID NO:4的3′端连接SEQ ID NO:5的5′端，并且The third strand (Y-Bottom-S), the 5' end of SEQ ID NO:4 contains P, used to connect the polynucleotide to be characterized, the 3' end of SEQ ID NO:4 is connected to the 5' of SEQ ID NO:5 ' end, and

SEQ ID NO:5的多核苷酸5’端的磷酸被阳离子寡聚物修饰，即被精胺修饰。The phosphate at the 5' end of the polynucleotide of SEQ ID NO: 5 is modified with a cationic oligomer, ie, with spermine.

Y-Top-1-NS：5’-Y-Top-1-NS: 5'-

333333333333333333333333333333GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCT GACT-3’33333333333333333333333333333GCGGAGTCAAACGGTAGAAGTCGTTTTTTTTTT-8888-ACTGCTCATTCGGTCCTGCT GACT-3’

Y-Top-2-NS：5'-CGACTTCTACCGTTTGACTCCGC-3’Y-Top-2-NS: 5'-CGACTTCTACCGTTTGACTCCGC-3'

Y-Bottom-S:5‘-P-GTCAG CAGGA CCGAA TGAGCAGTSSSAGTCCAGCACCGACC(S代表阳离子寡聚物精胺)Y-Bottom-S: 5'-P-GTCAG CAGGA CCGAA TGAGCAGTSSSAGTCCAGCACCGACC (S stands for cationic oligomer spermine)

测序结果如图6所示。测序过程中，文库的5’端进孔，解旋酶沿着5‘-3’移位并进行测序；待测物穿过纳米孔会引起电流变化，当运行到3’末端时(即YB-Dn链区域)，由于此区域带正电，过孔时不会引起电流变化，系统认为出现了堵孔现象，进而系统会施加反向电压踢孔，将测序完成的单链文库从反式(Trans)端踢回到顺式(Cis)端。The sequencing results are shown in Figure 6. During the sequencing process, the 5' end of the library enters the pore, and the helicase is shifted along the 5'-3' and sequenced; the passage of the analyte through the nanopore will cause a current change, and when running to the 3' end (i.e. YB -Dn chain region), because this region is positively charged, no current change will be caused when passing through the hole, the system thinks that there is a hole blocking phenomenon, and then the system will apply a reverse voltage to kick the hole, and the sequenced single-stranded library will be changed from trans The (Trans) end kicks back to the cis (Cis) end.

另外，本文中术语“和/或”，仅仅是一种描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。另外，本文中字符“/”，一般表示前后关联对象是一种“或”的关系。In addition, the term "and/or" in this article is only an association relationship to describe the associated objects, indicating that there can be three kinds of relationships, for example, A and/or B, it can mean that A exists alone, A and B exist at the same time, There are three cases of B alone. In addition, the character "/" in this document generally indicates that the related objects are an "or" relationship.

应理解，在本发明实施例中，“与A相应的B”表示B与A相关联，根据A可以确定B。但还应理解，根据A确定B并不意味着仅仅根据A确定B，还可以根据A和/或其它信息确定B。It should be understood that, in this embodiment of the present invention, "B corresponding to A" means that B is associated with A, and B can be determined according to A. However, it should also be understood that determining B according to A does not mean that B is only determined according to A, and B may also be determined according to A and/or other information.

以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求的保护范围为准。The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to this. Any person skilled in the art can easily think of various equivalents within the technical scope disclosed by the present invention. Modifications or substitutions should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

序列表sequence listing

<110> 成都齐碳科技有限公司<110> Chengdu Qi Carbon Technology Co., Ltd.

