WO2021209072A1 - 一种采用Aerolysin纳米孔道的蛋白质/多肽测序方法 - Google Patents
一种采用Aerolysin纳米孔道的蛋白质/多肽测序方法 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
- C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
- C07K1/128—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general sequencing
Definitions
- the invention belongs to the field of biotechnology, and specifically relates to a protein/peptide sequencing method based on Aerolysin nanopores.
- the technical problem to be solved by the present invention is: currently single-molecule protein sequencing is still facing huge challenges, it is urgent to develop a new principle for sensitive detection of 20 kinds of amino acid sequence information, and establish a precise measurement of the amino acid sequence and post-translational modification of a single protein molecule.
- innovative methods are used.
- the present invention provides a method for protein/peptide sequencing based on Aerolysin nanopores, which realizes the specific discrimination of natural amino acids and their post-translational modifications and the precise acquisition of single-molecule protein sequences, including the following Multiple steps:
- step (1) protein unfolding, a single protein molecule must unfold its high-level structure before nanopore sequencing and enter a single nanopore in a linear and linear manner, and a single protein will unfold through temperature regulation and pH regulation. .
- Step (2) Marking the starting point for terminal sequencing.
- Step (3) In the preliminary screening of chargeability, design the preliminary screening of the chargeability of nanopores.
- Step (4) In the unfolding of the tertiary structure of the polypeptide, in order to assist the preliminary screening of charges, further design the tertiary structure unfolding nanopores, that is, construct the unfolding domain at the entrance of the biological nanopores to further open the polypeptide molecular structure, that is, mutant T284 /F/Y/I/L/W or G214/F/Y/I/L/W.
- Step (5) In the orthogonal recognition of amino acids, the linear peptide molecule sequencing for each charged characteristic is used to design nanopores containing at least the following six orthogonal recognitions:
- mutant T218K/R/H/D/E, S278K/R/H/D/E, S276K/R/H/D/E, T274K/R/H/D/E , A224Q/N/D/E/R/K/H intends to realize the recognition of the first type of amino acids, including but not limited to H, R, K, E, D, Q, N, W;
- Step (6) In the restricted perturbation assisted amino acid identification, for the identification errors that may be introduced between some amino acids with small structural differences and isomer amino acids, with specific nanopores to further improve the sequencing accuracy, the introduction of alternating electric fields, optical
- the perturbation measurement system is designed, and the perturbation system perturbation amplifies the nanopore channel at the same time, and the specific nanopore channel is as follows:
- Step (7) The method of single-molecule protein sequence determination is:
- Sample pretreatment Use the method of increasing the temperature to 60-100°C and lowering the pH of the solution to 0-5 to destroy the internal hydrogen bonds of the protein, and at the same time use tris(2-carboxyethyl)phosphine (TCEP) or dithiothionine Sugar alcohol (DTT) reducing agent destroys the SS bond of the protein, releases and linearizes the polypeptide chain in a single protein;
- TCEP tris(2-carboxyethyl)phosphine
- DTT dithiothionine Sugar alcohol
- Nanopores are used to specifically identify amino acid sequences
- the ion current limit domain perturbation technology is used, combined with the specific design of nanopores to amplify the influence of temperature disturbances, alternating electric field disturbances and optical disturbances on ion mobility in the pores. Further identify the characteristics of ion migration frequency, thereby improving the amino acid recognition ability of the nanopore measurement interface, and accurately obtaining protein single-molecule sequence information;
- the present invention has the following beneficial effects:
- the present invention uses methods such as increasing the temperature and lowering the pH of the solution to destroy the internal hydrogen bonds of the protein, and at the same time uses reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) to destroy its SS. Bond, release and linearize multiple polypeptide chains in a single protein.
- TCEP tris(2-carboxyethyl)phosphine
- DTT dithiothreitol
- a polypeptide molecule can enter the nanopore from the N-terminus or the C-terminus. If the amino acid sequence is read out sequentially based on the characteristic signal of the time-series nanopore, it is urgent to determine the end position of a single polypeptide molecule entering the nanopore, that is, to determine the starting direction of sequencing.
