WO2022087804A1 - 一种嘌呤霉素连接子及其在体外核酸展示肽合成中的应用 - Google Patents

一种嘌呤霉素连接子及其在体外核酸展示肽合成中的应用 Download PDF

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WO2022087804A1
WO2022087804A1 PCT/CN2020/123835 CN2020123835W WO2022087804A1 WO 2022087804 A1 WO2022087804 A1 WO 2022087804A1 CN 2020123835 W CN2020123835 W CN 2020123835W WO 2022087804 A1 WO2022087804 A1 WO 2022087804A1
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mrna
sequence
nucleotides
puromycin
puromycin linker
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PCT/CN2020/123835
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French (fr)
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桑国芹
焦少灼
谢莹莹
徐猛
李宗文
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北京寻因生物科技有限公司
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Priority to PCT/CN2020/123835 priority Critical patent/WO2022087804A1/zh
Priority to CN202080066305.6A priority patent/CN114585736B/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention relates to the technical field of in vitro display, in particular to a puromycin linker and its application in the synthesis of nucleic acid display peptides in vitro.
  • Display technology is an analytical technology that specifically links genes and their expression products. It is essential for the isolation of specific high-affinity binding molecules (proteins, peptides, nucleic acids, etc.), which can be used in the diagnosis and treatment of cancer, infectious diseases, autoimmunity, neurodegenerative diseases, and inflammatory diseases. Demonstration technology applications also extend to other areas, such as antibody and enzyme engineering and discovery of protein-protein interactions.
  • Display technology mainly includes in vivo display technology and in vitro display technology.
  • In vitro display technologies such as ribosome display, mRNA display and cDNA display, have more advantages than in vivo display technologies represented by phage display systems, such as simple operation, short screening cycle, and higher library capacity (10 13 -10 15 ), with flexibility to incorporate non-natural residues into proteins/peptides and the ability to post-translationally modify.
  • In vitro display technology enables mRNA molecules to bind to their encoded protein products via ribosomes or puromycin molecules. Compared with ribosome display, mRNA and protein are covalently coupled to form a simpler and stronger complex in mRNA display.
  • the key to mRNA display technology is puromycin, which is similar in structure to aminoacyl-tRNA molecules and can easily enter the A site of the ribosome and is transferred to the nascent polypeptide chain by peptidyltransferase, so that the 3' end can be transferred to the nascent polypeptide chain.
  • the mRNA bound to the puromycin linker is covalently bound to the C-terminus of the nascent peptide through puromycin to form an mRNA-protein fusion molecule.
  • the application of mRNA display technology is severely limited due to the instability of mRNA in mRNA-protein fusion molecules.
  • the researchers converted the mRNA in the mRNA-protein fusion molecule into cDNA by optimizing the puromycin linker, and finally formed a cDNA-protein fusion molecule. This technology is cDNA display technology.
  • the puromycin linker is involved in every step in the preparation of cDNA-protein fusion molecules and is a key factor affecting the efficiency of cDNA display. Therefore, the design and synthesis of the puromycin linker is very important, which is very important for cDNA display.
  • Kurz first proposed a branched puromycin linker structure, including a psoralen-mediated linker covalently linked to mRNA; a puromycin arm covalently bound to a nascent peptide; A reverse transcription site that converts mRNA to cDNA.
  • the commonly used puromycin linker structure in the prior art is a branched structure, which includes a biotin purification site, an enzyme cleavage site and a fluorescent label in addition to an mRNA attachment site, a reverse transcription site and a puromycin arm.
  • the synthesis steps of the branched puromycin linker are: 1) synthesizing the modified skeleton chain and side chain separately, and the synthesis efficiency decreases with the increase of modification types; 2) chemically coupling the skeleton chain and the side chain through a reaction Covalently cross-linked into a branched structure, the chemical reaction step efficiency of covalently cross-linked backbone chain and side chain is low, and the efficiency of this step is only 0.5-4%.
  • the disadvantages of the above method are that the preparation cycle is long; the process is extremely cumbersome; and there are many types of primer modifications. These shortcomings limit the preparation efficiency of the puromycin linker and its application.
  • the application steps of the existing branched puromycin linker in displaying peptides in vitro are as follows: 1) template DNA is transcribed into mRNA; 2) mRNA and puromycin linker are photocrosslinked/enzymatically linked to form an mRNA-linker couple 3) The mRNA-linker conjugate is translated into mRNA-protein fusion in a cell-free system; 4) The mRNA-linker and mRNA-protein fusion product are immobilized on a streptavidin magnet by biotin modification.
  • the purpose of the present invention is to provide a puromycin linker, the synthesis method of the puromycin linker is simple, and the efficiency of obtaining key raw materials is greatly improved.
  • Another object of the present invention is to provide the application of the above-mentioned puromycin linker.
  • the present invention provides a puromycin linker, the puromycin linker is a modified unbranched single-stranded DNA, and the single-stranded DNA includes a first segment of nucleotides and a second segment of nucleotides.
  • the first segment of nucleotides comprises a segment of oligonucleotide sequences synthesized by dNTPs
  • the second segment of nucleotides comprises a segment of oligonucleotide sequences synthesized by reverse dNTPs
  • the 5' end of the first stretch of nucleotides and the 5' end of the second stretch of nucleotides are connected to form a linker structure comprising two 3' ends; the 3' end of the first stretch of nucleotides is modified There is puromycin; the second stretch of nucleotides includes an mRNA junction site and a reverse transcription site in the order of 5' to 3'.
  • the 5' end of the first stretch of nucleotides and the 5' end of the second stretch of nucleotides can be connected by a flexible linker; preferably, the flexible linker is Spacer , further preferably, described Spacer is selected from one or more combinations in Spacer C3, Spacer C6, Spacer C9, Spacer C12 and Spacer C18; Further preferably, described Spacer is selected from Spacer C18.
  • Spacer can provide the necessary space for oligonucleotide labeling to reduce the interaction between the labeling group and the oligonucleotide, and is mainly used in the study of DNA hairpin structure and double-stranded structure.
  • Spacer C3 is propane (see Figure 7 for the structural formula), which is mainly used to mimic the three-carbon spacer between the 3' and 5' hydroxyls of ribose, or to "replace" an unknown base in a sequence.
  • Spacer C6 is hexane (see Figure 8 for the structural formula) and is used to insert a 6-carbon spacer between nucleotides.
  • Spacer C9 is an ether (see Figure 9 for the structural formula), which is used to insert 9 atomic spacers (3 O, 6 C) between nucleotides.
  • Spacer C12 is dodecane (see Figure 10 for the structural formula), which is used to insert 12 C spacers between the nucleotide or oligo and the labeling group.
  • Spacer C18 is an ether (see Figure 11 for the structural formula), which is used to insert 18-atom spacers (6 Os, 12 Cs) between nucleotides, and is often used to form DNA stem-loop structures. Spacers can be labeled at any position of the oligonucleotide, or multiple Spacers can be connected to each other to form a larger space.
  • the first segment of nucleotides and the puromycin modified at the 3' end together form a puromycin arm, and the puromycin in the puromycin arm serves as a polypeptide binding site Covalently cross-linked to the displayed peptide.
  • the sequence of the first stretch of nucleotides from 5' to 3' includes TCTCTCCC
  • the sequence of the second stretch of nucleotides includes the sequence shown in SEQ ID NO.3 .
  • the first stretch of nucleotides further comprises a nucleotide sequence of 2-4 spacers and/or 1-18 bases to increase the length and flexibility of the puromycin arm
  • two spacers are connected between the 6-7 bases in the sequence of the first segment of nucleotides from 5' to 3'; further preferably, each spacer is independently selected Any one from Spacer C3, Spacer C6, Spacer C9, Spacer C12 and Spacer C18, preferably Spacer C18.
