CN118086286A - A DNA-peptide conjugate, ARCFU-like helicase and its application in detecting peptide segments - Google Patents
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Abstract
The invention discloses a DNA-peptide conjugate, ARCFU-like helicase and application thereof in detecting peptide fragments, and relates to the technical fields of gene sequencing, molecular detection and clinical detection. The DNA-peptide conjugate is capable of passing through the nanopore by a helicase on the DNA fragment, and the ARCFU-like helicase is used to control the rate at which the DNA, RNA or peptide fragment passes through the nanopore. The invention controls the speed of the peptide-DNA sample passing through the biological nano-pore by ARCFU-like helicase, thereby detecting the virus in the sample.
Description
Technical Field
The invention belongs to the technical fields of gene sequencing, molecular detection and clinical detection, and particularly relates to a DNA-peptide conjugate, ARCFU-like helicase and application thereof in detecting peptide fragments.
Background
Biological nanopores have been used as the basis for single molecule DNA sequencing technology that enables long reads and epigenetic marker detection on portable platforms at minimal cost. As described in patent CN111615560a, single-stranded DNA slowly passes step by step through a protein nanopore embedded in a membrane, partially blocking the current carried by the ions through the nanopore. DNA stepping is accomplished using DNA translocation sport enzymes that move DNA through the pore in discrete steps, creating a series of steps in the ion stream. Each ion current level characterizes the bases residing in the well in this step, and the level sequence can be decoded into a DNA base sequence.
Helicases are the most common rate controlling enzyme in controlling DNA or RNA passage through protein nanopores, and can be divided into 6 superfamilies according to the sequence, function and oligomerization status of the helicases, with helicase families 1 and 2 being non-hexameric helicases, which are most widely distributed and found in all genomes sequenced to date. These families have been under intense study in the last few years because they are associated with a number of diseases, including cancer, neurodegenerative diseases and infectious diseases.
Gene sequences are an important source of primary sequence information for proteins. However, neither the DNA genome nor the RNA transcriptome can fully describe the phenotype of the protein, as they do not directly encode the abundance of the protein or the post-translational modifications and splicing of the protein. A powerful method for directly identifying proteins and detecting post-translational modifications at the single molecule level would greatly facilitate proteomic studies, enabling quantification of low abundance proteins and distribution and correlation of post-translational modifications at the single cell level.
Therefore, the invention provides a peptide fragment detection method for the nanopore-based method, and can provide specific site information about the peptide primary sequence, thereby helping find application in single-molecule protein fingerprint and mutation identification and laying a good foundation for development of single-molecule protein fingerprint and analysis technology.
Disclosure of Invention
Aiming at the defects and problems in the prior art, the invention provides a DNA-peptide conjugate, ARCFU-like helicase and application thereof in detecting peptide fragments. The invention provides a proteomics tool capable of identifying single protein, which has important significance for research and application of cell biology, and also shows a single-molecule peptide reader based on a nanopore, which is sensitive to single amino acid substitution in single peptide, and the DNA helicase ARCFU-like provided by the invention pulls a DNA-peptide conjugate through an MspA mutant biological nanopore, and the single amino acid substitution can be identified in single reading through a nanometer Kong Douqu ion current signal.
The invention solves the technical problems by adopting the scheme that: a DNA-peptide conjugate consisting of a peptide linked to a ssDNA strand by a click chemistry linker, the DNA-peptide conjugate being extended with a typical nanopore linker consisting of an extension that is the helicase helication site, a10 base oligonucleotide complementary to a template and an extender, a short peg, enabling the two oligonucleotides to be effectively linked, and a complementary oligonucleotide complementary strand with 3' cholesterol modification.
Further, the click chemistry connector is DBCO-Azide linker.
The invention also provides ARCFU-like helicase, and the nucleotide sequence of the ARCFU-like helicase is shown as SEQ ID NO. 1.
Further, the amino acid sequence of ARCFU-like helicase is shown as SEQ ID NO. 2.
A recombinant plasmid, expression vector or cell comprising ARCFU-like helicase gene.
Further, the expression vector of ARCFU-like helicase gene is escherichia coli BL21.
The invention also provides application of the DNA-peptide conjugate in detecting the peptide fragment passing through the biological nano hole through the helicase on the DNA fragment.
Further, the DNA-peptide conjugate is capable of passing through a nanopore by a helicase on the DNA fragment, and the ARCFU-like helicase is used to control the rate at which the DNA, RNA, or peptide fragment passes through the nanopore.
