WO2017028548A1 - 镜像核酸复制体系 - Google Patents

镜像核酸复制体系 Download PDF

Info

Publication number
WO2017028548A1
WO2017028548A1 PCT/CN2016/079006 CN2016079006W WO2017028548A1 WO 2017028548 A1 WO2017028548 A1 WO 2017028548A1 CN 2016079006 W CN2016079006 W CN 2016079006W WO 2017028548 A1 WO2017028548 A1 WO 2017028548A1
Authority
WO
WIPO (PCT)
Prior art keywords
mirror
nucleic acid
image
polymerase
dna
Prior art date
Application number
PCT/CN2016/079006
Other languages
English (en)
French (fr)
Inventor
朱听
刘磊
王子谋
徐维亮
Original Assignee
清华大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 清华大学 filed Critical 清华大学
Priority to US16/092,757 priority Critical patent/US11371027B2/en
Priority to EP17781735.0A priority patent/EP3444341A4/en
Priority to CN201780026142.7A priority patent/CN109072203B/zh
Priority to PCT/CN2017/073573 priority patent/WO2017177759A1/zh
Publication of WO2017028548A1 publication Critical patent/WO2017028548A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07048—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase

Definitions

  • the present invention relates to the field of genetic engineering and, more particularly, to the replication and transcription of a mirror nucleic acid.
  • Chirality is the basic property of some molecules in three-dimensional space, that is, an object cannot coincide with its image in the mirror. Like the left and right hands of a person, the two are objects and mirrors, and they cannot overlap regardless of how they are flipped in three-dimensional space. This is the chirality of an object in space.
  • a mirror image of a molecule is called its enantiomer. They have the same physical and chemical properties, and have the same melting point, molecular weight, solubility, density, and NMR spectrum. The mutually different nature of the enantiomers is their optical nature - the direction of the plane of polarization of the rotation. Chirality is widespread in nature.
  • Biomacromolecules in living organisms such as proteins, polysaccharides, DNA and RNA are chiral. All organisms on the planet, whether large plants, mammals or microorganisms that are invisible to the naked eye, constitute the 20 amino acids of the protein. Except that glycine has no chirality, all 19 other amino acids are L-form; DNA and RNA, their ribose are all D-type.
  • the amino acid has a chiral carbon atom in the immediate vicinity of the carboxyl group.
  • the amino acids other than glycine have both L and D chiralities.
  • L refers to levorotatory or left-handed
  • D refers to dextrorotatory or right-handed.
  • type D is a chirality found in nature, and the chiral center of a nucleic acid molecule is located on its backbone.
  • the biological activity of two different chiral compounds in a living organism may be completely different. Enzymes and cell surface receptors in biological individuals are mostly chiral, and the two enantiomers are often absorbed, activated and degraded in different ways in the organism. For common chiral drugs, the two enantiomers may have equivalent pharmacological activities, or one may be active, the other inactive, or even the enantiomers. At present, it is believed that the molecules of proteins and nucleic acids present in living organisms have the characteristics of chirality. If a mirrored amino acid is incorporated into a native protein sequence, it destroys its own secondary structure (Krause et al., 2000) and has a severe impact on its protein function.
  • RNA is formed by organic molecules in the early Earth environment, and RNA evolves into a biologically functional RNA-self-replicating, self-shearing activity-RNA ribozyme;
  • proteases or peptides composed of amino acids begin to participate in the replication, unwinding, etc. of RNA, which may be the process of chiral selection at this stage, which leads to the chirality of the finally evolved complex organism. Uniformity.
  • mirrored genetic information replication and transcription illustrates the potential of mirrored life molecules and the potential for biological activity, and also lays the foundation for the future construction of mirror cells in the laboratory environment.
  • image replication system will also be used for biological methods to efficiently screen image nucleic acid drugs.
  • the present invention provides a method of replicating a mirror nucleic acid comprising: performing a reaction in the presence of a mirror nucleic acid polymerase, a mirror nucleic acid template, a mirror nucleic acid primer, and a mirror image dNTPs/rNTPs to obtain the mirror nucleic acid.
  • the present invention provides a method of performing mirror image PCR comprising: performing a reaction in the presence of a mirror nucleic acid polymerase, a mirror nucleic acid template, a mirror nucleic acid primer, and mirror image dNTPs/rNTPs to obtain a mirror nucleic acid.
  • the invention provides a method for screening a mirror nucleic acid molecule, comprising: contacting a library of random image nucleic acid sequences with a target molecule under conditions allowing binding of the two; obtaining a mirror nucleic acid molecule that binds to the target molecule; and amplifying the image by mirror image amplification A mirror nucleic acid molecule that binds to a target molecule.
  • the present invention also provides D-type ASFV pol X, the sequence of which is shown in SEQ ID NO: 17, wherein all amino acids except the chiral glycine are D-type amino acids.
  • Figure 1 shows the chemical synthesis route of D-ASFV Pol X.
  • 174 amino acid ASFV pol X is synthesized by peptide 1: Met1-Lys85, peptide 2: Cys86-Leu105 and peptide 3: Ala106-Leu174 And connected. Cys86 was first protected with Acm, Cys86-Leu105 was synthesized, and peptide 3: Cys106-Glu107-Leu174 was synthesized. After activation and ligation, the ligation product of peptide 2 and peptide 3 was obtained, and then Cys106 was desulfurized to form Ala106. Recombination of peptide 1 catalyzes the removal of the Acm protecting group of peptide 2, activation and ligation, to obtain full-length ASFV pol X polymerase.
  • Figure 2 shows the detection of the full length product of D-ASFV pol X synthesis.
  • a Folded D-ASFV pol X HPLC spectrum. HPLC analysis used a 214 nm absorption wavelength, Vydac C18 (4.6 x 250 mm) liquid chromatography column.
  • b ESI-MS spectrum By analyzing and calculating the ion peak map, the size of the main synthetic product was observed to be 20317.0 Da, and the theoretical value of ASFV pol X was 20316.0 Da.
  • Figure 3 shows L-type and D-type ASFV pol X detection.