  • the first segment of nucleotides further comprises a nucleic acid purification tag and/or chemical modification; preferably, the nucleic acid purification tag comprises a polyadenylation sequence (polyA) or any other
  • the base sequence can be used both to purify the conjugate from the lysate and to extend the puromycin arm to increase the fusion efficiency;
  • the chemical modification includes a modified label for nucleic acid purification or binding with other ligands and /or fluorescent label, further preferably, the modified label used for nucleic acid purification or binding with other ligands includes biotin labeling; further preferably, the fluorescent label includes FAM, FITC, Cy dye or other fluorescence, preferably FAM;
  • the site to which the fluorescent label is attached includes the 3rd base in the sequence from 5' to 3' of the first stretch of nucleotides.
  • the mRNA attachment site is a segment of an oligonucleotide sequence comprising an artificially synthesized nucleic acid, 3-cyano-vinylcar-bazole ( cnv K) modified , which is used for the covalent cross-linking of puromycin linker and mRNA, which can ensure fast, simple and efficient acquisition of mRNA-puromycin linker conjugate;
  • the second nucleotide sequence is from the 1st to the 7th base in the 5' to 3' sequence, and the 7th base is an artificial synthetic base cnv K.
  • the reverse transcription site is a reverse oligonucleotide sequence, which is complementary to the 3' end of the mRNA, and the length is preferably 1-15 bases; the reverse transcription site The site is used to form a stable cDNA-protein fusion, and the cDNA reverse transcribed from the mRNA is covalently linked to the protein it encodes; further preferably, the reverse transcription site includes nucleotides located in the second segment from 5 The 8th to 19th base sequence of the 'to 3' sequence.
  • the nucleic acid purification tag is used to purify the fusion product from the expression system; the fluorescent tag is used to detect the mRNA-puromycin linker conjugate and the mRNA/cDNA-protein fusion. form.
  • the present invention also provides the application of the above-mentioned puromycin linker in the synthesis of nucleic acid display peptides in vitro, comprising the following steps: (1) providing template DNA; (2) transcribing and purifying the template DNA in vitro to obtain a single (3) annealing the mRNA product after mixing it with the puromycin linker, and irradiating it with light of a certain wavelength (ultraviolet light wave) to obtain the mRNA-puromycin linker conjugate; (4) irradiating the mRNA product with the puromycin linker; The mRNA-puromycin linker conjugate is translated in an expression system to bind a peptide corresponding to the mRNA sequence at the polypeptide binding site of the puromycin linker, thereby forming an mRNA-protein fusion.
  • the step (4) further includes the following steps: (5) immobilizing the mRNA and the mRNA-protein fusion on streptavidin magnetic beads; Reverse transcription to form reverse transcription product: mRNA/cDNA-protein fusion.
  • the step (5) further includes the following steps: (6) using a protein purification tag to separate and purify the reverse transcription product to obtain a cDNA-protein fusion.
  • the sequence of the template DNA from 5' to 3' comprises a promoter, a translation enhancer, a Kozak sequence, a target gene, a spacer sequence (Spc), a protein purification tag , spacer sequence (Spc) and Y tag;
  • the promoter includes T7 promoter, SP6 promoter or T3 promoter, preferably T7 promoter or SP6 promoter;
  • the translation enhancer such as 5' leader sequence ( ⁇ sequence) of tobacco mosaic virus or Xenopus ⁇ -globin untranslated sequence or other sequences available in the prior art; further preferably, protein purification tags such as His tag, Flag tag, etc.;
  • the spacer sequence (Spc) is selected from one or more combinations of the nucleotide sequences encoding amino acids GGS, GGGS, GGGASG4SG4S, (G4S) 2 and GGGASGGGGS; further preferably, the Y tag
  • the length of the template DNA depends on the length of the nucleic acid coding sequence of the displayed peptide, preferably, the length of the template DNA is 50-1000 nucleotides, further preferably, The length of the template DNA is 200-500 nucleotides, more preferably 200-400 nucleotides, and the synthesis of the template DNA can be done by whole gene synthesis, fusion PCR or the like.
  • the mRNA purification method includes column purification or magnetic bead purification.
  • mRNA is obtained by in vitro transcription quickly, conveniently and with high precision.
  • In vitro transcription kits include T7 RiboMAXTM Express Large Scale RNA Production System (Promega), RiboMAXTM Large Scale RNA Production Systems—SP6 and T7, MEGAscriptTM T7 Transcription Kit (Thermo) or other conventionally available transcription kits; specifically, step (2) ), the mRNA was obtained by using an in vitro transcription kit such as T7 RiboMAX TM Express Large Scale RNA Production System (Promega).
  • RNA was purified using TIANSeq RNA purification magnetic beads.
  • the mRNA acquisition method is to construct a transcription system in a conventional manner, such as in a reaction system comprising T7 transcription buffer, 25mM each of rATP, rCTP, rGTP, rTTP and transcriptase , add template DNA, react at 37°C for 1-4h, then add 1-4 ⁇ l of DNase, and react at 37°C for 15-30min.
  • a reaction system comprising T7 transcription buffer, 25mM each of rATP, rCTP, rGTP, rTTP and transcriptase , add template DNA, react at 37°C for 1-4h, then add 1-4 ⁇ l of DNase, and react at 37°C for 15-30min.
  • step (3) the molar ratio of the mRNA product to the puromycin linker is 1:(1-1.5).
  • the wavelength of the ultraviolet light wave is 330-400nm, preferably 345-390nm, irradiated for 0.5-6min, and the equipment used can be a gel imager, an ultraviolet crosslinker or other devices in this wavelength range.
  • the expression system used is a cell-free expression system; preferably, the cell-free expression system includes a rabbit reticulum expression system, a wheat embryo expression system or an E. coli expression system system.
  • step (4) the mRNA-protein fusion is incubated at 25-37°C for 0.5-1.5h under the conditions of 0.3-1.6M KCl and 40-170mM MgCl 2 (final concentration) Forming.
  • the specific steps of step (5) include: (a) nucleic acid purification, separating the mRNA-puromycin linker conjugate and the mRNA-protein fusion from the translation system; (b) ) to carry out reverse transcription reaction of mRNA; (c) after the completion of reverse transcription, add RNaseH to digest mRNA (if the existence of mRNA does not affect subsequent experiments, this step can be omitted).
  • the specific steps of step (5) include the following: (1) use oligo dT magnetic beads or DNA purification magnetic beads to mix with the expression system, and incubate for 30 min; (2) perform mRNA reversal on all beads (3) After the reaction, add RNase H and react at 37°C for 15-30min.
  • the nucleic acid purification method includes oligo dT magnetic beads, magnetic beads containing a sequence complementary to the nucleotide sequence used for purification in the puromycin linker, DNA purification magnetic beads or streptavidin Hesu Magnetic Beads.
  • the reverse transcription reaction system can be set arbitrarily without limitation; commercially available kits can be used, such as ReverTra Ace (TOYOBO), SuperScript IV kit (Thermo), M-MLV Reverse Transcriptase (Promega) or other similar products.
  • commercially available kits can be used, such as ReverTra Ace (TOYOBO), SuperScript IV kit (Thermo), M-MLV Reverse Transcriptase (Promega) or other similar products.
  • the protein purification tag includes a His tag or a Flag tag.
  • the present invention also provides the in vitro nucleic acid display peptide prepared by the above application.
  • the above application of the present invention focuses on the optimization of steps 4)-7) in the background technology "application of puromycin linker with existing branch structure in in vitro display of peptides", using different DNA purification magnetic beads to replace streptavidin
  • the advantage of avidin magnetic beads is that, in the design of the puromycin linker, the modification of biotin and enzyme cleavage sites is reduced, and the types of modifications are reduced; again, in the process of in vitro peptide display, the enzyme cleavage steps are reduced, The reduction of yield and the prolongation of operation time caused by the problem of enzyme cleavage reaction efficiency are avoided; finally, the present invention reduces the use of restriction endonucleases, and simultaneously reduces biotin modification and enzyme cleavage in the process of linker synthesis Point modification reduces the cost of in vitro peptide display and linker synthesis.