Compared with the prior art, the invention has the beneficial effects that:
The invention utilizes the advantages of peptide segment detection through the via hole of the protein nanopore, provides a DNA-peptide conjugate, ARCFU-like helicase and application thereof in peptide segment detection after combining and hybridizing, provides a proteomic tool capable of identifying single protein, has important significance for research and application of cell biology, also displays a single-molecule peptide reader based on the nanopore, is sensitive to single amino acid substitution in single peptide, and pulls the DNA-peptide conjugate through the MspA mutant biological nanopore by the DNA helicase ARCFU-like provided by the invention, and can identify single amino acid substitution in single reading by a nanometer Kong Douqu ion current signal, thereby laying a good foundation for development of single-molecule protein fingerprint and analysis technology. The invention provides a novel ARCFU-like helicase, which is used for controlling the speed of a peptide fragment-DNA sample passing through a biological nano hole in the process of detecting the peptide fragment passing through the biological hole, namely ARCFU-like helicase is used for controlling the speed of the peptide fragment-DNA sample passing through the biological nano hole, so that viruses in the sample are detected.
Drawings
FIG. 1 is a schematic diagram of a hybridization structure of a DNA-peptide conjugate;
FIG. 2 is a schematic structural view of a peptide fragment perforation;
FIG. 3 is a schematic representation of the signal of a peptide fragment passing through a biological nanopore with ARCFU-like helicase;
FIG. 4 is a diagram showing SDS-PAGE results;
FIG. 5 is an electrophoresis chart of the result of ARCFU-like helicase experiments;
FIG. 6 is a graph of the detection signal of peptide fragments passing through a nanopore in the absence of ARCFU-like helicase.
In the figure: ① -peptide fragment; ② -ssDNA strands; ③ -a template expander; ④ -short nails; ⑤ -an oligonucleotide complementary strand.
Detailed Description
The invention will be further described with reference to the drawings and examples.
The DNA-peptide conjugate hybridization structure used in the experiments consisted of four parts:
(1) Peptide fragment detection template strand (N '-DEDDDEDEDEDDDEDEDDEDEDDDD-C' - (linker) -5- 'GCATGCTAGCTACGTACGATCGATCGATCGATCGATCGTAGC-3'): consists of a 42 base nucleotide sequence linked at the 5' end to the C-terminus of a 25 amino acid peptide by azide-DBCO-C5 linkage. This strand is electrophoretically pulled into the nanopore and read by a sequencer.
(2) Complementary strand （C3C3C3C3C3C3C3C3C3C3C3C3C3C3C3C3C3C3C3C3-5'-TGATCAAGCATGCTACGATCGATCGATCGAGATGTACTTTTTTTTTTTTTTTT- 3' -(cholesterol)）： complementary strand to a portion of the template strand, has 3 functions:
(a) 3' cholesterol allows it to bind to the bilayer, increasing the frequency of DNA-pore interactions;
(b) The 5' overhang provides an adhesive end to attach the template extender;
(c) On the hybridization construct, it prevents ARCFU-lik from unwinding along the template strand and processing using ATP until the template strand enters the pore, the complementary region being cleaved by helicase.
(3) Template expander gene
((Phosphate) 5 'CATCCACAGTGAATTGATCAGGACTGTAGGT'): a template expander comprising 31 bases, which binds to the cohesive 5 'end of the complementary region and extends from the cohesive 3' end of itself 10 bases, serves as a binding site for helicase. The length of the DNA-peptide hybrid is increased by the ligation of the expander to avoid the problem that the hybrid is too short in length to be effectively captured by the well.
(4) Staple (5 'GGATGTAATATG'): 10 base oligonucleotides complementary to the template strand and the expander allow for efficient ligation of the two oligonucleotides. Short staples are used to prepare the structure, have no function once assembled in the structure, and are sheared off like complement by helicase upon capture.
As shown in fig. 1, the DNA-peptide conjugate consisted of ① peptides linked to ② ssDNA strands by a click chemistry linker (linker) that was extended with a typical nanopore linker consisting of a ③ extension, ④ short spike ("stable"), 10 base oligonucleotide complementary to template and extender, enabling efficient ligation of the two oligonucleotides, and a complementary strand of complementary oligonucleotide ⑤ with 3' cholesterol modification. Common examples of such click chemistry connectors include tris (3-hydroxypropyl triazidomethyl) amine, diphenyl azide phosphate, DBCO-Cy5, and 3-azido-1-propanol, and the like, with DBCO-Azide linker being used in the present invention.