  • A SDS-PAGE was used to detect ASFV pol X polymerase expressed in Escherichia coli, chemically synthesized L-type and chemically synthesized D-type on 15% SDS-PAGE gel, and detected by silver staining. M, protein molecular weight marker.
  • b CD detection L-type and D-type chemical synthesis ASFV pol X was tested on an Applied Photophysics Pistar-180 CD spectrometer, and the absorption curve was the average of 3 independent tests after deducting the background.
  • Figure 4 shows mirror image DNA detection.
  • HPLC detection provided by Chemgenes
  • HPLC detection for the purification and analysis of four L-dNTPs by HPLC.
  • the results of L-dATP and L-dGTP have almost no peaks, while L-dTTP and L-dCTP have obvious impurities. peak.
  • Figure 5 shows a mirror image polymerase catalyzing DNA extension.
  • Natural and mirror DNA replication system schematics Natural DNA replication systems contain L-polymerases, D-DNA and D-dNTPs; mirror DNA replication systems contain D-polymerases, L-DNA and L-dNTPs
  • Natural and mirrored polymerizations Enzyme-catalyzed DNA extension (12 nt primer, 18 nt template) buffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, and a corresponding chiral 0.7 ⁇ g ASFV pol was added to a 10 ul natural or mirror reaction system.
  • Natural and mirror polymerase catalyzed DNA extension (15 nt primer) , 21 nt template) reaction system was carried out in 50 mM Tris-HCl pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, 2.5 ⁇ M 15-nt L-primer 15 (without FAM modification), 2.5 ⁇ M 21-nt L-template 21 0.2 mM L-dNTPs (each concentration), and 1.4 ⁇ g D-ASFV pol X. After 12 hours of reaction at 37 ° C, 20% PAGE gel separation was performed and detected by Sybr Gold staining.
  • Figure 6 shows a schematic representation of multi-cycle amplification of mirror DNA.
  • the reverse 11 primer without FAM was amplified to obtain a double-stranded template
  • the second cycle of the FAM-labeled primer 11 was only complementary to the template (orange-red) amplified in the first cycle to obtain a full-length product.
  • the third cycle produces three times the fluorescent full length product of the second cycle.
  • Figure 7 shows multi-cycle amplification of a mirror DNA replication system.
  • Cycle1 amplification yields the full length product of reverse11, which can be used as a template by subsequent cycles.
  • Samples were separated on 8 M urea denatured 20% PAGE gel and scanned for fluorescence with Typhoon Trio+. All products detected were FAM-labeled DNA.
  • Figure 8 shows the base complementary pairing specificity of the mirror DNA extension. Add one of the corresponding chiral 0.2 mM dATP, dTTP, dCTP, and dGTP in the natural or mirror image system. The next base of the template is A, T, C, and G (white on white) Box labeled), buffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, 0.7 ⁇ g ASFV pol X, 2.5 ⁇ M primer, 2.5 ⁇ M template 18, corresponding to chirality.
  • the reaction was carried out at 37 ° C for 30 minutes, electrophoresed on a 20% PAGE gel and the fluorescent signal was scanned with a Typhoon Trio+ scanner. "*" indicates the 5'FAM mark. "-” indicates a control group to which D or L-dNTPs were not added.
  • Figure 9 shows the chiral specificity of mirror image DNA extension.
  • buffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, 0.7 ⁇ g ASFV pol X, 2.5 ⁇ M primer, 2.5 ⁇ M template18, chirality of protein, primer-template, dNTPs were added.
  • the reaction was carried out at 37 ° C for 12 hours, electrophoresed on a 20% PAGE gel and the fluorescent signal was scanned with a Typhoon Trio+ scanner. "*" indicates the 5'FAM mark.
  • Figure 10 shows the reaction of the natural and mirror system in the same solution.
  • Two buffered 0.7 ⁇ g ASFV pol X, 2.5 ⁇ M native 5' end Cy5 labeled primer20, 2.5 ⁇ M native template26 were added in buffer conditions of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl.
  • Figure 11 shows the enzyme synthesis and activity assay of native and mirror DNAzymes.
  • a Zn2+-dependent DNAzyme secondary structure
  • a 12 nt primer sequence was added to the 5' end of the 44 nt DNAzyme with a full length of 56 nt. The secondary structure is generated by the mfold server (Zuker, 2003).
  • b Enzymatic synthesis of DNAzyme
  • Both natural and mirrored DNAzymes were reacted in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl buffer, and 66 nt DNAzyme template and 12 nt primers were added at 37 ° C. The full length product was obtained after 36 hours.
  • M marker is a chemically synthesized 56 nt sequence as a molecular weight standard.
  • c The extended full-length DNAzyme was excised from the PAGE gel, spread in a buffer overnight, and precipitated by a Tianenze PAGE gel recovery kit. The precipitated DNA was dissolved in buffer 1:50 mM HEPES, pH 7.0 and 100 mM NaCl, and heated at 90 ° C for 2 min, and then cooled on ice for 5 min.
  • Figure 12 shows native and mirror system DNA template-dependent RNA transcription.
  • Native and mirrored ASFV pol X catalyzed RNA transcription buffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, 0.7 ⁇ g ASFV pol X, 2.5 ⁇ M primer was added to the 10 ul reaction system.
  • a 2.5 ⁇ M template and four 0.4 mM rNTPs were added to the natural system with 2unit RNase inhibitor. The reaction was terminated after reacting at 37 ° C for 60 hours.
  • b Native and mirrored ASFV pol X catalyzed RNA transcription buffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, 0.7 ⁇ g ASFV pol X, 2.5 ⁇ M primer was added to the 10 ul reaction system.
  • the natural system to which rNTP was added was subjected to a reaction at 37 ° C for 36 h to obtain a full length product.
  • ASFV pol X and RNase inhibitors were inactivated by heating to 75 ° C for 10 min. 1 ⁇ g/ ⁇ l, 0.1 ⁇ g/ ⁇ l, 0.01 ⁇ g/ ⁇ l of RNase A were added to each of the three experiments, followed by incubation at 23 ° C for 10 min.
  • the degradation reaction was terminated by the addition of a 20unit RNase inhibitor and a loading buffer was added.