  • the puromycin linker synthesis method of the novel structure of the present application is simple, the efficiency is as high as 53%, the efficiency of obtaining key raw materials is greatly improved, the system efficiency is further improved, and the synthesis cost of mRNA/cDNA-protein fusion products is reduced.
  • the mRNA connection site in the structure is to covalently cross-link the puromycin linker and mRNA by means of photocrosslinking, and the structure is more stable; secondly, the experimental process of the prior art method is optimized, and the operation is simple; A wide range of base materials are used and are very inexpensive.
  • Figure 1 is a flow chart of the preparation of the cDNA-protein fusion in Example 1 of the present invention.
  • Example 2 is a schematic structural diagram of a novel puromycin linker in Example 1 of the present invention.
  • FIG. 3 is a schematic diagram of the combination of puromycin linker and mRNA by photocrosslinking in Example 1 of the present invention.
  • FIG. 4A is the PAGE detection result of the photocrosslinking product of mRNA and fluorescently labeled puromycin linker in Example 1 of the present invention.
  • Fig. 4B is the SYBR Green staining gel image of the photocrosslinking product of mRNA and puromycin linker in Example 1 of the present invention.
  • FIG. 5 is a fluorescence image of urea SDS-PAGE of the cDNA-protein fusion of BDA gene in Example 1 of the present invention.
  • FIG. 6 is a schematic diagram of the structural composition of the template DNA in Example 1 of the present invention.
  • Fig. 7 is the structural formula of Spacer C3 of the present invention.
  • Fig. 8 is the structural formula of Spacer C6 of the present invention.
  • Fig. 9 is the structural formula of Spacer C9 of the present invention.
  • Fig. 10 is the structural formula of Spacer C12 of the present invention.
  • Fig. 11 is the structural formula of Spacer C18 of the present invention.
  • Fig. 12 is a fluorescence image of urea SDS-PAGE of the cDNA-protein fusion of PDO gene in Example 2 of the present invention.
  • Figure 13 is a fluorescence image of urea SDS-PAGE of the cDNA-protein fusion of the anti-GFP VHH gene in Example 3 of the present invention.
  • This example provides a method for synthesizing an in vitro nucleic acid display peptide using the B domain of protein A (BDA for short) as a target gene.
  • BDA protein A
  • the whole process of preparation of cDNA-protein fusion of BDA protein is shown in Figure 1.
  • the nucleic acid sequence of the target gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.
  • the structure of the puromycin linker is shown in Figure 2.
  • the puromycin linker is a modified unbranched single-stranded DNA, and the single-stranded DNA includes a first segment of nucleotides and a second segment of nucleotides;
  • the 3' end of a segment of nucleotides is modified with puromycin, which together form a puromycin arm, and the first segment of nucleotides is also modified with a fluorescent label;
  • the second segment of nucleotides is arranged according to 5' to 3 ' sequence includes the mRNA junction site and the reverse transcription site.
  • sequence of the first segment of nucleotides is TCTCTCCC from 5' to 3'
  • sequence of the second segment of nucleotides is shown in SEQ ID NO.3.
  • the nucleotide sequence of the puromycin linker is synthesized by a primer synthesis company, and the nucleotide sequence length of the puromycin linker is 27 bp, which is further sequenced from 5' to 3' in the first segment of nucleotides.
  • the third base of the modified fluorescent label is modified, and the first segment of nucleotides and the second segment of nucleotides are connected by a flexible linker Spacer C18 (referred to as spc18), in the first segment of nucleotides from 5' to Two Spacer C18s were inserted between bases 6-7 of the 3' sequence.
  • spc18 flexible linker Spacer C18
  • the mRNA connection site is located at the 1st to 7th base sequence of the second stretch of nucleotides from 5' to 3', and the reverse transcription site is located at The second stretch of nucleotides is from the 8th to the 19th base sequence in the 5' to 3' sequence.
  • the connection information of this puromycin linker is as follows:
  • the synthesis method of the puromycin linker synthesized by the above method is simple, the efficiency is as high as 53%, the efficiency of obtaining key raw materials is greatly improved, the system efficiency is further improved, and the synthesis cost of the cDNA-protein fusion product is reduced.
  • the mRNA attachment site of the puromycin is covalently cross-linked with the mRNA in the form of photo-cross-linking, and the structure is more stable.
  • the template DNA structure is shown in Figure 6.
  • the sequence of the template DNA from 5' to 3' consists of T7 promoter, translation enhancer, Kozak sequence, target gene, spacer sequence (Spc), His tag, spacer sequence (Spc) and Y tag (sequence that is complementary to the puromycin linker part); direct chemical synthesis of the full-length sequence such as SEQ ID NO.4, and then PCR amplification to obtain a sufficient amount of DNA.
  • nucleic acid sequence of T7 promoter-translation enhancer in the above-mentioned template DNA is shown in SEQ ID NO.5
  • nucleotide sequence of the protein purification tag (His tag) is shown in SEQ ID.6
  • spacer sequence Including a first spacer sequence (located between the target gene and the His tag) and a second spacer sequence (located between the His tag and the Y tag) wherein the nucleic acid sequence of the first spacer sequence is shown in SEQ ID NO.7
  • nucleotide sequence of the second spacer sequence from the 5' to the 3' sequence is: GGCGGAAGC
  • nucleotide sequence of the Y tag is shown in SEQ ID NO.8.
  • Reaction system 50 ⁇ l: 0.1-1ng DNA, 0.5-1 ⁇ l Q5 high-fidelity DNA polymerase (2unit/ ⁇ l), 10ul 5 ⁇ buffer, 0.4 ⁇ l dNTPs (25mM), 0.2ul forward primer F (nucleic acid sequence as SEQ ID NO.9), 0.2ul reverse primer R (the nucleic acid sequence is shown in SEQ ID NO.10), and the rest of the RNase free water is supplemented to 50ul.
  • PCR reaction conditions a, 98°C (1-3min), b, 98°C (5-45s), c, 55-70°C (10-60s), d, 72°C (10-60s), e, 72°C (1-5min), cycle steps b-d 25 to 35 times.
  • DNA purification magnetic beads or gel recovery can be used for purification.
  • the DNA obtained in the above step was used as a template, and was transcribed using RiboMAX TM Express Large Scale RNA Production System-T7 (Promega).
  • the 20 ⁇ l reaction mixture includes 10 ⁇ l 2 ⁇ T7 transcription buffer, 2 ⁇ l mixed enzyme, 0.2-1 ⁇ g double-stranded DNA, and the rest is RNase free water. First, react at 37°C for 30 minutes, then add 0.5-1 ⁇ l RQ1 RNase free DNase to the reaction mixture and react at 37°C for 15 minutes. After the reaction, the reaction mixture was purified using DNA purification magnetic beads.
  • the mRNA and the puromycin linker were added to the hybridization buffer (see Table 1 for the formula) in a molar ratio of 1:1 for annealing; after annealing, 1 ⁇ l of the sample was taken as a reserve sample, and the remaining samples were placed under a UV lamp with a wavelength of 365 nm. Direct illumination for 60s to obtain mRNA-puromycin linker conjugate;
  • Annealing conditions 90°C for 1min (-0.4°C/s, that is, 0.4°C decrease per second), 70°C 1min (-0.1°C/s, that is, 0.1°C decrease per second), 25°C to stop.
  • the mRNA-Linker conjugate (ie, the mRNA-puromycin linker conjugate) obtained in the above step was added to the rabbit reticulum translation system for translation coupling.
  • Rabbit reticulocyte lysate 17.5 ⁇ l Amino acid mixture does not contain isoleucine, 1mM 0.25 ⁇ l Amino acid mixture, does not contain methionine, 1mM 0.25 ⁇ l ribonuclease inhibitor 0.5 ⁇ l mRNA-puromycin linker conjugate 3pmol Nuclease-Free Water Make up to 25 ⁇ l
  • the reaction was carried out at 30°C for 20 min, and then final concentrations of 900 mM KCl and 80 mM MgCl 2 were added, and incubated at 37° C. for 60 min under high salt conditions.