- the present invention specifically modifies the peptide nucleic acid PNA (such as a PNA sequence containing multiple adenines), oligonucleotides, polypeptide chains or organic functional groups (such as FAM) at the N-terminus of the polypeptide to make it
- PNA peptide nucleic acid
- oligonucleotides such as a PNA sequence containing multiple adenines
- polypeptide chains such as FAM
- organic functional groups such as FAM
- the present invention adopts denaturing agents (such as guanidine hydrochloride GdHCl) and methods such as designing and constructing "tertiary structure unfolding nanopores" to realize the unfolding of the tertiary structure of the polypeptide.
- denaturing agents such as guanidine hydrochloride GdHCl
- the "tertiary structure unfolding nanopore” design it is planned to biomimize the central amino acid environment of the proteasome 19S domain at the entrance of the Aerolysin nanopore (such as mutant T210Y&S213W, etc.) to enhance the specificity of the entrance of the nanopore with the peptide Non-covalent interactions, and with the help of electrophoresis, electroosmotic flow, dielectrophoresis, and other electric driving forces, gradually destroy the weak interactions within the polypeptide molecules, drive them into the restricted pores and achieve linear unfolding, thereby overcoming The tertiary structure of peptides poses a major challenge to the sequencing of nanopore peptides.
- the present invention designs functionalized Aerolysin nanopores that can drive polypeptides with different charged properties, and initially screens the charged properties of the polypeptides to match the selection of the next orthogonal sequencing nanopores.
- the present invention designs at least 4 kinds of "electric preliminary screening nanopores” to specifically capture 4 types of charged polypeptides, namely, negatively charged polypeptides, positively charged polypeptides, electrically neutral and positively and negatively charged mutually shielding polypeptides, and electrically neutral and positively negatively charged polypeptides. Separate peptides.
- the design of 4 kinds of "electrical preliminary sieve nano-pores" is as follows:
- the present invention constructs at least 6 types of polypeptides due to each charged characteristic Orthogonal recognition nanopores are used to specifically recognize amino acid sequences, as shown below:
- (I) Based on electrostatic interaction, introduce charged amino acids into the existing current sensing area in the channel, such as mutant T218K/R/H/D/E, S278K/R/H/D/E, S276K/R/ H/D/E, T274K/R/H/D/E, A224Q/N/D/E/R/K/H, etc.
- the introduction of charged amino acids will enhance the hydrogen bond, salt bond and cation-p interaction between the channel and the amino acid to be tested, so as to realize the recognition of the first type of amino acids, including but not limited to H, R, K, E , D, Q, N, W.
- Regions such as mutant D222W/H/F/Y, S276F/Y, A224K/R/W, S272W/H, T274W/H/F/Y, etc., enhance the pp interaction between this sensitive region and specific amino acids, cation- P bond interaction, pp interaction, etc., to realize the recognition of the fourth type of amino acids, including but not limited to W, P, F, Y, H, I, L, and V.
- the present invention introduces amino acids that can easily form hydrogen bonds in the region near the entrance of each orthogonal recognition nanopore, and adjusts the pore structure of this region, thereby designing and constructing a polypeptide secondary structure tag domain.
- the amino acid residues in the pores will form specific regular hydrogen bond interactions with the polypeptides of different secondary structures, thereby inducing specific ion current blocking and specific ion mobility changes, forming secondary structure tag ions
- the flow characteristics are used to calibrate the electrochemical signal of a single protein sequencing ion to reduce noise during data processing.
- the present invention is based on the applicant’s team’s previous research on the ion current migration trajectory in the pores, using ion current limit domain perturbation technology, combined with specifically designed nanopores to amplify temperature disturbances , The influence of alternating electric field perturbation and optical perturbation on ion mobility in the pores, to further identify the characteristics of ion migration frequency, so as to improve the amino acid recognition ability of the nanopore measurement interface, and accurately obtain protein single-molecule sequence information.