<120> 用于表征目标多核苷酸的方法及衔接体<120> Methods and adapters for characterizing target polynucleotides

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<212> DNA<212> DNA

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Claims (14)

Translated fromChinese

1.表征目标多核苷酸的方法，包括：1. A method of characterizing a polynucleotide of interest, comprising:(a)使目标多核苷酸穿过纳米孔移动，(a) moving the target polynucleotide through the nanopore,其中，所述目标多核苷酸的测序终端包含修饰部分，所述修饰部分使所述纳米孔堵塞；Wherein, the sequencing terminal of the target polynucleotide comprises a modified part, and the modified part blocks the nanopore;(b)随着所述目标多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。(b) obtaining one or more electrical and/or optical measurements as the target polynucleotide moves relative to the pore, wherein the measurements represent one or more characteristics of the target polynucleotide, and thereby characterize the target polynucleotide.2.根据权利要求1所述的方法，其中，2. The method of claim 1, wherein,所述纳米孔包括固态纳米孔和/或生物纳米孔；所述生物纳米孔包括跨膜孔；所述跨膜孔包括跨膜蛋白孔。The nanopores include solid state nanopores and/or biological nanopores; the biological nanopores include transmembrane pores; the transmembrane pores include transmembrane protein pores.3.根据权利要求1或2所述的方法，还包括：3. The method of claim 1 or 2, further comprising:(c)使所述目标多核苷酸相对于所述纳米孔反向移动，回到所述纳米孔的起始侧。(c) moving the target polynucleotide in the opposite direction relative to the nanopore, back to the start side of the nanopore.4.根据权利要求3所述的方法，其中，步骤(c)不包括：4. The method of claim 3, wherein step (c) does not include:随着所述目标多核苷酸相对于所述孔移动，获取一个或多个电和/或光测量值，其中所述测量值代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸；和/或As the target polynucleotide moves relative to the pore, one or more electrical and/or optical measurements are taken, wherein the measurements represent one or more characteristics of the target polynucleotide, and thereby characterizing the target polynucleotide; and/or步骤(c)中，所述使所述目标多核苷酸相对于所述孔反向移动通过包括如下至少一种方式实现：施加反向电压，原子力显微镜牵引和/或使所述目标多核苷酸相对于所述纳米孔反向移动的反向酶的牵引。In step (c), the reverse movement of the target polynucleotide relative to the pore is achieved by including at least one of the following methods: applying a reverse voltage, pulling and/or making the target polynucleotide by an atomic force microscope. Traction of the reverse enzyme moving in the opposite direction relative to the nanopore.5.根据权利要求1至4中任一项所述的方法，其中，步骤(a)包括：5. The method of any one of claims 1 to 4, wherein step (a) comprises:将包含所述修饰部分的衔接体附接到所述目标多核苷酸，以使所述目标多核苷酸的测序终端包含所述修饰部分；和/或attaching an adaptor comprising the modified moiety to the target polynucleotide such that the sequencing terminus of the target polynucleotide comprises the modified moiety; and/or所述修饰部分包含侧链不带电荷或侧链带正电荷的修饰部分；和/或The modified moiety comprises a modified moiety with an uncharged side chain or a positively charged side chain; and/or所述侧链不带电荷的修饰部分包括PNA、多肽和磷酸骨架烷基化修饰的核苷酸中任一种或任两种以上的组合；和/或The modified moieties with uncharged side chains include any one or a combination of any two or more of PNAs, polypeptides, and phosphate backbone alkylation-modified nucleotides; and/or所述侧链带正电荷的修饰部分包括磷酸骨架阳离子寡聚物修饰的核苷酸；和/或The side chain positively charged modified moiety comprises a phosphate backbone cationic oligomer modified nucleotide; and/or所述阳离子寡聚物包括精胺、亚精胺、腐胺任一种或任两种以上的组合；和/或The cationic oligomer includes spermine, spermidine, putrescine, or any combination of any two or more; and/or所述修饰部分包含相互结合的配基和配体，所述配基和配体包括链霉亲和素和生物素，和/或The modified moiety comprises a ligand and a ligand that bind to each other, the ligand and ligand including streptavidin and biotin, and/or抗原和抗体。