  • the above supernatant was incubated with 20 ⁇ l of His-tag purification magnetic beads, and 200 ⁇ l of His-tag elution buffer (recipe shown in Table 5) was used for elution to obtain a cDNA-protein fusion, which was detected by urea-SDS-PAGE. Formation.
  • the samples obtained in each step of the entire cDNA-protein fusion preparation process were subjected to electrophoresis analysis with retained samples.
  • the mRNA-puromycin linker conjugate (lane1) is translated into mRNA-protein fusion (lane2, the upper band is the fusion product; the lower band corresponds to the mRNA-puromycin connection that is not coupled to the polypeptide Subconjugate), and then bound to DNA purification magnetic beads (to detect the binding effect, analyze the supernatant lane3).
  • This embodiment provides a method for synthesizing a POU-specific DNA binding domain (PDO for short) as a target gene in vitro nucleic acid display peptide.
  • PDO POU-specific DNA binding domain
  • the difference from Example 1 is only the part of the target gene in step 2 and the part of the synthesis efficiency of the cDNA-protein fusion in step 7.
  • the nucleic acid sequence of the PDO target gene is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12.
  • the cDNA in vitro display peptide of PDO protein was prepared according to steps 1-7 in Example 1, and the formation of cDNA-protein fusion was detected by urea SDS-PAGE. As shown in Figure 12:
  • the mRNA-puromycin linker conjugate (lane1) is translated into mRNA-protein fusion (lane2, the upper band is the fusion product; the lower band corresponds to the mRNA-puromycin connection that is not coupled to the polypeptide Subconjugate), and then bound to DNA purification magnetic beads (to detect the binding effect, analyze the supernatant lane3).
  • the purified magnetic beads were used for reverse transcription, and then the mRNA was digested with RNaseH to form a cDNA-protein fusion (lane4, the upper band is the cDNA-protein fusion, and the lower band corresponds to the cDNA of uncoupled protein).
  • This embodiment provides a method for synthesizing an anti-green fluorescent protein alpaca antibody gene (anti-GFP VHH for short) as a target gene in vitro nucleic acid display peptide.
  • anti-GFP VHH anti-green fluorescent protein alpaca antibody gene
  • the difference from Example 1 is only the part of the target gene in step 2 and the part of the synthesis efficiency of the cDNA-protein fusion in step 7.
  • the nucleic acid sequence of the anti-GFP VHH target gene is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14.
  • the cDNA in vitro display peptide of anti-GFP VHH antibody was prepared according to steps 1-7 in Example 1, and the formation of cDNA-protein fusion was detected by urea SDS-PAGE. As shown in Figure 13:
  • the mRNA-puromycin linker conjugate (lane1) is translated into mRNA-protein fusion, then combined with DNA purification magnetic beads, reverse transcribed with the above purified magnetic beads, and then RNaseH digests mRNA to form cDNA-protein fusion (lane2, the upper band is the cDNA-protein fusion, the lower band corresponds to the cDNA of the unconjugated protein). Further purification with His-tag purification magnetic beads (lane3, His-tag purification supernatant) yielded a single cDNA-protein fusion (lane4, His-tag purification eluate). The total formation efficiency of cDNA-protein fusions (from mRNA-puromycin linker conjugate to His-tag purification eluate) was calculated to be about 0.9% based on band intensity.