  • the reaction products were separated on 8M urea denatured 20% PAGE gel and imaged using the Typhoon Trio+ system.
  • Sample 1 Control group, extension 0h
  • Sample 2 D-primer12 36 hours extended full length product
  • Sample 3 full length product heated at 75 ° C for 10 min, inactivated RNase inhibitor and ASFV pol X
  • Sample 4-6 full length The product was extended, heated at 75 ° C for 10 min, 0.01 ⁇ g / ⁇ l, 0.1 ⁇ g / ⁇ l, 1 ⁇ g / ⁇ l of RNase A, placed at 23 ° C for 10 min, and the reaction was terminated by the addition of 20unit RNase inhibitor. Electrophoresis was performed on a 20% PAGE gel and the fluorescent signal was scanned with a Typhoon Trio+ scanner. "*" indicates the 5'FAM mark.
  • Figure 13 shows the base complementary pairing specificity of native and mirror system RNA transcription.
  • one of the corresponding chiral 0.2 mM rATP, rUTP, rCTP, and rGTP is added, and the next base of the template is A, T, C, and G (white box on black background).
  • the buffer conditions were 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl, and 0.7 ⁇ g ASFV pol X, 2.5 ⁇ M primer, 2.5 ⁇ M template 18 corresponding to chirality were added.
  • the reaction was carried out at 37 ° C for 12 hours, electrophoresed on a 20% PAGE gel and the fluorescent signal was scanned with a Typhoon Trio+ scanner. "*" indicates the 5'FAM mark. "-” indicates a control group to which D or L-dNTPs were not added.
  • the present invention provides a method of replicating a mirror nucleic acid comprising: performing a reaction in the presence of a mirror nucleic acid polymerase, a mirror nucleic acid template, a mirror nucleic acid primer, and a mirror image dNTPs/rNTPs to obtain the mirror nucleic acid.
  • mirror image refers to an isomer that is mirrored to the natural material in chirality.
  • image nucleic acid refers to an L-type nucleic acid that is in mirror image relationship with a natural nucleic acid (ie, a D-type nucleic acid).
  • Mirror nucleic acids include L-form DNA and L-form RNA.
  • mirror DNA is used interchangeably with "L-type DNA” or "L-DNA.”
  • image nucleic acid polymerase or "mirror polymerase” as used herein refers to a D-type polymerase that is in mirror image relationship with a native polymerase (ie, an L-type polymerase).
  • mirror polymerase is used interchangeably with “D-type polymerase” or “D-polymerase.”
  • D-Dpo4 refers to D-type Dpo4 polymerase which is mirror image of native L-type Dpo4 polymerase.
  • Polymerases particularly suitable for the present invention include D-ASFV pol X, D-Dpo4, D-Taq polymerase, and D-Pfu polymerase.
  • Dpo4 (Sulfolobus solfataricus P2DNA polymerase IV, Sulfolobus sp. P2 DNA polymerase IV) is a thermostable polymerase that also synthesizes DNA at 37 °C. Its mismatch rate is between 8 ⁇ 10 -3 -3 ⁇ 10 -4 . It is a polymerase that can replace Taq for multi-cycle PCR reactions. Its amino acid sequence length is within the reach of current chemical synthesis techniques.
  • Taq polymerase is a thermostable polymerase discovered by Chien and colleagues in the hot spring microbe Thermus aquaticus in 1976. It can remain active at DNA denaturation temperatures, so it is used in PCR instead of E. coli polymerase.
  • the optimum temperature for Taq is between 75 ° C and 80 ° C and the half-life at 92.5 ° C is about 2 h.
  • Pfu polymerase is found in Pyrococcus furiosus, and its function in microorganisms is to replicate DNA during cell division. It is superior to Taq in that it has 3 '-5' exonuclease activity and can cleave the mis-added nucleotides on the extended strand during DNA synthesis.
  • the commercial Pfu mismatch rate is around 1.3 million.
  • the mirror nucleic acid, the mirror nucleic acid template, the mirror nucleic acid primer, and the mirror dNTPs/rNTPs are L-form, and the mirror nucleic acid polymerase is D-form.
  • different types of templates, primers, or dNTPs/rNTPs may be mixed in the reaction system (for example, a portion of D-type template primers or dNTPs/rNTPs may be mixed) without causing serious interference with the reaction.
  • the nucleic acid replication reaction can be carried out in only one cycle or in multiple cycles. This can be determined by the technician based on actual needs.
  • multiple cycles refers to 2 or more cycles, such as 3, 4, or 10 cycles.
  • replication includes obtaining one or more copies of a target DNA in the presence of a DNA template and dNTPs; it also includes obtaining one or more copies of the target RNA in the presence of a DNA template and rNTPs (this process may also be referred to as For the "transcription" of RNA).
  • the template and the primer are usually DNA. If the target nucleic acid is DNA, dNTPs should be added to the reaction system; if the target nucleic acid is RNA, rNTPs should be added to the reaction system.
  • the image nucleic acid is an L-form DNA, such as an L-DNAzyme. In other embodiments, the image nucleic acid is an L-form RNA.
  • the reaction is a polymerase chain reaction.
  • PCR as used herein has a meaning as known in the art and refers to a polymerase chain reaction (Polymerase Chain Reaction).
  • the reaction is carried out in the following buffer: 50mM Tris-HCl, pH 7.5,20mM MgCl 2, 1mM DTT, 50mM KCl.
  • the present invention also provides a method of performing mirror image PCR comprising: performing a reaction in the presence of a mirror nucleic acid polymerase, a mirror nucleic acid template, a mirror nucleic acid primer, and a mirror image dNTPs/rNTPs to obtain a mirror nucleic acid.
  • the invention also provides a method for screening a mirror nucleic acid molecule, comprising: contacting a library of random image nucleic acid sequences with a target molecule under conditions allowing binding of the two; obtaining a mirror nucleic acid molecule that binds to the target molecule; and amplifying by mirror image PCR The image nucleic acid molecule that binds to the target molecule.
  • the target molecule is immobilized on a solid phase medium, which may be more advantageous for separation and purification.