- the measurement time of the same kind of polypeptide molecules in the six types of orthogonal recognition nanopores shown in step (6) is different, and the residence time of different amino acids in the sensing sensitive area in each kind of pore is different, resulting in different pores.
- the present invention introduces tag amino acids in the recognition of six major types of amino acids. ), (IV), (VI) orthogonal recognition of isoleucine (I) in nanopores, (II), (III), (V) orthogonal recognition of cysteine in nanopores" C, Types (II), (IV), (VI) orthogonal recognition of tyrosine Y in nanopores, etc.
- Most amino acids to be tested have at least two types of orthogonal recognition nanopores for specific recognition, thus Correct and unify the sequencing time axis in different channels from multiple angles to realize the crossover, correction and precise integration of the ion current signals measured in the six channels.
- the post-translational modification of amino acids in this project can be detected at the same time as the amino acid sequence determination.
- the phosphorylation modification of serine S, threonine T, and histidine H can be identified in the type (I) and (II) orthogonal recognition nanopores shown in step (6).
- the methylation modification of aspartic acid D and glutamic acid E can be identified in the type (I) orthogonal recognition nanopores shown in step (6).
- Asparagine N, threonine T, and serine S The glycosylation modification can be recognized in the type (I), (II), (V) orthogonal recognition nanopores shown in step (6). Therefore, this project can recognize the amino acid sequence of the peptide at the same time, and is expected to be at the same time Realize the precise determination of the type, quantity, and position of post-translational modifications of specific amino acids.
- the temperature disturbance in step (7) is to use temperature changes to significantly change the random vibration of molecules and the interaction between molecules, etc.
- the present invention achieves maximum stimulation by precisely regulating the temperature of the experimental system within the range of 0-40°C. The degree of change in ion mobility caused by the interaction, thereby improving the signal-to-noise ratio of the frequency characteristics of a single amino acid.
- the present invention designs highly confined nanopores to enable diverse interaction types in charge-sensitive areas, such as mutant S236W/H, K238I/L, S256Y/F/W, P249W, V250I/L/F/Y/W, etc., so as to use specific frequency infrared (10000-25000nm) or ultraviolet light (180-400nm) to perturb the weak interaction between the peptide to be tested and the specific amino acid in the channel, such as hydrogen bond, pp Interaction, pp interaction, etc., improve the recognition of amino acids with similar weak interactions and isomers, such as serine S and threonine T, leucine L and isoleucine I, etc.
- one, two, three, four, or five or more characteristics of the protein or polypeptide are measured.
- One or more features are preferably selected from: (i) the sequence of the protein or polypeptide; (ii) whether the protein or polypeptide is modified and the type, position, and number of modified amino acids; (iii) the length of the protein or polypeptide; (iv) The identity of the protein or polypeptide; (v) The conformation of the protein or polypeptide; (vi) The secondary structure of the protein or polypeptide.
- Figure 1 Flow chart of protein/peptide sequencing method.
- Figure 2 The original current traces of the Glu-Gly-Cys and Glu-Cys-Gly tripeptide molecules were detected by the Aerolysin nanopores of the N226Q electrical preliminary screening.
- FIG. 3 T232K electrical preliminary screening Aerolysin nanopore channel detects the original current trajectory of the mixture of Glu-Gly-Cys and Glu-Cys-Gly two tripeptide molecules.
- FIG. 4 T232K/K238Q peptide sequencing Aerolysin nanopores detect the original current trajectories of Glu-Gly-Cys and Glu-Cys-Gly two tripeptide molecules, respectively.
- Figure 5 Wild-type electrical preliminary screening Aerolysin nanopores detect the original current trajectories of the template polypeptide molecule and the two phosphorylated polypeptide molecules respectively.
- FIG. 6 T232K/K238Q phosphorylation detection Aerolysin nanopores detect the original current traces of the template polypeptide molecule and the two phosphorylated polypeptide molecules, respectively.