Antigens and Antibodies.6.根据权利要求5所述的方法，其中，所述衔接体为包含双链区和至少一个单链区的Y型衔接体，或6. The method of claim 5, wherein the adaptor is a Y-shaped adaptor comprising a double-stranded region and at least one single-stranded region, or包含双链区并且不包含单链区的E型衔接体。E-type adapters that contain double-stranded regions and do not contain single-stranded regions.7.根据权利要求6所述的方法，其中，所述衔接体为Y型衔接体，所述Y型衔接体的修饰部分位于所述Y型衔接体的悬突部分或所述Y型衔接体的修饰部分形成所述Y型衔接体的悬突部分；7. The method of claim 6, wherein the adaptor is a Y-shaped adaptor, and the modified portion of the Y-shaped adaptor is located in the overhanging portion of the Y-shaped adaptor or the Y-shaped adaptor The modified portion forms the overhang portion of the Y-shaped adaptor;和/或，所述修饰部分共价地连接到所述Y型衔接体，或所述修饰部分通过点击化学反应连接到所述Y型衔接体。And/or, the modified moiety is covalently attached to the Y-type adaptor, or the modified moiety is attached to the Y-type adaptor through a click chemistry reaction.8.根据权利要求6所述的方法，其中，所述衔接体为E型衔接体，所述修饰部分位于没有附接至所述目标多核苷酸的一端。8. The method of claim 6, wherein the adaptor is an E-type adaptor, and the modified moiety is located at the end not attached to the target polynucleotide.9.根据权利要求1至8中任一项所述的方法，其中，所述衔接体包含阻断链，所述阻断链具有与所述多核苷酸不同的结构，用于阻滞马达蛋白。9. The method of any one of claims 1 to 8, wherein the adaptor comprises a blocking strand having a different structure from the polynucleotide for blocking a motor protein .10.用于表征目标多核苷酸的衔接体，其特征在于，所述衔接体包含修饰部分，所述修饰部分用于结合到所述目标多核苷酸的测序终端，所述修饰部分能够使纳米孔堵塞。10. An adaptor for characterizing a target polynucleotide, characterized in that the adaptor comprises a modified portion for binding to the sequencing terminal of the target polynucleotide, the modified portion capable of enabling nano- The hole is blocked.11.用于表征目标多核苷酸的构建体，所述构建体包含衔接体，和目标多核苷酸；11. A construct for characterizing a polynucleotide of interest, the construct comprising an adaptor, and a polynucleotide of interest;所述衔接体包含修饰部分，所述修饰部分结合到所述目标多核苷酸的测序终端，所述修饰部分能够使纳米孔堵塞；the adaptor comprises a modified moiety that binds to the sequencing terminus of the target polynucleotide, the modified moiety capable of clogging the nanopore;所述目标多核苷酸为双链多核苷酸。The target polynucleotide is a double-stranded polynucleotide.12.用于表征目标多核苷酸的复合物，所述复合物包含衔接体或构建体，和马达蛋白，其中，12. A complex for characterizing a polynucleotide of interest, said complex comprising an adaptor or construct, and a motor protein, wherein,所述构建体包含所述衔接体和目标多核苷酸，The construct comprises the adaptor and a polynucleotide of interest,所述衔接体包含修饰部分，所述修饰部分结合到所述目标多核苷酸的测序终端，所述修饰部分能够使纳米孔堵塞；the adaptor comprises a modified moiety that binds to the sequencing terminus of the target polynucleotide, the modified moiety capable of clogging the nanopore;所述马达蛋白为能够结合到所述目标多核苷酸并且控制其移动穿过孔的蛋白。The motor protein is a protein capable of binding to the target polynucleotide and controlling its movement through the pore.13.根据权利要求12所述的复合物，其中，所述马达蛋白选自聚合酶、核酸外切酶、解旋酶和拓扑异构酶中的一种或多种；13. The complex of claim 12, wherein the motor protein is selected from one or more of a polymerase, an exonuclease, a helicase, and a topoisomerase;所述解旋酶选自Hel308解旋酶、RecD解旋酶、Tral解旋酶、TrwC解旋酶、XPD解旋酶和DDA解旋酶中的一种或多种。The helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase and DDA helicase.14.用于纳米孔表征多核苷酸的试剂盒，所述试剂盒的组成包含：14. A kit for nanopore characterization of polynucleotides, the kit comprising:1)衔接体，所述衔接体包含修饰部分，所述修饰部分用于结合到所述目标多核苷酸的测序终端，所述修饰部分能够使纳米孔堵塞；和1) an adaptor comprising a modified moiety for binding to the sequencing terminus of the target polynucleotide, the modified moiety capable of clogging the nanopore; and2)马达蛋白。2) Motor proteins.

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