The assembly process of the DNA-peptide conjugate hybridization structure comprises the following steps:
First, after mixing equal amounts of template, short staple and template extender, i.e., 1:1:1, they were heated to 95℃for 10 minutes in a thermocycler (10 mM NaCl in binding buffer, 5mM HEPES buffer, pH 7.5) and then slowly cooled to room temperature. The mixture was then incubated with T4 DNA ligase (NEB, M0202T) for 1 hour in the buffer provided by the manufacturer. Finally, 1.1 times of the ligation product was mixed with 1. Mu.M of the complementary sequence and annealed in an annealing buffer (annealing buffer 10mM NaCl, 5mM HEPES buffer, pH 7.5).
The DNA-peptide conjugate designed by the invention combines DNA and peptide, and can be used for detecting peptide fragments related to human respiratory viruses; the use of the click chemistry connector enables the conjugate to have the characteristic of bioconjugation, and can be used for identifying, positioning and characterizing new and old biomolecules, so that a novel tool is provided for detecting related viruses, and a novel path is provided for detecting and researching biomolecules.
The invention clones and expresses ARCFU-like helicase of a helicase family 1. Because the DNA, RNA or peptide fragments pass through the nanopore too fast, the speed control enzyme is needed to control the speed, the novel helicase has a nucleotide sequence shown as SEQ ID NO. 1:
gtgaaagagttcatagaggggttgatcaggctaacagaggttgagagagatgctcagatttccgcaatgatggatgagataaggcggttgagcggagagaagagagaaaggaaggggagggctgttctggggctgagaggtaaggtggttggagaagagcttggcttcaagcttgtaaggtacgggaggaggaaagcaatagagaccgagatttctgttggagatgaagtgctaatcagcagaggtgacccgctgaaaagcgatttgagaggagttgtggtagagaaggggagcagataccttaccgtctctctcgaatccgttccggagtgggcgctgagggatgtaagaattgatttgtatgccagcgacctcaccttcaagaggtggattgaaaaccttgagaatctgacggaaaatggaaagagggcgctgaaatttgcccttggccttgaagagccctccaaaacagaatgtgaagacttcaagcctttcgactcttccttaaacagagcgcagctcaaagctgttggctgtgccgtctcaactgacgacttcttcctcattcacggcccctttggaacaggaaagacgagaacagttgttgaggtggtaaggcagctcgttaagaggggtgagagagttctcgttacagctgaaagcaacaccgccgttgacaacctcgttgagcttctctcagacatgaaaatcgtcaggctcggccacccttcgagggttgagaaaaggcttaaggagcacacccttgcctccctcgtcctcaaccaccccgattacaagaggattgaggaaattaaggggaagattgaggagattgaaagaagaatggaaaggctgaccaagccatccccccaattgaggcgtggtctgagcgatgaggagattttaaggcttgccagaagcaacagaggggcgagaggtgttgcagctaaaaaaataaggtcgatggcagagtggattgaggcgagaaaggctcttgaccagctttacacggagatgaaggaagaggaggagagaatcgttaaggagatcattgaggagagcgatgttgtgatttcgacaaactcctccgcctttttgctcgaagaaagctttgatactgcagtaattgacgaagcaagtcaggcaacgattccaagcgttctaataccaataaacagggcgaggaagttcatccttgctggagaccaccgacagctcccgccaacggtaatgaaggcagagaagctttcagagactctcttcgagaagctaattgagctctaccctgaaaaatcccagcttctaaacgttcagtacaggatgaatgagaaactgatggagtttccaagcagggagttctacggcggaaggatagtggcgcatgagagctgcacagcgatagccttaagccagatagcgaagagagaggctgagaagctgagagagatacttggagatgagcccctggtgttcatagacacctcaaaatgcaagaacaggtgggagggaaagcttgcggattcaacgtctaggtacaacaggcttgaggcagaaattgtgacagaaatcgttaccgagttgctgaaaatgggcttaaaaaaggagcagattggagttataaccccctacgatgaccaggttgacctgctgagggagaaggttgacgtggaggtcagcagcgttgacggatttcaagggagggagaaggaggttataatcatttcattcgtcagaagcaacaggaagagagagattggcttccttgatgacctgagaaggctgaacgtttcactgacgagggcgagaaggaagctgataatggttggcgattcggaaactctaagtgttaatggcacctatgcaaggctgatagatcacgtcaaaagaaagggtgtttacgttgagctggataaaaatgggaagcttggtggcaatccgaagggc（SEQ ID NO.1）
The amino acid sequence of the helicase is shown as SEQ ID NO. 2:
MKEFIEGLIRLTEVERDAQISAMMDEIRRLSGEKRERKGRAVLGLRGKVVGEELGFKLVRYGRRKAIETEISVGDEVLISRGDPLKSDLRGVVVEKGSRYLTVSLESVPEWALRDVRIDLYASDLTFKRWIENLENLTENGKRALKFALGLEEPSKTECEDFKPFDSSLNRAQLKAVGCAVSTDDFFLIHGPFGTGKTRTVVEVVRQLVKRGERVLVTAESNTAVDNLVELLSDMKIVRLGHPSRVEKRLKEHTLASLVLNHPDYKRIEEIKGKIEEIERRMERLTKPSPQLRRGLSDEEILRLARSNRGARGVAAKKIRSMAEWIEARKALDQLYTEMKEEEERIVKEIIEESDVVISTNSSAFLLEESFDTAVIDEASQATIPSVLIPINRARKFILAGDHRQLPPTVMKAEKLSETLFEKLIELYPEKSQLLNVQYRMNEKLMEFPSREFYGGRIVAHESCTAIALSQIAKREAEKLREILGDEPLVFIDTSKCKNRWEGKLADSTSRYNRLEAEIVTEIVTELLKMGLKKEQIGVITPYDDQVDLLREKVDVEVSSVDGFQGREKEVIIISFVRSNRKREIGFLDDLRRLNVSLTRARRKLIMVGDSETLSVNGTYARLIDHVKRKGVYVELDKNGKLGGNPKG（SEQ ID NO.2）