- a method for sequencing peptide molecules using cysteine-specific Aerolysin nanopores uses Glu as the guide chain, and the amino acid sequences of the two peptide molecules are Glu-Gly-Cys and Glu-Cys-Gly, respectively. Specific steps are as follows:
- Aerolysin monomer protein After the formation of a stable phospholipid bilayer, a voltage of 200mV is applied, and 1 ⁇ L of Aerolysin monomer protein is added to the cis detection cell. Aerolysin monomer self-assembles to form a heptamer and inserts into the phospholipid membrane to form a stable nanopore. At the same time, the ion current jumps, and a stable opening current is obtained at a voltage of 100mV.
- the T232K/K238Q double mutant peptide sequencing channel was further designed, and the mutant Proerolysin protein was expressed and purified using site-directed mutagenesis for channel construction.
- the two peptide molecules produced two blocking signals in the T232K/K238Q double mutant peptide sequencing channel.
- the characteristic double-step blocking signal with a long blocking time is the blocking signal generated by the N-terminal peptide molecule pulled by the guide chain Glu into the pore, while the signal with a short blocking time is the non-guide chain pulling into the pore.
- the blocking signal generated by the peptide molecule can be distinguished according to the shape of the blocking signal, the blocking time and the degree.
- the wild-type electrical preliminary screening channel was designed, and the wild-type Proerolysin protein was expressed and purified for channel construction. The specific steps are as described in the patent CN202010131704.8.
- Aerolysin monomer protein After the formation of a stable phospholipid bilayer, a voltage of 200mV is applied, and 1 ⁇ L of Aerolysin monomer protein is added to the cis detection cell. Aerolysin monomer self-assembles to form a heptamer and inserts into the phospholipid membrane to form a stable nanopore. At the same time, the ion current jumps to obtain a stable opening current.
- the T232K/K238Q double mutant phosphorylation detection channel was further designed, and the mutant Proerolysin protein was expressed and purified using site-directed mutagenesis for channel construction.