  • the image nucleic acid molecule that does not bind to the target molecule can be removed by washing to obtain a mirror nucleic acid molecule that binds to the target molecule.
  • the image nucleic acid molecule can be L-form DNA or L-form RNA.
  • the image nucleic acid polymerase used in the mirror image PCR may be D-ASFV pol X, D-Dpo4, D-Taq polymerase or D-Pfu polymerase.
  • the mirror image PCR is carried out in a buffer of 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl.
  • the present invention also provides D-type ASFV pol X, the sequence of which is shown in SEQ ID NO: 17, wherein all amino acids except the chiral glycine are D-type amino acids.
  • D-amino acids are present in mammals.
  • Hoeprich and colleagues detected D-Ala in the blood of guinea pigs and mice, the first time researchers have discovered D-amino acids in mammals (Corrigan, 1969).
  • D-Ala has been found in the brains and pituitaries of various mammals, and D-Ala excretion has been found in the urine.
  • D-Pro and D-Leu were found in seven regions of the mouse brain, indicating that D-amino acids are Pituitary and pineal bodies have relatively high concentrations (Hamase et al., 2001).
  • D-Ser and D-Ala are also detected in the brain and blood of mammals such as humans and mice (Hashimoto et al., 1993; Hashimoto et al., 1995).
  • D-amino acids were first thought to be synthesized by microorganisms, plants, and invertebrates, and recent studies have shown that D-Ser and D-Asp can be synthesized by mammalian tissues. It was found by isotopic labeling experiments that radioactive L-Ser can be transformed into D-Ser in rat and mouse brains. In 1999, Ser racemase was cloned and purified. The brain can catalyze the catalysis of L-Ser to D-Ser (Wolosker et al., 1999). Another source of large D-amino acids in mammals is exogenous foods and microorganisms.
  • the mirrored amino acid has been confirmed to exist in a natural state of life, the natural state of the mirrored amino acid is mainly monomeric, and also exists in short peptide fragments, such as the tetrapeptide side chain amino acid d of Gram-positive bacteria.
  • -C-d-valley-rl-ly-d-c no mirror image protein composed of functional mirror amino acids in nature has been found yet.
  • the translation in the central rule is that ribosomes and tRNAs participate in the synthesis of proteins according to mRNA genetic information.
  • the process of mirroring amino acids was not used as a substrate in this process. It is generally believed that the transcription, translation and realization of major biological functions of the genetic information of living organisms depend on the natural chiral L-amino acids.
  • L-type and D-type HIV-1 proteases have the same mass spectral molecular weight, the same HPLC retention time, and the opposite circular dichroism spectral curve. In terms of activity, the L-type HIV-1 protease can cleave the L-peptide substrate, while the D-type protease can cleave the D-form substrate.
  • DapA is a protein that relies on the molecular chaperone GroEL/ES and can be folded into a functional conformation upon expression with the aid of a molecular chaperone. GroEL/ES can fold both D and L chiral DapAs, but the folding efficiency of native L-DapA is higher than that of mirrored DapA (Weinstock et al., 2014).
  • RNA in this study was obtained by chemical synthesis. It can generate a full-length RNA-polymerizing ribozyme opposite to its own chirality by linking 11 oligonucleotide fragments, and amplification of the two RNAs can be performed without interference in the same reaction system. This is an early stage of life, where two chiral RNA molecules coexist and provide a theoretical possibility for amplification by RNA-polymerizing ribozymes.
  • DNA/RNA was first thought to be a portable tool for genetic information and is considered to be much simpler than the structure of a protein. But in fact DNA/RNA can also be folded into a tertiary structure, which has a series of potential physiological functions. Originally in 1990, researchers discovered that RNA structures can specifically bind to small molecule substrates. These RNA structures, like antibodies, bind selectively to the substrate and have a high affinity. These can bind to specific substrate RNA structures and are referred to as aptamers. Later, DNA aptamers were also discovered by researchers.
  • In vitro screening techniques utilize random DNA/RNA sequence libraries to find nucleic acid aptamers that bind to specific target molecules.
  • In vitro screening requires a library of random sequences, either DNA or RNA.
  • the library typically contains a random sequence of 30-80 nucleotides and two primer regions on either side to facilitate PCR amplification.
  • a plurality of cycles of the screening process are performed, the target small molecule substrate is fixed on a substrate, and the random sequence is added to the substrate. After washing, the unbound DNA or RNA molecules flow through the immobilized substrate. The sequence with the binding ability that is screened will remain on top.
  • These special sequences are then eluted for PCR amplification, and after multiple cycles of enrichment and screening, one or several nucleotide sequences that specifically bind to the substrate can be obtained.
  • the image nucleic acid sequence has a specific secondary and tertiary structure according to its sequence, and can bind the target molecule in close and high specificity. If an in vitro screening strategy is applied to screen the target molecules of the mirror and then synthesize the mirrored nucleotide sequence, a mirrored nucleotide molecule capable of binding to the native target can be obtained.
  • the researchers obtained a mirrored L-RNA aptamer that binds D-adenosine and L-arginine by in vitro screening (Klusmann et al., 1996; Nolte et al., 1996). DPBartel was obtained in 1997.
  • L-DNA aptamers that bind vasopressin (Williams et al., 1997).
  • the image nucleotide molecule has the advantages of being stable in the body, being non-degradable, non-toxic, not causing an immune reaction, and having a relatively low production cost, and has a good application prospect as a drug molecule.
  • Mirror replication and transcription system can realize the amplification of mirror DNA and RNA by polymerase, indicating that mirror polymerase can catalyze the synthesis of DNA and RNA like natural chiral polymerase, and prove that mirror biomolecule has effective biological activity;
  • the mirror copying and transcription system implements two key steps in the mirror center rule, laying a technological foundation for the mirroring of the original cell synthesis;
  • the natural nucleic acid molecules obtained by the in vitro screening technology at present, as a drug have serious defects in the body that are easily hydrolyzed.
  • special methods are needed for screening of image nucleic acids.
  • the existing in vitro screening technology screens the mirror target by a natural random library to obtain an effective nucleic acid sequence, and then chemically synthesizes the image nucleic acid molecule, so that the obtained image nucleic acid molecule can bind the natural target, that is, the potential Mirrored drug.