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Abstract

提供了一种嘌呤霉素连接子及其在体外核酸展示肽合成中的应用。所述嘌呤霉素连接子为含修饰无分支的单链DNA，该单链DNA包括第一段核苷酸和第二段核苷酸；所述第一段核苷酸的5'端和所述第二段核苷酸的5'端相连接形成包含两个3'末端的连接体结构；所述第一段核苷酸的3'端修饰有嘌呤霉素；所述第二段核苷酸按照5'至3'的顺序包括mRNA连接位点和逆转录位点。所述嘌呤霉素连接子合成方法简单，效率高达53％，极大地提高了关键原料获取的效率，进一步提高系统效率及缩减了cDNA-蛋白的合成成本。

Description

一种嘌呤霉素连接子及其在体外核酸展示肽合成中的应用 技术领域
本发明涉及体外展示技术领域，具体涉及一种嘌呤霉素连接子及其在体外核酸展示肽合成中的应用。
背景技术
展示技术是一种将基因与其表达产物之间特异性连接的分析技术。其对于分离特定的高亲和力结合分子(蛋白质，多肽，核酸等)至关重要，可用于癌症、传染病、自身免疫、神经退行性疾病以及炎症性疾病等的诊断和治疗。展示技术应用范围也扩展到其他领域，例如抗体和酶工程以及蛋白质与蛋白质相互作用的发现。展示技术主要包括体内展示技术和体外展示技术。体外展示技术，如核糖体展示、mRNA展示及cDNA展示，与以噬菌体展示系统为代表的体内展示技术相比更具优势，如操作简单，筛选周期短，更高的文库容量(10 13-10 15)，且具备将非天然残基整合到蛋白质/肽中的灵活性以及翻译后修饰的能力。
体外展示技术使得mRNA分子能够通过核糖体或嘌呤霉素分子与其编码的蛋白质产物结合。相比于核糖体展示，mRNA展示中mRNA与蛋白质以共价方式进行偶联，形成一个更简单更牢固的复合物。mRNA展示技术的关键是嘌呤霉素，它的结构类似于氨基酰基-tRNA分子，很容易进入核糖体A位点，并通过肽基转移酶转移到新生的多肽链上，因此可将3’末端和嘌呤霉素连接子结合的mRNA通过嘌呤霉素与新生肽的C-末端共价结合，形成mRNA-蛋白质融合分子。然而，由于mRNA-蛋白质融合分子中mRNA的不稳定性，严重限制了mRNA展示技术的应用。为了克服这个问题，研究者通过优化嘌呤霉素连接子将mRNA-蛋白质融合分子中的mRNA转换为cDNA，最终形成cDNA-蛋白质融合分子，该技术即为cDNA展示技术。
嘌呤霉素连接子参与cDNA-蛋白质融合分子制备过程中每一个步骤，是影响cDNA展示效率的关键因素，因此对嘌呤霉素连接子的设计与合成非常重要，这对cDNA展示至关重要。2001年，Kurz最早提出了一种分支嘌呤霉素连接子结构，包含补骨脂素介导的连接子与mRNA进行共价连接的连接位点；与新生肽共价结合的嘌呤霉素臂；将mRNA转换为cDNA的逆转录位点。近年来，为了提高效率、减少操作时间等问题，研究者们在原连接子结构的基础上进行了不断的优化，相继提出了酶连接法和cnvK介导mRNA与连接子偶联的光交联法。现有技术中的常用嘌呤霉素连接子结构为分支结构， 除包含mRNA连接位点、逆转录位点和嘌呤霉素臂外，还包含生物素纯化位点、酶切位点以及荧光标记。分支结构嘌呤霉素连接子的合成步骤为：1)单独合成带有修饰的骨架链及侧链，合成效率随着修饰种类的增加而降低；2)将骨架链与侧链通过化学偶联反应共价交联成分支结构，共价交联骨架链和侧链的化学反应步骤效率低，此步效率仅有0.5-4％。上述方法的缺陷在于制备周期长；过程极其繁琐；引物修饰种类多。这些缺点限制了嘌呤霉素连接子的制备效率及其应用。
现有分支结构的嘌呤霉素连接子在体外展示肽中的应用步骤如下：1)模板DNA转录成mRNA；2)mRNA与嘌呤霉素连接子进行光交联/酶连接形成mRNA-连接子偶联物；3)mRNA-连接子偶联物在无细胞体系中翻译成mRNA-蛋白融合物；4)将mRNA-连接子及mRNA-蛋白融合产物通过生物素修饰固定在链霉亲和素磁珠上；5)用上述磁珠作为模板进行逆转录反应，形成mRNA/cDNA-蛋白融合物；6)RNaseH消化mRNA部分，形成cDNA-蛋白融合物；7)通过限制性酶切反应将cDNA-蛋白融合物从磁珠上释放；8)进一步用His标签纯化磁珠纯化得到单一的cDNA-蛋白融合物。该应用中以链霉亲和素与生物素系统作为核酸纯化方式，在此基础上引入了限制性内切酶的使用导致了产量的进一步降低，并且步骤较多，操作复杂，并不利于广泛地推广使用。
发明内容
为了解决上述技术问题，本发明的目的在于提供一种嘌呤霉素连接子，该嘌呤霉素连接子合成方法简单，极大的提高了关键原料获取的效率。
本发明的另一个目的在于提供上述嘌呤霉素连接子的应用。
为达上述目的，一方面，本发明提供了一种嘌呤霉素连接子，所述嘌呤霉素连接子为含修饰无分支的单链DNA，该单链DNA包括第一段核苷酸和第二段核苷酸；其中，所述第一段核苷酸包含一段dNTP合成的寡核苷酸序列，所述第二段核苷酸包含一段反向dNTP合成的寡核苷酸序列，所述第一段核苷酸的5’端和所述第二段核苷酸的5’端相连接形成包含两个3’末端的连接体结构；所述第一段核苷酸的3’末端修饰有嘌呤霉素；所述第二段核苷酸按照5’至3’的顺序包括mRNA连接位点和逆转录位点。
本发明上述新型嘌呤霉素连接子结构的设计与合成，首先，简化了现有技术的合成步骤，仅需一步序列合成即可，避免了骨架链与侧链化学偶联带来的效率的降低及操作时间的增加；再次，减少了连接子中的修饰种类，进一步避免了修饰带来的效率的降低。
根据本发明的一些具体实施方案，所述第一段核苷酸的5’端和所述第二段核苷酸的5’端可通过柔性接头相连接；优选地，所述柔性接头为Spacer，进一步优选地，所述Spacer选自Spacer C3、Spacer C6、Spacer C9、Spacer C12和Spacer C18中的一种或两种以上的组合；进一步优选地，所述Spacer选自Spacer C18。
上述Spacer可为寡核苷酸标记提供必要的间隔以减少标记基团与寡核苷酸间的相互作用，主要应用于DNA发夹结构和双链结构研究。其中Spacer C3为丙烷(结构式见图7)，主要用于模仿核糖的3'和5'羟基间的三碳间隔，或“替代”一个序列中未知的碱基。Spacer C6为己烷(结构式见图8)，用于在核苷酸之间插入6碳间隔。Spacer C9为醚(结构式见图9)，用于核苷酸间插入9个原子间隔(3个O、6个C)。Spacer C12为十二烷(结构式见图10)，用于在核苷酸或oligo与标记基团间插入12个C间隔。Spacer C18为醚(结构式见图11)，用于在核苷酸之间插入18原子间隔(6个O、12个C)，常用于形成DNA茎环结构。Spacer可标记于寡核苷酸任意位置，也可以多个Spacer互相连接，以形成更大间隔。
根据本发明的一些具体实施方案，所述第一段核苷酸及其3’末端修饰的嘌呤霉素共同组成嘌呤霉素臂，所述嘌呤霉素臂中的嘌呤霉素作为多肽结合位点与被展示肽进行共价交联。
根据本发明的一些具体实施方案，所述第一段核苷酸的序列自5’至3’的顺序包括TCTCTCCC，所述第二段核苷酸的序列包括如SEQ ID NO.3所示序列。
根据本发明的一些具体实施方案，所述第一段苷酸中还包含2-4个间隔子和/或1-18个碱基的核苷酸序列，以增加嘌呤霉素臂的长度及柔韧性；优选地，在所述第一段核苷酸自5’至3’顺序的第6-7位碱基之间连接有两个间隔子；进一步优选地，每个间隔子各自独立地选自Spacer C3、Spacer C6、Spacer C9、Spacer C12和Spacer C18中的任意一种，优选为Spacer C18。
根据本发明的一些具体实施方案，所述第一段核苷酸中还包含核酸纯化标签和/或化学修饰；优选地，所述核酸纯化标签包括多聚腺苷酸序列(polyA)或其他任意碱基序列，既可用于从裂解物中纯化结合物也可用于延长嘌呤霉素臂，增加融合效率；进一步优选地，所述化学修饰包括用于核酸纯化或与其他配体结合的修饰标记和/或荧光标记，进一步优选地，用于核酸纯化或与其他配体结合的修饰标记包括生物素标记；进一步优选地，所述荧光标记包括FAM、FITC、Cy染料或其他荧光，优选为FAM；进一步优 选地，所述荧光标记所连接的位点包括位于所述第一段核苷酸自5’至3’顺序的第3位碱基。