further, the cloning and expression process of ARCFU-like helicase comprises the following steps:
(1) Transformation of the general biosynthetic pET29a (+) recombinant plasmid: after codon optimization, the recombinant plasmid is transformed into BL21 (DE 3) competent cells by a heat shock method, and the competent cells are inversely cultured overnight at 37 ℃ after plating.
(2) Expression identification, optimization and solubility analysis
Selecting a monoclonal to culture in a 5-tube LB culture medium at 37 ℃ until the OD600 of the thalli is 0.6-0.8, adding IPTG to a final concentration of 0.5mM, culturing for 4 hours at 37 ℃, centrifuging, collecting bacteria, preparing a sample, and analyzing SDS-PAGE and Western Blot;
Adding IPTG to the 4-tube culture which is inoculated with the seed retaining bacteria and cultured until the bacterial OD600 is 0.6-0.8, wherein the final concentration is 0.2mM and 1mM respectively, culturing for 4 hours and 16 hours at 37 ℃ and 15 ℃ respectively and 220 rpm, and inducing the expression of fusion proteins, and performing SDS-PAGE analysis under various conditions;
and (3) taking bacterial liquid under each condition in the previous step, centrifugally collecting bacterial bodies, crushing (2 mM Tris-0.5M NaCl buffer, pH 8.0), and respectively preparing supernatant precipitates for sample preparation and SDS-PAGE analysis.
The analysis results were as follows:
(1) Expression identification: the target protein is expressed by SDS-PAGE and Western Blot detection.
(2) Solubility analysis: the target protein was soluble as determined by SDS-PAGE.
The invention uses the heterologous expression system to clone the gene into the artificial vector, and the invention selects the escherichia coli BL21 as the expression vector of the helicase, has simple gene operation, easy escherichia coli culture, low cost and good tolerance of a plurality of foreign proteins, and can be expressed at a high level. Heterologous expression systems also allow simple modification of the protein to optimize expression, mutation analysis of specific sites within the protein, and facilitate purification of the protein using engineering affinity tags. In addition, some purification of the target protein is required for functional analysis. The invention selects GST label for purification, and fusion of protein and carrier protein can increase solubility, change positioning, provide means for affinity purification or cause reaction in immunological analysis, and various cleavable fusion labels are helpful for purification and detection.
The invention clones and expresses ARCFU-like helicase of helicase family 1 and uses the helicase to open double chains and control the time of template chains passing through protein nanopores, and the helicase can help control the speed of DNA, RNA or peptide fragments passing through the nanopores, so that researchers can more accurately detect and analyze related viruses, and bring new prospects for biomedical research and clinical diagnosis.
The present invention is based on a DNA-peptide conjugate consisting of an 80 nucleotide DNA strand covalently linked to an amino acid synthetic peptide via a DBCO click linker located at the 5' end of the DNA with a peptide c-terminal azide modification by means of helicase on the DNA fragment to pass through a biological nanopore, see fig. 2. A negatively charged peptide sequence consisting essentially of aspartic (D) and glutamic (E) residues is selected so that the electrophoretic forces assist in pulling the peptide into the well.