  • the limitation of this method is that many common drug targets in living organisms have proteins of more than 300 amino acids, and it is impossible to synthesize mirror images by chemical methods.
  • Mirror in vitro screening if performed directly with natural target molecules and mirrored random libraries, will greatly enhance the ubiquity of this technology and screen for drug molecules in a wider range of diseases.
  • the bottleneck of mirror drug screening is that mirror PCR cannot be realized.
  • Our image replication and transcription system can achieve mirrored PCR.
  • PCR efficiency needs further optimization and improvement, it still provides a theoretical and practical basis for mirror drug screening.
  • RP-HPLC semi-preparative grade reversed-phase high performance liquid chromatography
  • the natural chemical linkage of the polypeptide fragments was carried out as follows.
  • the hydrazide group-containing polypeptide fragment was first dissolved in a buffer (6 M guanidine hydrochloride, 200 mM disodium hydrogen phosphate, pH 3.0).
  • a buffer solution mixed with 40 equivalents of 4-mercaptophenylacetic acid (MPAA), an equivalent N-terminal cysteine, and a pH 7.0 buffer solution was added.
  • MPAA 4-mercaptophenylacetic acid
  • N-terminal cysteine an equivalent N-terminal cysteine
  • a pH 7.0 buffer solution was added.
  • the pH of the system was adjusted to 7.0 and the reaction was carried out for 12 hours.
  • 80 mM trichloroethyl phosphate (TCEP) buffer was added to dilute the system concentration.
  • the target product was finally isolated using semi-preparative grade RP-HPLC.
  • the acetamidomethyl (Acm) protecting group of the Cys86 side chain was removed as follows. 0.5 ⁇ mol of the polypeptide fragment 4 was dissolved in 1 ml of 50% aqueous acetic acid. Then 5 mg of silver acetate was added and stirred at 30 ° C overnight. Then 2.5 mmol of mercaptoethanol was added and the system was diluted 2 fold with 6 M aqueous guanidine hydrochloride solution. The precipitate was removed by centrifugation, and the supernatant was separated by RP-HPLC to obtain the objective product 5.
  • D-ASFV pol X F-folding renaturation of D-ASFV pol X was carried out as follows. 5 mg of D-ASFV pol X was dissolved in 10 ml of 6 M guanidine hydrochloride solution, and the solution was placed in a 3 K Da dialysis bag. The dialysis bag was then immersed in a buffer system containing 4 M guanidine hydrochloride (50 mM Tris-HCl, 40 mM KCl, 6 mM magnesium acetate, 0.01 M EDTA and 16% glycerol) for 10 hours, then the guanidine hydrochloride concentration was gradually reduced to 2M. 1M and 0M.
  • 4 M guanidine hydrochloride 50 mM Tris-HCl, 40 mM KCl, 6 mM magnesium acetate, 0.01 M EDTA and 16% glycerol
  • the dialysis bag was immersed in each concentration of guanidine hydrochloride solution for 10 hours. It was confirmed by circular dichroism and mass spectrometry that D-ASFV pol X was correctly folded and a disulfide bond was formed by air oxidation between D-Cys81 and D-Cys86.
  • DNA polymerization method Polymerase reaction buffer, 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl was placed. 0.7 ⁇ g of ASFV pol X, 2.5 ⁇ M primer, 2.5 ⁇ M template and 4 0.4 mM dNTPs were added to the 10 ul reaction system. The reaction system was placed at 37 ° C for 4 hours, and the reaction was terminated by adding 0.5 ⁇ M of EDTA. The reaction yields a DNA fragment complementary to the template.
  • RNA polymerization method Polymerase reaction buffer, 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl was placed. 0.7 ⁇ g of ASFV pol X, 2.5 ⁇ M primer, 2.5 ⁇ M template and 4 0.4 mM rNTPs were added to the 10 ul reaction system. The reaction system was placed at 37 ° C for 60 hours, and the reaction was terminated by adding 0.5 ⁇ M of EDTA. The reaction yields a complex of primer DNA and RNA.
  • the reaction system was: 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, and 50 mM KCl, 28.9 ⁇ g D-ASFV pol X, 5 ⁇ M primer12 primer, 5 ⁇ M DNAzymeTemplate template and 1.6 mM dNTPs.
  • the reaction was carried out at 37 ° C for 36 hours.
  • the reaction product was applied to a loading buffer, and the strips were separated on a 12% PAGE gel using 300 V for 3 hours.
  • the DNAzymeTemplate template was designed to be 10 nucleotides longer than the full-length product.
  • the full-length product sequence was developed and excised.
  • the gel pieces were treated as described above, diffused overnight and ethanol precipitated to recover the DNA.
  • the precipitated product was dissolved in buffer 1:50 mM HEPES, pH 7.0 and 100 mM NaCl, and heated at 90 °C for 2 min, and then cooled on ice for 5 min.
  • Polymerase reaction buffer 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl were placed.
  • To the reaction system were added 2.672 ⁇ g of D-ASFV pol X, 2.5 ⁇ M L-FAM-primer, 2.5 ⁇ M L-template and 4 0.4 mM L-dNTPs.
  • the reaction system was placed at 37 ° C for 4 hours, and the reaction was terminated by adding 0.5 ⁇ M of EDTA. Since ASFV pol X has a strong ability to bind DNA, it cannot be dissociated even under the condition of heat denaturation at 95 °C.
  • RNase A digests natural RNA polymer products
  • the L-ASFV pol X RNA reaction system (see 2.2.3) was inactivated by a reaction at 37 ° C for 36 h and heating to 75 ° C for 10 min to attenuate ASFV pol X and RNase inhibitors. 1 ⁇ g/ ⁇ l, 0.1 ⁇ g/ ⁇ l, 0.01 ⁇ g/ ⁇ l of RNase A were added to each of the three experiments and incubated at 23 ° C for 10 min. The reaction was stopped by the addition of a 20 unit RNase inhibitor and a loading buffer was added. The reaction products were separated on 8M urea denatured 20% PAGE gel and imaged using the Typhoon Trio+ system.