根据本发明的一些具体实施方案，所述mRNA连接位点是一段包含一个人工合成核酸三氰基乙烯基甲基咔唑(3-cyano-vinylcar-bazole， cnvK)修饰的寡核苷酸序列，其用于嘌呤霉素连接子与mRNA的共价交联，可保证快速、简便、高效的获得mRNA-嘌呤霉素连接子偶联物；优选地，所述mRNA连接位点包括位于所述第二段核苷酸自5’至3’顺序的第1位到第7位碱基序列，该第7位碱基为人工合成碱基 cnvK。
根据本发明的一些具体实施方案，所述逆转录位点为一段反向寡核苷酸序列，其与mRNA的3’端互补，长度范围优选为1-15个碱基；所述逆转录位点用于形成稳定的cDNA-蛋白融合物，所述mRNA逆转录出的cDNA与其编码的蛋白共价连接；进一步优选地，所述逆转录位点包括位于所述第二段核苷酸自5’至3’顺序的第8位到第19位碱基序列。
根据本发明的一些具体实施方案，所述核酸纯化标签用于从表达体系中纯化融合产物；所述荧光标记用于检测mRNA-嘌呤霉素连接子偶联物及mRNA/cDNA-蛋白融合物的形成。
另一方面，本发明还提供了上述嘌呤霉素连接子在体外核酸展示肽合成中的应用，包含如下步骤：(1)提供模板DNA；(2)将所述模板DNA体外转录、纯化得到单一的mRNA产物；(3)将所述mRNA产物与嘌呤霉素连接子混合后进行退火，使用一定波长的光(紫外光波)照射，获取mRNA-嘌呤霉素连接子偶联物；(4)将所述mRNA-嘌呤霉素连接子偶联物在表达体系中进行翻译，以在嘌呤霉素连接子的多肽结合位点上结合对应于mRNA序列的肽，从而形成mRNA-蛋白融合物。
可选地，所述步骤(4)之后还包括如下步骤：(5)将mRNA及mRNA-蛋白融合物固定在链霉亲和素磁珠上；对所述mRNA-蛋白融合物中的mRNA进行逆转录以形成逆转录产物：mRNA/cDNA-蛋白融合物。
进一步可选地，所述步骤(5)之后还包括如下步骤：(6)利用蛋白纯化标签对所述逆转录产物进行分离纯化，获取cDNA-蛋白融合物。
根据本发明的一些具体实施方案，步骤(1)中，所述模板DNA自5’至3’的顺序包含启动子、翻译增强子、Kozak序列、目标基因、间隔序列(Spc)、蛋白纯化标签、间隔序列(Spc)以及Y标签；优选地，所述启动子包括T7启动子、SP6启动子或T3启动子，优选为T7启动子或SP6启动子；进一步优选地，所述翻译增强子如烟草花叶 病毒的5’前导序列(Ω序列)或非洲爪蟾β-珠蛋白非翻译序列或其他现有技术可用序列；进一步优选地，蛋白纯化标签例如His标签，Flag标签等；进一步优选地，所述间隔序列(Spc)选自编码氨基酸GGS、GGGS、GGGASG4SG4S、(G4S) 2及GGGASGGGGS的核苷酸序列中的一种或两种以上的组合；进一步优选地，所述Y标签是与嘌呤霉素连接子部分互补配对的序列。
根据本发明的一些具体实施方案，步骤(1)中，模板DNA的长度依赖于被展示肽的核酸编码序列的长度，优选地，模板DNA长度为50-1000个核苷酸，进一步优选地，模板DNA长度为200-500个核苷酸，进一步优选为200-400个核苷酸，所述模板DNA的合成可以采用全基因合成、融合PCR方式等。
根据本发明的一些具体实施方案，步骤(2)中，mRNA纯化方式包括柱纯化或磁珠纯化。
根据本发明的一些具体实施方案，步骤(2)中，mRNA通过体外转录快速、方便、高精度的获得。体外转录试剂盒包括T7 RiboMAXTM Express Large Scale RNA Production System(Promega)、RiboMAXTM Large Scale RNA Production Systems—SP6 and T7、MEGAscript TM T7 Transcription Kit(Thermo)或其他常规可用转录试剂盒；具体地，步骤(2)中，mRNA获取方式通过体外转录试剂盒如T7 RiboMAX TM Express Large Scale RNA Production System(Promega)，根据试剂盒说明书将0.2-1μg的模板DNA加入20μl反应体系中，37℃反应30min，然后加入0.5-1μl RQ1DNase I，37℃反应15min。使用TIANSeq RNA纯化磁珠纯化mRNA。
根据本发明的一些具体实施方案，步骤(2)中，mRNA获取方式通过常规方式构建转录体系，如在包含T7转录缓冲液、各25mM的rATP、rCTP、rGTP、rTTP及转录酶的反应体系中，加入模板DNA，37℃反应1-4h，然后加入1-4μl的DNase，37℃反应15-30min。使用DNA纯化磁珠纯化mRNA。
根据本发明的一些具体实施方案，步骤(3)中，所述mRNA产物与嘌呤霉素连接子的摩尔数比为1：(1-1.5)。
根据本发明的一些具体实施方案，步骤(3)中，所述紫外光波的波长为330-400nm，优选为345-390nm，照射0.5-6min，所用设备可以是凝胶成像仪、紫外交联仪或其他在此波长范围内的设备。
根据本发明的一些具体实施方案，步骤(4)中，所用的表达体系为无细胞表达体系；优选地，所述无细胞表达体系包括兔网织红表达体系、小麦胚表达体系或大肠杆菌表达体系。
根据本发明的一些具体实施方案，步骤(4)中，mRNA-蛋白融合物是在0.3-1.6M KCl及40-170mM MgCl 2(终浓度)的条件下，25-37℃孵育0.5-1.5h形成的。
根据本发明的一些具体实施方案，步骤(5)的具体步骤包含：(a)核酸纯化，将mRNA-嘌呤霉素连接子偶联物及mRNA-蛋白融合物从翻译体系中进行分离；(b)进行mRNA的逆转录反应；(c)逆转录结束后，加入RNaseH消化mRNA(若mRNA的存在不影响后续实验，此步骤可省略)。
根据本发明的一些具体实施方案，步骤(5)的具体步骤包括如下：(1)使用oligo dT磁珠或DNA纯化磁珠与表达体系混合，孵育30min；(2)将全部珠子进行mRNA的逆转录反应；(3)反应结束后，加入RNase H，37℃反应15-30min。
根据本发明的一些具体实施方案，所述核酸纯化方式包括oligo dT磁珠、含与嘌呤霉素连接子中用于纯化的核苷酸序列互补序列的磁珠、DNA纯化磁珠或链霉亲和素磁珠。
根据本发明的一些具体实施方案，所述逆转录反应体系可以任意设置，不受限制；可以使用市场在售试剂盒，如ReverTra Ace(TOYOBO)，SuperScript IV kit(Thermo)，M-MLV Reverse Transcriptase(Promega)或其他类似产品。
根据本发明的一些具体实施方案，步骤(6)中，所述蛋白纯化标签包括His标签或Flag标签。
再一方面，本发明还提供了上述应用所制备出的体外核酸展示肽。
本发明的上述应用重点对背景技术“现有分支结构的嘌呤霉素连接子在体外展示肽中的应用”中的步骤4)-7)进行了优化，利用不同的DNA纯化磁珠代替链霉亲和素磁珠的优势在于，首先在嘌呤霉素连接子的设计上减少了生物素及酶切位点的修饰，降低了修饰种类；再次在体外肽展示过程中，减少了酶切步骤，避免了由酶切反应效率问题带来的产量的降低及操作时间的延长；最后，本发明减少了限制性内切酶的使用，同时在连接子合成过程中减少了生物素修饰及酶切位点修饰，降低了体外肽展示及连接子的合成成本。
本申请新型结构的嘌呤霉素连接子合成方法简单，效率高达53％，极大的提高了关键原料获取的效率，进一步提高系统效率及缩减了mRNA/cDNA-蛋白融合产物的合成 成本，并且该结构中的mRNA连接位点是以光交联的方式将嘌呤霉素连接子与mRNA进行共价交联，结构更加稳定；其次，优化了现有技术方法的实验流程，操作简单；再次，所使用的基础材料范围广泛，并且价格非常便宜。
附图说明
图1为本发明实施例1中cDNA-蛋白融合物的制备流程图。
图2为本发明实施例1中新型嘌呤霉素连接子的结构示意图。
图3为本发明实施例1中嘌呤霉素连接子与mRNA通过光交联结合的示意图。
图4A为本发明实施例1中mRNA与荧光标记的嘌呤霉素连接子光交联产物的PAGE检测结果。
图4B为本发明实施例1中mRNA与嘌呤霉素连接子光交联产物的SYBR Green染胶图。
图5为本发明实施例1中BDA基因的cDNA-蛋白融合物的尿素SDS-PAGE的荧光图。
图6为本发明实施例1中模板DNA结构组成示意图。