The invention establishes a single MspA mutant well in the lipid bilayer. Briefly, a1, 2-bis phytanyl-n-glycol-3-phosphocholine (Avanti Polar Lipids) lipid bilayer was formed over a Teflon horizontal pore diameter of about 20 μm in diameter. The bilayer was flanked by 60. Mu.L of compartments containing 0.4M KCl, 1 mM EDTA, 1 mM DTT, 10mM ATP and 10mM HEPES/KOH buffer, pH 8.0.+ -. 0.05. An axiopatch 200B integrated patch clamp amplifier (Axon Instruments) applies a 180 mV voltage (reverse positive) across the bilayer and measures the ion current through the aperture. MspA mutants were added to the grounded cis chamber at a concentration of 2.5ng/ml. Once inserted into one well, the compartment is rinsed with assay buffer to avoid further insertion. The peptide fragment-DNA concentration in this experiment was 1. Mu.M. ARCFU-like helicase was added to the cis chamber at a concentration of 0.75. Mu.M. All experiments reported herein were completed at room temperature (23.+ -. 1 ℃). The analog signal is low pass filtered with a4 pole Bessel filter at 100kHz and digitized at 500 kHz. Data acquisition was controlled with a clampox 11.2.
The present invention uses a mutant nanopore MspA with a cup-like shape that distinguishes helicase from shrinkage of the pore where ion current blocking occurs by about 10nm. For DNA translocation sport enzymes, ARCFU-like helicase was used, which pulls single stranded DNA through protein biological pores in an observable step of 0.33: 0.33 nm of half nucleotides.
Experimental example: samples were taken through a throat swab, washed 3 times with 0.01M potassium chloride solution (0.01 mol/LPBS buffer, 10mM HEPES, pH 7.5), followed by filtration to remove impurities with 450. 450 nm. 200. Mu.L of the filtered sample was exposed to the peptide fragment of the protein at 100℃for 10min, and the constructed peptide fragment-DNA was ligated with the sample using T4 ligase (ligation buffer: 100mM KCl,10 MmM HEPES, 1mM DTT, pH 8.0) and incubated at room temperature for 15 min. After purification using magnetic beads, 20 μl of the purified product was added to a fluid pool to be examined. During the experimental process, ARCFU-like helicase and ARCFU-like helicase-free group were set to verify the rate controlling effect.
As shown in FIGS. 3 to 6, proteins were separated by size using SDS-PAGE, and FIG. 4 shows SDS-PAGE results, as can be seen from FIG. 4, lane M: protein Marker; lane 1: uninduced samples; lanes 2-6: post induction samples. Lane 2:0.2mM IPTG induction for 4 hours at 37 ℃; lane 3:0.2mM IPTG 16℃for 16 hours; lane 4:1.0mM IPTG induction for 4 hours at 37 ℃; lane 5:1.0mM IPTG induction at 16℃for 16 hours; lane 6:1.0mM IPTG was induced at 15℃for 24 hours. The protein is transferred to a membrane by using a western-blot experiment, then the membrane is detected by using a His antibody, and the result ARCFU-like helicase of the western-blot experiment is expressed normally and purified.
Referring to fig. 5, the solubility analysis was performed from the electropherogram of fig. 5, and the results are shown in the following table:
note that: induction was carried out at 37℃for 4 hours and at 15℃for 16 hours.
The best expression of ARCFU-like helicase was obtained by precipitation after induction with 1.0mM IPTG for 4h at 37 ℃.
FIG. 3 is a schematic representation of the signal of a peptide fragment passing through a biological nanopore when it contains ARCFU-like helicase; FIG. 6 is a graph of the detection signal of peptide fragments passing through a nanopore in the absence of ARCFU-like helicase, and by comparison, it was found that no via signal was detected in the absence of ARCFU-like helicase.
According to the experiment, compared with the prior method for detecting the respiratory tract virus in the sample, the method provided by the invention mainly adopts a fluorescence PCR method, the whole amplification process needs about 1 hour to obtain a result, and the biological nano-pores can obtain a result within 15 minutes, so that the effect of rapid detection is achieved. Meanwhile, as the DNA, RNA and peptide fragments pass through the biological nano holes too fast, the speed of the helicase is required to be controlled, and the novel ARCFU-like helicase provided by the invention can control the speed of the peptide fragment-DNA sample passing through the biological nano holes, so that viruses in the sample can be detected.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (8)
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| US20150191709A1 (en) * | 2012-07-19 | 2015-07-09 | Oxford Nanopore Technologies Limited | Modified helicases |
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2024
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| WO2026050906A1 (en) * | 2024-09-03 | 2026-03-12 | 深圳华大生命科学研究院 | Binary complex containing chaperone molecule and use thereof in polypeptide sequencing |
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