  • the amino acids constituting the protein are almost L-form except for the chiral glycine, and the ribose in the nucleic acid is D-type.
  • Proteins and nucleic acids are characterized by chirality.
  • the erroneous addition of one or several mirrored amino acids to natural proteins may alter the secondary structure of the protein or even lose its biological activity (Krause et al., 2000).
  • the organism has a strict chirality.
  • researchers have not been able to find clear evidence of why the mirrored chirality is lost in evolution, we can still study the properties of mirrored proteins and nucleic acids by chemical synthesis, and try to construct the biological primitives needed to mirror the living organisms of the cells.
  • DNA replication and transcribed RNA are the key research directions.
  • DNA replication requires long strands of DNA as a template, short DNA strands as primers, DNA polymerase, dATP, dCTP, dTTP, dGTP, and suitable solution conditions such as suitable pH and Mg 2+ ion.
  • suitable solution conditions such as suitable pH and Mg 2+ ion.
  • Mirror proteins and nucleic acid molecules do not exist in natural living organisms.
  • the chemical synthesis of proteins is mainly through the solid phase synthesis of peptides of about 60 amino acids, and the natural chemical linkages in the solution to link the deprotected peptides one by one.
  • the upper limit of the protein that can be synthesized is about 350 amino acids depending on the sequence.
  • the size of E. coli polymerase I is 928 amino acids.
  • the commonly used taq polymerase is 832 amino acids, which is 487 amino acids more than the T4 DNA ligase, which is generally considered to be a simpler polymerase function. These enzymes are beyond the scope of our synthesis.
  • literature search we selected a 174 amino acid polymerase ASFV pol X, African swine fever virus polymerase X.
  • ASFV encodes two DNA polymerases, one is a DNA polymerase of the eukaryotic type family B for replication of the viral genome; and the other is a DNA polymerase of family X, named ASFV pol X (Oliveros et al) ., 1997).
  • ASFV Pol X is the smallest DNA polymerase found today (Showalter and Tsai, 2001), consisting of 174 amino acids and a size of 20 kDa.
  • ASFV Pol X is a template-dependent polymerase with very low fidelity, lacks 3'-5' exonuclease activity, and has very poor recognition ability for dideoxynucleotides (Oliveros et al., 1997).
  • ASFV Pol X Aligning the gene sequence of ASFV Pol X with other representative family X polymerases, See ASFV Pol X and human, cattle, rat TdT, human, mouse pol ⁇ and so on have a certain relationship between structure and function.
  • the three-dimensional structure of ASFV Pol X has been solved by NMR. Unlike other polymerases, Pol X has only one palm structure and one C-terminal domain (Macieiewski et al., 2001). In eukaryotic Pol ⁇ , there is generally an N-terminal domain responsible for DNA binding. Pol X does not have this critical domain, and its ability to bind DNA is stronger than Pol ⁇ .
  • ASFV Pol X can bind to a single nucleotide cleavage repair (BER) intermediate and efficiently repair a single nucleotide gap (Showalter et al., 2001).
  • BER nucleotide cleavage repair
  • Pol X repairs the gap and is easy to introduce mutations.
  • a new synthetic strand with a mismatch is ligated into the genome by a fault-tolerant ligase encoded by ASFV.
  • Pol X introduces new mutations when repairing the genome, which helps the virus to be produced in order to survive in an environment with stress.
  • the chemical total synthesis method of D-ASFV PolX uses the solid phase peptide synthesis technology (SPPS) proposed by Merrifield in 1963, which is currently the most effective method for peptide synthesis.
  • SPPS solid phase peptide synthesis technology
  • Boc tert-butoxycarbonyl
  • Fmoc fluorenylmethoxycarbonyl
  • the lyophilized polymerase dry powder was added to 10 ml dialysate containing 6 M guanidine hydrochloride.
  • the dialysate contained 50 mM Tris-HCl, pH 7.4, 40 mM. KCl, 6 mM (AcO) 2 Mg, 0.01 M EDTA and 16% glycerol.
  • the dialysis process continuously reduced the concentration of guanidine hydrochloride in the dialysate from 4M, 2M to 0, and the dialysis was stirred at 4 ° C for 10 hours each time.
  • a disulfide bond is formed between D-Cys81 and D-Cys86 by air oxidation.
  • E. coli expression synthesized L-form and synthetic D-form ASFV pol X by SDS-PAGE, and their band positions were consistent (Fig. 3).
  • Fig. 3 We also observed a small number of peptides that were not completely joined.
  • L and D ASFV pol X by circular dichroism spectroscopy (CD).
  • D-ASFV pol X cannot be degraded by common proteases (such as trypsin and pepsin, the experimental results are not listed) and cannot be sequenced, we only analyzed the sequence of L-ASFV pol X by mass spectrometry and covered it in the test results. The 100% pol X sequence was reached (Table 3.1).
  • image nucleic acid replication system After the successful synthesis of D-ASFV pol X polymerase, the image nucleic acid replication system is still lacking in the image nucleic acid.
  • image DNA fragments such as L-primer12 and L-template18 (see 2.1.3 for the sequence) and four L-dNTPs from Chemgenes.
  • the extended 6 bases are ACTGAG, indicating that all four dNTPs can be used as substrates to synthesize DNA strands in the mirror image system.
  • This conclusion still requires subsequent complementary pairing verification.
  • the molecular weight of the full-length extension product of the image was 5516.9 Da (theoretical value 5517.6 Da) by ESI-MS.
  • the full-length product of 18 nt was considered to be of correct quality within the error range.
  • ASFV pol X is not a thermostable enzyme, we can still perform multiple cycles of amplification by enzymatic addition per cycle.
  • the binding of ASFV pol X to DNA blocks the next cycle of amplification, so we removed the protein by phenol chloroform extraction at the end of each cycle and added the new ASFV pol X.
  • the extraction of each cycle resulted in a large DNA loss (recovery efficiency of approximately 40%), and we only performed amplification for 3 cycles.
  • the mirror DNA replication system also follows the principle of complementary pairing and has a certain degree of fidelity.