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Abstract
Description
Claims (15)
- 一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,包括以下步骤:(1)蛋白质解折叠;(2)端位测序起点标记;(3)带电性初步筛选;(4)多肽三级结构解折叠;(5)氨基酸正交识别;(6)限域微扰辅助氨基酸识别;(7)单分子蛋白质序列测定。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,所述步骤(1)蛋白质解折叠中,单个蛋白质分子在纳米孔道测序前必须解开其高级结构以线性直链的方式进入单个纳米孔道,单个蛋白质在经由温度调控和pH调控的方法将去折叠。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,步骤(2)端位测序起点标记利用特定序列的肽核酸、寡聚核苷酸、多肽链或有机功能团标记去折叠多肽链的N端或C端为测序起点,从而获得离子流起点标签信号。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,步骤(3)带电性初步筛选中,设计电性的初步筛选纳米孔道。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,步骤(4)多肽三级结构解折叠中,为辅助电荷初筛,进一步设计三级结构解折叠纳米孔道,即在生物纳米孔道入口处构建解折叠域以进一步打开多肽分子结构,即突变型T284/F/Y/I/L/W或G214/F/Y/I/L/W。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,步骤(5)氨基酸正交识别中,采用针对每一种带电特性的线性多肽分子测序,设计至少包含以下六种正交识别的纳米孔道:(a)基于静电相互作用,即突变型T218K/R/H/D/E、S278K/R/H/D/E、S276K/R/H/D/E、T274K/R/H/D/E、A224Q/N/D/E/R/K/H拟实现对第一类氨基酸的识别,包括但不仅限于H、R、K、E、D、Q、N、W;(b)基于氢键及亲水作用,即突变型T218N/Q、Q212R/K/H、D209S/T、S276Q/N、D222G/A/S或A224E/D,拟实现对第二类氨基酸的识别,包括但不仅限于Q、N、Y、T、S、C、G、H;其中,组氨酸His中R基的pKa为7,可通过微调pH使其不带电,从而基于其与关键区域的氢键相互作用使其该特异性纳米孔道中实现区分;(c)基于范德华相互作用,即突变型R220S/T/A、D222G/A、S236I/L/V、G270I/L、T232I/L/V、T274G/A/I/L或K238F/Y/W,拟实现对第三类氨基酸的识别,包括但不仅限 于I、L、M、V、P、A、C、G;(d)基于部分氨基酸侧链的大p键,即突变型D222W/H/F/Y、S276F/Y、A224K/R/W、S272W/H或T274W/H/F/Y,拟实现对第四类氨基酸的识别,包括但不仅限于W、P、F、Y、H、I、L、V;(e)基于小位阻效应,即突变型S276F/Y/I/L、S278F/Y/I/L/P、T274W/P、S236W或K238G/W/I/L/F/Y/P,拟实现对第五类小体积氨基酸的识别,包括但不仅限于A、C、G、S、T、V;(f)基于大位阻效应,即突变型T218G/A、S276G/A、S278G/A、T274G/A、N226D/E、Q268S/T/G/A,拟实现第六类大体积氨基酸的识别,包括但不仅限于W、H、I、K、R、Y。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,步骤(6)限域微扰辅助氨基酸识别中,针对部分结构差异较小的氨基酸及异构体氨基酸之间可能引入的识别误差,配合特定纳米孔道进一步提高测序准确性,引入交变电场、光学微扰测量体系,同时设计扰动体系微扰放大纳米孔道,所述特定纳米孔道如下所示:(a)突变型S236D/E/K/H/R、A260D/E/K/H/R、K238H/R/D/E、T240D/E、S256H/R/W结合交变电场微扰体系;(b)突变型S236W/H、K238I/L、S256Y/F/W、P249W、V250I/L/F/Y/W结合光学微扰体系。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,步骤(7)单分子蛋白质序列测定的方法为:(a)使所述蛋白或多肽与所述孔接触,使得所述蛋白或多肽相对于所述孔移动;(b)在所述蛋白或多肽相对于所述孔移动时,测量穿过所述孔的电流,其中所述电流指示所述蛋白或多肽的一或多个特征,包含电流信号的形状、幅值、持续时间,根据数学变换解析出该电流信号的特征,建立多肽数据库进行数据的相互校正,从而表征所述蛋白或多肽。