图7为本发明Spacer C3的结构式。
图8为本发明Spacer C6的结构式。
图9为本发明Spacer C9的结构式。
图10为本发明Spacer C12的结构式。
图11为本发明Spacer C18的结构式。
图12为本发明实施例2中PDO基因的cDNA-蛋白融合物的尿素SDS-PAGE的荧光图。
图13为本发明实施例3中anti-GFP VHH基因的cDNA-蛋白融合物的尿素SDS-PAGE的荧光图。
具体实施方式
下面结合具体实施例对本发明进行详细的描述。以下实施例将有助于本领域技术人员进一步理解本发明。实施例中未注明具体条件的方法，采用所属领域中的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
本实施例提供的是一种以蛋白A的B结构域(简称BDA)为目标基因体外核酸展示肽的合成方法。BDA蛋白的cDNA-蛋白融合物制备的整个流程见图1。其中所述目标基因的核酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。
以下为整个流程的详细流程及参数：
1、嘌呤霉素连接子的合成
嘌呤霉素连接子结构如图2所示，该嘌呤霉素连接子为含修饰无分支的单链DNA，该单链DNA包括第一段核苷酸和第二段核苷酸；所述第一段核苷酸的3’末端修饰有嘌呤霉素，共同组成嘌呤霉素臂，所述第一段核苷酸上还修饰有荧光标记；所述第二段核苷酸按照5’至3’的顺序包括mRNA连接位点和逆转录位点。
其中，所述第一段核苷酸的序列自5’至3’顺序为TCTCTCCC，所述第二段核苷酸的序列如SEQ ID NO.3所示。所述嘌呤霉素连接子的核苷酸序列经由引物合成公司合成，该嘌呤霉素连接子的核苷酸序列长度为27bp，进一步于所述第一段核苷酸自5’至3’顺序的第3位碱基修饰荧光标记，并将第一段核苷酸与第二段核苷酸由柔性接头Spacer C18(简称spc18)相连接，在所述第一段核苷酸自5’至3’顺序的第6-7位碱基之间插入两个Spacer C18。在该嘌呤霉素连接子中，所述mRNA连接位点位于所述第二段核苷酸自5’至3’顺序的第1位到第7位碱基序列，所述逆转录位点位于所述第二段核苷酸自5’至3’顺序的第8位到第19位碱基序列。该嘌呤霉素连接子的连接信息如下：
3’-反向(TGCCCCCCGCCG cnvKACCTTT)(spc18)TC(FAM-dT)CTC(spc18)(spc18)CC-嘌呤霉素-3’
mRNA与嘌呤霉素连接子共价交联及cnvK结构示意图见图3。
通过上述方法合成的嘌呤霉素连接子合成方法简单，效率高达53％，极大的提高了关键原料获取的效率，进一步提高系统效率及缩减了cDNA-蛋白融合产物的合成成本，并且该结构中的mRNA连接位点是以光交联的方式将嘌呤霉素连接子与mRNA进行共价交联，结构更加稳定。
2、模板DNA的准备
模板DNA结构如图6所示，该模板DNA自5’至3’的顺序由T7启动子、翻译增强子、Kozak序列、目标基因、间隔序列(Spc)、His标签、间隔序列(Spc)以及Y标签(与嘌呤霉素连接子部分互补配对的序列)组成；直接化学合成全长序列如SEQ ID NO.4，后通过PCR扩增获取足够量DNA。其中上述模板DNA中T7启动子-翻译增强子的核酸序列如SEQ ID NO.5所示，所述蛋白纯化标签(His标签)的核苷酸序列如SEQ  ID.6所示，所述间隔序列包括第一间隔序列(位于目标基因与His标签之间)和第二间隔序列(位于His标签与Y标签之间)，其中所述第一间隔序列的的核酸序列如SEQ ID NO.7所示，所述第二间隔序列自第5’至3’顺序的核苷酸序列为：GGCGGAAGC，所述Y标签的核酸序列如SEQ ID NO.8所示。
取上述0.1-1ng化学合成基因序列制备PCR反应体系，进行PCR反应。反应体系(50μl)：0.1-1ng DNA、0.5-1μl Q5高保真DNA聚合酶(2unit/μl)、10ul 5×缓冲液、0.4μl dNTPs(25mM)、0.2ul正向引物F(核酸序列如SEQ ID NO.9所示)、0.2ul反向引物R(核酸序列如SEQ ID NO.10所示)，其余RNase free水补至50ul。PCR反应条件：a、98℃(1-3min)，b、98℃(5-45s)，c、55-70℃(10-60s)，d、72℃(10-60s)，e、72℃(1-5min)，循环步骤b-d 25到35次。PCR结束后，可使用DNA纯化磁珠或者胶回收进行纯化。
3、体外转录获取目标mRNA
上述步骤获得的DNA作为模板，使用RiboMAX TM Express Large Scale RNA Production System-T7(Promega)进行转录。20μl反应混合物包括10μl 2×T7转录缓冲液,2μl混合酶,0.2-1μg的双链DNA，其余为RNase free水。首先，37℃反应30min，然后，将0.5-1μl RQ1 RNase free DNase加入到反应混合物中，37℃反应15min。反应结束后，使用DNA纯化磁珠对反应混合物进行纯化。
4、mRNA与嘌呤霉素连接子的共价偶联
mRNA与嘌呤霉素连接子按照摩尔数1:1的比例加入到杂交缓冲液(配方见表1)中，进行退火；退火后，取1μl样本留样，其余样本在波长365nm的紫外灯下，直接光照60s，获得mRNA-嘌呤霉素连接子偶联物；
退火条件：90℃1min(-0.4℃/s，即每秒钟降低0.4℃)70℃1min(-0.1℃/s，即每秒钟降低0.1℃)25℃停止。
将上述1μl退火后样本(不光照，阴性对照)及1μl光照后样本通过变性聚丙烯酰胺凝胶在60℃电泳条件下检测偶联。在荧光条件下，不光照样本无荧光条带，光照样本有特异性荧光条带，具体结果如图4A所示。在SYBR Green染胶条件下，不光照样本，仅有一条mRNA带；光照样本，除mRNA条带外，其上方出现一条特异性的条带，对应于荧光条件下出现的荧光条带位置，即为mRNA-嘌呤霉素连接子偶联物，具体结果如图4B所示。通过mRNA-嘌呤霉素连接子偶联物与不光照泳道中mRNA的条带亮度比，计算偶联效率大于90％。
表1
Tris-HCl PH7.5 25mM
氯化钠 100mM
5、mRNA-Linker-蛋白融合物的形成
将上述步骤获得的mRNA-Linker偶联物(即，mRNA-嘌呤霉素连接子偶联物)加入到兔网织红翻译体系中，进行翻译偶联。按表2配制反应体系：
表2
兔网织红细胞裂解液 17.5μl
氨基酸混合物，不包含异亮氨酸,1mM 0.25μl
氨基酸混合物，不包含甲硫氨酸,1mM 0.25μl
核糖核酸酶抑制因子 0.5μl
mRNA-嘌呤霉素连接子偶联物 3pmol
Nuclease-Free水 补至25μl
30℃反应20min，然后加入终浓度900mM KCl及80mM MgCl 2，在高盐条件下，37℃孵育60min。
6、逆转录
使用DNA纯化磁珠纯化表达产物，将全部磁珠加入到逆转录反应体系(见表3)，42℃反应60min。逆转录结束后，向上述反应液中加入等体积的2×His标签结合缓冲液(配方见表4)，同时加入1μl RNaseH，37℃反应30min(在此过程中避免磁珠沉降)，反应结束后，将反应管置于磁力架上，待溶液澄清后，小心转移上清液到新的1.5ml离心管中，此过程中避免碰到磁珠。
表3
Figure PCTCN2020123835-appb-000001
表4
Figure PCTCN2020123835-appb-000002
7、His标签蛋白纯化
上述上清液与20μl His标签纯化磁珠孵育，使用200μl His标签洗脱缓冲液(配方见表5)进行洗脱，得到cDNA-蛋白融合物，通过尿素-SDS-PAGE检测cDNA-蛋白融合物的形成。
表5
Figure PCTCN2020123835-appb-000003