  • L-dTTP inhibits human DNA polymerase ⁇ , ⁇ , bovine thymus terminal transferase, but does not inhibit human DNA polymerase ⁇ , but DNA polymerases ⁇ , ⁇ , ⁇ cannot use L-dTTP as a substrate.
  • the addition of L-nucleoside at the 3' end renders DNA difficult to degrade by 3'-5' exonuclease (Focher et al., 1995). Therefore, some polymerases are inhibited by mirrored dNTPs when adding two substrates, and some may not.
  • Our study hopes to demonstrate whether there is chiral specificity for mirroring ASFV pol X and whether its polymerase activity is inhibited by natural chiral dNTPs. In our research, we first tried to add a combination of different chiral polymerases, primer-templates, and dNTPs separately, and then tried to react both the natural and mirrored systems in the same solution.
  • L-ASFV pol X, D-primer-template, L-dNTPs natural polymerase cannot add mirrored dNTPs to the 3' end of the natural primer; for L-ASFV pol X, L-primer-template, D- dNTPs, natural polymerases cannot add native dNTPs to the 3' end of mirrored primers; D-ASFV pol X, L-primer-templates, D-dNTPs, mirrored polymerases cannot add native dNTPs to mirrored primers The 3' end; for D-ASFV pol X, D-primer-template, L-dNTPs, the mirrored polymerase cannot add mirrored dNTPs to the 3' end of the native primer. Therefore, in the mirror and natural systems, the primers of the wrong chiral-template and dNTP can not be used as substrates to participate in the extension reaction, and the natural and mirrored DNA replication systems have good
  • mirror DNA replication systems can rely on templates to synthesize DNA with a certain base-complementary pairing specificity. Furthermore, we hope to verify that the mirror DNA sequence synthesized by the image polymerase is biologically active.
  • in vitro selection was created and applied.
  • This technology can be designed and screened by a complex library of random DNA or RNA sequences to obtain functional DNA or RNA molecules, mainly aptamers and catalytically active ribozymes (ribozyme) that can bind to specific targets. ) and deoxyribozyme (DNAzyme).
  • ribozyme catalytically active ribozymes
  • DNAzyme deoxyribozyme
  • the random library interacts with the target molecule under suitable conditions to capture DNA or RNA molecules capable of binding to the target, and then these molecules are subjected to PCR amplification.
  • the affinity of these amplified molecules relative to the original random library for the target is enhanced, using it as a secondary library, followed by a second round of screening of the target molecule.
  • a high affinity nucleic acid aptamer sequence can be screened by repeated amplification.
  • DNAzymes synthesized by both natural and image enzymes can achieve high-efficiency self-shearing in the presence of 2 mM Zn 2+ ions, and cannot be self- cleared in control experiments lacking Zn 2+ or adding Mg 2+ (Fig. 11c) ). Therefore, the DNAzyme sequence synthesized by mirroring ASFV pol X has self-shearing activity.
  • the mirrored DNA polymerase can perform primer extension according to the DNA template, and it is hoped that the mirror DNA can be transcribed into a mirror RNA.
  • the known X-family DNA polymerase has poor selectivity for dNTPs and rNTPs.
  • pol ⁇ can add dNTPs or rNTPs to the DNA strand according to the template. Quantitatively, its selectivity for rNTPs is 1000 times lower than that of pol ⁇ (Sa and Ramsden). , 2003).
  • ASFV pol X has not been verified to be able to utilize rNTP.
  • RNase A specifically catalyzes the cleavage of the next nucleotide 5' phosphodiester bond of the ribose of RNA at the C and U residues, which recognizes the 3',5' phosphodiester bond and does not cleave the 2', 5' bond Forming a 3'C or 3'U oligonucleotide having a 2',3'-cyclic phosphate derivative.
  • L-rNTPs can also be added to the primer strands, albeit at a slower rate than native systems (Fig. 12a). We also believe that this may be due to the insufficient purity of the mirror template, primers and rNTPs (the peaks can be observed in HPLC, the template is found in the CD, and the amount of primers is less than the nominal value).
  • RNA transcription system follows the base-complementing pairing rules according to the template.
  • Four kinds of rNTPs were separately added to the reaction system, and the 5'-end FAM-labeled primer12 was used as a primer.
  • the next base of the template was A, T, C, and G
  • primer12 and four different templates with the 13th base at the 3' end of the A, T, C, and G sequences, respectively.
  • the extension reaction was carried out by adding A, AC, and ACT with primer12, and the PAGE glue was recovered to obtain primer13, primer14, and primer15.
  • the bases on the next template are T, G, A, and C, respectively.
  • rNTPs can be efficiently added to the 3' end of the primer sequence under the correct pairing conditions of A:U, U:A, C:G, G:C4.
  • Some obvious mismatches occur in the mirroring system, such as T:rG, A:rC, etc., but the efficiency of false nucleoside addition is significantly lower than the correct pairing ( Figure 13). Therefore, the image RNA transcription system follows the principle of complementary pairing and has a certain degree of fidelity.
  • a mirrored chiral genetic information replication and transcription system containing core components D-ASFV pol X, L-primer, L-template, L-dNTPs/rNTPs, which can be used to amplify and transcribe RNA according to the template. It is shown that the mirrored chiral polymerase interacts with DNA. Using ASFV pol X polymerase, we achieved theoretical multi-cycle amplification of DNA, achieving 2.3-fold DNA amplification in the second to third cycles. In addition, we verified the use of native and mirror systems to add dNTP or rNTP extension primer strands, following the principle of base-pair pairing, and good fidelity.
  • the polymerase, primer template and dNTP have chiral specificity in the reaction system. Only when the three are all natural or all mirror molecules can be extended, the other combinations can not achieve amplification. Two chiral DNA replication systems of the same concentration were added to the same solution system, and the two systems were allowed to carry out extension reactions separately without serious mutual hindrance. We extended the 44 nt Zn 2+ -dependent DNAzyme sequence with the mirror ASFV pol X. The purified single-stranded L-DNAzyme has self-shearing biological activity in a buffer environment containing 2 mM Zn 2+ .