- 根据权利要求1所述采用Aerolysin纳米孔道蛋白质/多肽测序的方法,具体步骤如下所示:样本前处理:采用升高温度至60-100℃、降低溶液pH至0-5的方法破坏蛋白质内部氢键,同时采用三(2-羧乙基)膦(TCEP)或二硫苏糖醇(DTT)还原剂破坏蛋白质其S-S 键,释放并线性化单个蛋白质中的多肽链;通过在多肽链的N端特异性修饰肽核酸PNA、寡聚核苷酸、多肽链或有机功能团使其在进入纳米孔道的起始或终止产生特异性的离子流阻断台阶信号或荧光信号,从而确定单个多肽分子纳米孔道测序的起点,并为多个正交识别纳米孔道并行测序信号的互相校正提供起始时间标签;采用变性剂以及设计构建“三级结构解折叠纳米孔道”实现多肽三级结构的解折叠,其中,对于“三级结构解折叠纳米孔道”的设计为在Aerolysin纳米孔道入口处仿生构建蛋白酶体19S域的中心氨基酸环境,用于增强纳米孔道入口处与多肽的特异性非共价相互作用,并借助电驱动力,逐步破坏多肽分子内部的弱相互作用,驱动其进入限域孔道内部并实现线性解折叠,从而克服多肽三级结构对纳米孔道多肽测序的一大挑战;设计可驱动不同带电性质多肽的功能化Aerolysin纳米孔道,初步筛选多肽的电荷性质以匹配选择下一步正交测序纳米孔道;基于静电作用、氢键及亲水作用、范德华相互作用、氨基酸大p键相互作用、大位阻效应和小位阻效应,针对由于每一种带电特性的多肽构建6类正交识别纳米孔道用以专一性识别氨基酸序列;在每一个正交识别纳米孔道入口区域引入易形成氢键的氨基酸,并调节该区域限域孔道结构,从而设计构建多肽二级结构标签域,在该区域中,孔道内部氨基酸残疾将与不同二级结构的多肽形成特定规则的氢键相互作用,从而诱导特异性的离子流阻断及特定离子迁移率的变化,形成二级结构标签离子流特征,用以数据处理时对单个蛋白质测序离子流电信号的校准降噪;针对氨基酸正交识别可能存在的氨基酸识别误差,采用离子流限域微扰技术,结合特定设计的纳米孔道放大温度扰动、交变电场扰动及光学扰动对孔道内离子迁移率的影响,进一步识别离子迁移频率特征,从而提高纳米孔道测量界面的氨基酸识别能力,精准获得蛋白质单分子序列信息;在所述蛋白质或多肽相对于所述Aerolysin纳米孔道移动时,进行一或多个测量,测量和分析穿过所述孔的电流,包括电流幅值、频率、形状和持续时间等特征,从而测定所述分析物是否存在或其一或多个特征,根据数学变换解析出该电流信号的特征,建立多肽数据库进行数据的相互校正,从而表征所述蛋白或多肽。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在 于,所述步骤(2)中的有机功能团为FAM、VIC、CY5、HEX、ROX。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,所述步骤(3)中采用的变性剂为盐酸胍或GdHCl。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,所述步骤(3)中所述电驱动力为电泳力、电渗流、介电泳力。
- 根据权利要求1所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,所述步骤(3)中设计可驱动不同带电性质多肽的功能化Aerolysin纳米孔道具体方法为:采用4种“电性初筛纳米孔道”分别针对性捕获4类带电性质多肽即负电荷多肽、正电荷多肽、电中性且正负电荷相互屏蔽多肽以及电中性且正负电荷分离多肽。
- 根据权利要求5所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,所述步骤4种“电性初筛纳米孔道”分别为特异性识别带负电多肽的“电性初筛纳米孔道”、特异性识别带正电多肽的“电性初筛纳米孔道”、特异性识别电中性且正负电荷相互屏蔽多肽的“电性初筛纳米孔道”、特异性识别电中性且正负电荷分离多肽的“电性初筛纳米孔道”;这4钟孔道的设计分别如下:(i)特异性识别带负电多肽的“电性初筛纳米孔道”:通过调节Aerolysin纳米孔道内部关键区域直径或转移孔道内电荷至直径较宽区域,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型T274N/Q/I/L、T232D/E、K238H/D/R/F/A/C/G/Q/E/K/L/M/N/S/Y/T/I/W/P/V或S280T/N/Q/H/I/L,控制其纳米孔道内部由孔道最窄处电荷决定的电渗流为零,并减小介电泳力,使单个带负电多肽由电泳力驱动进入纳米孔道;(ii)特异性识别带正电多肽的“电性初筛纳米孔道”:通过引入或增大Aerolysin纳米孔道内负电荷的分布,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型T274D/E、T218D/E、S276D/E、S278D/E、K238A/N/D/E/Q、R282D/E/S/T/N/Q/A或R220D/E/S/T/N/Q/A,从而调节由孔道内部阳离子决定的电渗流;在实验中,施加反向电压,从而实现对单个正电荷多肽的高效捕获;(iii)特异性识别电中性且正负电荷相互屏蔽多肽的“电性初筛纳米孔道”:对于电中性且其中正负电荷互相屏蔽的多肽,通过在Aerolysin纳米孔道内直径较小区域引入正电荷氨基酸,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型T218K/R/H/N/Q、S276K/R/H、S278K/R/H/N/Q、S274K/R/H、N226K/R/H、S272K/R/H、G270K/R/H、 