如图5所示，将整个cDNA-蛋白融合物制备过程中得到每一步样本进行留样电泳分析。将mRNA-嘌呤霉素连接子偶联物(lane1)翻译成mRNA-蛋白融合物(lane2，上面的条带为融合产物；下面的条带对应为未与多肽偶联的mRNA-嘌呤霉素连接子偶联物)，然后与DNA纯化磁珠结合(为检测结合效果，分析上清液lane3)。用上述纯化磁珠进行反转录，形成mRNA/cDNA-蛋白融合物(lane4，上面的条带为mRNA/cDNA-蛋白融合物，下面条带对应为未偶联蛋白的mRNA/cDNA杂交链)，RNaseH消化mRNA(lane5，上面的条带为cDNA-蛋白融合物，下面条带对应为未偶联蛋白的cDNA)。用His标签纯化磁珠进一步纯化(lane6为结合后上清液)，得到单一的cDNA-蛋白融合物(lane7，His标签纯化洗脱液)。根据条带强度计算分析cDNA-蛋白融合物的总形成效率(从mRNA-嘌呤霉素连接子偶联物到His标签纯化洗脱液)约为15％左右。
实施例2
本实施例提供的是一种POU特异性DNA结合结构域(简称PDO)为目标基因体外核酸展示肽的合成方法。与实施例1的差异仅在于步骤2中目标基因部分及步骤7中cDNA-蛋白融合物合成效率的部分。其中所述PDO目标基因的核酸序列如SEQ ID NO.11所示，氨基酸序列如SEQ ID NO.12所示。
根据实施例1中的步骤1-7制备PDO蛋白的cDNA体外展示肽，通过尿素SDS-PAGE检测cDNA-蛋白融合物的形成。如图12所示：
将mRNA-嘌呤霉素连接子偶联物(lane1)翻译成mRNA-蛋白融合物(lane2，上面的条带为融合产物；下面的条带对应为未与多肽偶联的mRNA-嘌呤霉素连接子偶联物)， 然后与DNA纯化磁珠结合(为检测结合效果，分析上清液lane3)。用上述纯化磁珠进行反转录，然后RNaseH消化mRNA，形成cDNA-蛋白融合物(lane4，上面的条带为cDNA-蛋白融合物，下面条带对应为未偶联蛋白的cDNA)。用His标签纯化磁珠进一步纯化(lane5为His标签纯化上清液)，得到单一的cDNA-蛋白融合物(lane6，His标签纯化洗脱液)。根据条带强度计算分析cDNA-蛋白融合物的总形成效率(从mRNA-嘌呤霉素连接子偶联物到His标签纯化洗脱液)约为7.5％左右。
实施例3
本实施例提供的是一种抗绿色荧光蛋白的羊驼抗体基因(简称anti-GFP VHH)为目标基因体外核酸展示肽的合成方法。与实施例1的差异仅在于步骤2中目标基因部分及步骤7中cDNA-蛋白融合物合成效率的部分。其中所述anti-GFP VHH目标基因的核酸序列如SEQ ID NO.13所示，氨基酸序列如SEQ ID NO.14所示。
根据实施例1中的步骤1-7制备anti-GFP VHH抗体的cDNA体外展示肽，通过尿素SDS-PAGE检测cDNA-蛋白融合物的形成。如图13所示：
将mRNA-嘌呤霉素连接子偶联物(lane1)翻译成mRNA-蛋白融合物，然后与DNA纯化磁珠结合，用上述纯化磁珠进行反转录，然后RNaseH消化mRNA，形成cDNA-蛋白融合物(lane2，上面的条带为cDNA-蛋白融合物，下面条带对应为未偶联蛋白的cDNA)。用His标签纯化磁珠进一步纯化(lane3，His标签纯化上清液)，得到单一的cDNA-蛋白融合物(lane4，His标签纯化洗脱液)。根据条带强度计算分析cDNA-蛋白融合物的总形成效率(从mRNA-嘌呤霉素连接子偶联物到His标签纯化洗脱液)约为0.9％左右。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (14)

  1. 一种嘌呤霉素连接子，所述嘌呤霉素连接子为含修饰无分支的单链DNA，该单链DNA包括第一段核苷酸和第二段核苷酸；
    其中，所述第一段核苷酸包含一段dNTP合成的寡核苷酸序列，所述第二段核苷酸包含一段反向dNTP合成的寡核苷酸序列，所述第一段核苷酸的5'端和所述第二段核苷酸的5'端相连接形成包含两个3’末端的连接体结构；
    所述第一段核苷酸的3’末端修饰有嘌呤霉素；所述第二段核苷酸按照5’至3’的顺序包括mRNA连接位点和逆转录位点。
  2. 根据权利要求1所述的嘌呤霉素连接子，所述第一段核苷酸的序列自5’至3’顺序包括TCTCTCCC，所述第二段核苷酸的序列包括如SEQ ID NO.3所示序列。
  3. 根据权利要求1所述的嘌呤霉素连接子，所述第一段苷酸中还包含2-4个间隔子和/或1-18个碱基的核苷酸序列；优选地，每个间隔子各自独立地选自Spacer C3、Spacer C6、Spacer C9、Spacer C12和Spacer C18中的任意一种，优选为Spacer C18。
  4. 根据权利要求1所述的嘌呤霉素连接子，所述第一段核苷酸中还包含核酸纯化标签和/或化学修饰；优选地，所述核酸纯化标签包括多聚腺苷酸序列或任意碱基序列；进一步优选地，化学修饰包括用于核酸纯化或与其他配体结合的修饰标记和/或荧光标记；进一步优选地，用于核酸纯化或与其他配体结合的修饰标记包括生物素标记；进一步优选地，所述荧光标记包括FAM、FITC或Cy染料；进一步优选地，所述荧光标记所连接的位点包括位于所述第一段核苷酸自5’至3’顺序的第3位碱基。
  5. 根据权利要求1所述的嘌呤霉素连接子，所述mRNA连接位点是一段包含人工合成核酸三氰基乙烯基甲基咔唑修饰的寡核苷酸序列；优选地，所述mRNA连接位点包括位于所述第二段核苷酸自5’至3’顺序的第1位到第7位碱基序列。
  6. 根据权利要求1-5任一项所述的嘌呤霉素连接子，所述逆转录位点为一段寡核苷酸序列，长度范围优选为1-15个碱基；进一步优选地，所述逆转录位点包括位于所述第二段核苷酸自5’至3’顺序的第8位到第19位碱基序列。
  7. 权利要求1-6任一项所述的嘌呤霉素连接子在体外核酸展示肽合成中的应用，所述应用包含如下步骤：
    (1)提供模板DNA；
    (2)将所述模板DNA体外转录、纯化得到单一的mRNA产物；
    (3)将所述mRNA产物与权利要求1-6任一项所述的嘌呤霉素连接子混合后进行退火，使用紫外光波照射，获取mRNA-嘌呤霉素连接子偶联物；
    (4)将所述mRNA-嘌呤霉素连接子偶联物在表达体系中进行翻译，以在嘌呤霉素连接子上结合对应于mRNA序列的肽，从而形成mRNA-蛋白融合物；
    可选地，在步骤(4)之后还包括如下步骤：
    (5)对所述mRNA-蛋白融合物中的mRNA进行逆转录以形成逆转录产物：
    mRNA/cDNA-蛋白融合物；
    进一步可选地，在步骤(5)之后还包括如下步骤：
    (6)利用蛋白纯化标签对所述逆转录产物进行分离纯化，获取cDNA-蛋白融合物。
  8. 如权利要求7所述的应用，步骤(1)中，所述模板DNA自5’至3’的顺序包含启动子、翻译增强子、Kozak序列、目标基因、间隔序列、蛋白纯化标签、间隔序列以及Y标签；优选地，所述启动子包括T7启动子、SP6启动子或T3启动子；进一步优选地，所述翻译增强子包括烟草花叶病毒的5’前导序列或非洲爪蟾β-珠蛋白非翻译序列；进一步优选地，所述蛋白纯化标签包括His标签或Flag标签；进一步优选地，所述间隔序列选自编码氨基酸GGS、GGGS、GGGASG4SG4S、(G4S) 2及GGGASGGGGS的核苷酸序列中的一种或两种以上的组合；进一步优选地，所述Y标签是与嘌呤霉素连接子部分互补配对的序列。
  9. 根据权利要求7或8所述的应用，步骤(1)中，模板DNA的长度依赖于被展示肽的核酸编码序列的长度；优选地，模板DNA长度为50-1000个核苷酸，进一步优选地，模板DNA长度为200-500个核苷酸。
  10. 根据权利要求7-9任一项所述的应用，步骤(3)中，所述mRNA产物与嘌呤霉素连接子的混合摩尔数比为1：(1-1.5)；所述紫外光波的波长为330-400nm。
  11. 如权利要求7所述的应用，步骤(4)中，所述表达体系为无细胞表达体系；优选地，所述无细胞表达体系包括兔网织红表达体系、小麦胚表达体系或大肠杆菌表达体系。
  12. 如权利要求7所述的应用，步骤(5)的具体步骤包含：
    (a)核酸纯化，将mRNA及mRNA-蛋白融合物从翻译体系中进行分离；
    (b)进行mRNA的逆转录反应；
    (c)逆转录结束后，选择性加入RNaseH消化mRNA。
  13. 根据权利要求7所述的应用，步骤(6)中，蛋白纯化标签包括His标签或Flag标签。
  14. 权利要求7-13任一项所述的应用所制备出的体外核酸展示肽。
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