S228K/R/H、Q268K/R/H、T230K/R/H、A266K/R/H、T232K/R/H、S264K/R/H、G234K/R/H、N262K/R/H、S236K/R/H、A260K/R/H或S280N/Q,用以增强孔道内部由阴离子决定的电渗流,从而增强纳米孔道对于电中性且正负电荷相互屏蔽多肽的捕获效率,获得特异性离子流响应;(iiii)特异性识别电中性且正负电荷分离多肽的“电性初筛纳米孔道”;对于电中性且其中正负电荷分离的多肽,通过增强Aerolysin纳米孔道入口处的电势梯度,即将将Aerolysin纳米孔道中的Aerolysin蛋白变为突变型S280Q/N/A、T284Q/N/A或G214Q/N/A,调控于非线性的电场强度,从而利用介电泳力驱动单个电中性且正负电荷分离多肽进入孔道。
- 根据权利要求5所述一种采用Aerolysin纳米孔道蛋白质/多肽测序方法,其特征在于,所述步骤(5)中6类正交识别纳米孔道构建方法如下所示:(I)基于静电相互作用,在孔道内现有电流传感区域中引入带电荷氨基酸,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型T218K/R/H/D/E、S278K/R/H/D/E、S276K/R/H/D/E、T274K/R/H/D/E或A224Q/N/D/E/R/K/H;带电荷氨基酸的引入会增强孔道与待测氨基酸之间的氢键、盐键和阳离子-p相互作用,从而实现对第一类氨基酸的识别,包括但不仅限于H、R、K、E、D、Q、N、W;(II)基于氢键及亲水作用,调控孔道内电流传感区域的电势梯度,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型T218N/Q、Q212R/K/H、D209S/T、S276Q/N、D222G/A/S或A224E/D,用以加快带电荷氨基酸通过该区域的速度并延长极性不带电氨基酸在此区域内的停留时间,从而实现对第二类氨基酸的识别,包括但不仅限于Q、N、Y、T、S、C、G、H;其中,组氨酸H中R基的pKa约7,可通过微调pH使其不带电,从而基于其与纳米孔道关键传感区域的氢键相互作用,使其在该特异性纳米孔道中实现特征区分;(Ⅲ)基于范德华相互作用,调控孔道整体的电势分布和孔道立体结构分布,引入疏水氨基酸域,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型R220S/T/A、D222G/A、S236I/L/V、G270I/L、T232I/L/V、T274G/A/I/L或K238F/Y/W,使得电流传感区域从野生型孔道的静电敏感域转移到突变型孔道的疏水域中,从而通过相互作用延长特定氨基酸在该区域的停留时间,获得特征离子流信号,从而拟实现对第三类氨基酸的识别,包括但不仅限于I、L、M、V、P、A、C、G;(IV)基于部分氨基酸大p键相互作用,在调控孔道立体结构和电势分布的基础上,重构Aerolysin纳米孔道内的电流传感区域组成,构建以正电荷氨基酸及输水氨基酸为主导的灵敏区域,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型D222W/H/F/Y、S276F/Y、A224K/R/W、S272W/H或T274W/H/F/Y,用以增强该灵敏区域与特定氨基酸的p-p相互作用、阳离子-p键相互作用、p-p相互作用,从而实现对第四类氨基酸的识别,包括但不仅限于W、P、F、Y、H、I、L、V;(V)基于大位阻效应,进一步减小孔道内电流传感区域的限域空间,增大该区域的位阻,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型S276F/Y/I/L、S278F/Y/I/L/P、T274W/P、S236W或K238G/W/I/L/F/Y/P,从而延长所有氨基酸通过该区域的时间,增强小体积氨基酸的离子流信号电流幅值,并使得大体积氨基酸造成近乎全阻断离子流台阶,从而特异性区分氨基酸体积,实现对第五类小体积氨基酸的识别,包括但不仅限于A、C、G、S、T、V;(VI)基于小位阻效应,调控孔道内的立体结构,增大电流关键区域的尺寸,即将Aerolysin纳米孔道中的Aerolysin蛋白改为突变型T218G/A、S276G/A、S278G/A、T274G/A、N226D/E或Q268S/T/G/A,并基于多肽整体的带电荷类型减小纳米孔道内的电渗流,从而进一步减小小体积氨基酸的电流响应,增强大体积氨基酸的电流差异,实现第六类大体积氨基酸的识别,包括但不仅限于W、H、I、K、R、Y。
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