  • mirror DNA replication system add 0.7 ⁇ g D-ASFV pol X polymerase, 2.5 ⁇ M L-template, 2.5 ⁇ M L-primer and 0.2 mM L-dNTPs (each concentration) under the above buffer conditions. Confirmed in multiple template primer combinations (primer12-template18,primer15-template21, The primer12-DNAzymeTemplate) copy extension of the mirror DNA can be performed. Moreover, the newly synthesized DNA is complementary to the template strand during DNA replication, and the mirror replication process follows the base complementary pairing rule and has good fidelity.
  • a 44 nt Zn 2+ -dependent DNAzyme sequence was amplified using a mirror DNA replication system and a full-length DNA strand was obtained after 36 hours of extension.
  • the single-stranded DNAzyme was isolated by PAGE gel recovery.
  • the L-DNAzyme had self-shearing biological activity, while the control group could not be sheared under 20 mM Mg 2+ .
  • the mirror DNA replication system can amplify long-chain, active L-DNA sequences.
  • the mirror image transcription system contains D-ASFV pol X, L-template, L-primer and L-rNTP, and L-rNTP can be added under 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT, 50 mM KCl.
  • An RNA sequence was synthesized at the 3' end of the template.
  • D-ASFV pol X template-dependent transcriptional RNA reaction rate is lower than that of replicating DNA, and transcription also follows the principle of base-complementary pairing, and some mismatches are produced.
  • the invention has two main fields of application: one is to use a mirror DNA replication system for in vitro screening to obtain nucleic acid drugs, and the other is to attempt to construct a complete mirror original cell.
  • nucleic acid drugs In vitro screening of nucleic acid drugs is a promising future. Both natural and mirrored nucleic acid sequences can be folded into complex and diverse structures due to their diverse sequences, and some can be bound to a specific target molecule DNA or RNA called an aptamer.
  • the method for obtaining an aptamer is an in vitro screening technique, and in vitro screening uses DNA or RNA containing usually 30-80 random sequences, generally achieving a complexity of 10 14 or more, and a fixed primer complementary region at both ends is convenient for PCR amplification. increase.
  • the target molecule is usually fixed by different methods, and the random sequence library is combined with the target molecule, and then the nucleic acid sequence not bound to the target molecule is separated by the washing liquid, and then the nucleic acid with stronger affinity is obtained.
  • Molecules sequences enriched for high affinity by PCR amplification. After multiple cycles of screening, one or several nucleic acid sequences that bind more strongly to the target molecule can be obtained.
  • the difficulty of mirroring in vitro screening is that there is currently no way to perform mirrored PCR amplification in each round of screening.
  • Our mirrored PCR technology will do just that. Synthesize the image random sequence library by DNA synthesizer, directly use it for the combination of natural drug targets, and then directly obtain the image nucleic acid aptamer sequence through multiple cycles by rinsing, eluting, and amplifying the high affinity sequence. Optimization and clinical trials as potential drug molecules.
  • mirrored PCR was performed using ASFV pol X per cycle plus enzyme.
  • thermostable polymerase The reaction rate of ASFV pol X is very low (mainly used for repair of genomic gaps in viruses), and it is not a thermostable polymerase, and it loses activity at 50 ° C for 1 minute under 3.5 M proline-protected conditions.
  • thermostable polymerases such as 352 amino acid Dpo4 (Sulfolobus solfataricus P2DNA polymerase IV), which is reported in the literature.
  • Dpo4 can be used for PCR amplification, and mirrored Dpo4 protein can be used in the future. In vitro screening of PCR and mirroring.
  • ribosomes and tRNAs are the primary biological primitives for translation, and current state of the art still tends to be constructed using chemical synthesis methods.
  • the ribosomes of bacteria contain rRNA, usually with 50-80 ribosomal proteins (Wilson et al., 2009), most of which are shorter than 240 amino acids.
  • Current protein synthesis technology can be achieved, and there are 557
  • the amino acid rpsA protein needs to be improved by future improved protein synthesis techniques.
  • Each component is synthesized and then renatured and assembled in vitro to become a functional mirror ribosome.
  • RNA molecules that function as mirror cells such as DNA ligase, helicase , pyruvate dehydrogenase and the like. If the protein and nucleic acid components required for mirror cells are synthesized as much as possible, further technological advances will make it possible to construct simple mirror cells, or mirror cells to produce mirrored drugs or mirrored biomaterials.
  • d-Serine is an endogenous Ligand for the glycine site of the N-methyl-d-aspartate receptor. Proceedings of the National Academy of Sciences of the United States of America 97,4926.
  • Polymerase mu is a DNA-directed DNA/RNA polymerase. Molecular and Cellular Biology 23, 2309-2315.
  • D-serine an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proceedings of the National Academy of Sciences of the United States of America 92,3948-3952.
  • Ribosomal Proteins Role in Ribosomal Functions, Vol 2009.
  • Serine racemase A glial enzyme synthesizing d-serine to regulate glutamate-N-methyl-d-aspartate neurotransmission.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了对镜像核酸进行复制的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得所述镜像核酸。

Description

镜像核酸复制体系 技术领域
本发明涉及基因工程领域，更具体地说，涉及镜像核酸的复制和转录。
背景技术
手性是三维空间中一些分子的基本属性，也就是一个物体不能和它在镜中的影像重合。像人的左手和右手一样，二者是物体及镜像的关系，而无论在三维空间中如何翻转都不能重叠。这就是物体在空间中的手性，一个分子的镜像被称为它的对映体。它们有着相同的物理和化学性质，熔点、分子量、溶解度、密度、NMR谱等均相同。对映体之间相互不同的性质是它们的光学性质——旋转平面偏振光方向。手性在自然界中广泛存在。生命体中的生物大分子，如蛋白质、多糖、DNA和RNA等具有手性。地球上所有生物体，无论是大型的植物、哺乳动物或是肉眼不可见的微生物，组成蛋白质的20种氨基酸，除了甘氨酸没有手性以外，其他19种氨基酸全都是L型；而承载遗传信息的DNA和RNA，它们的核糖全都是D型。
氨基酸在紧邻羧基的位置有一个手性碳原子，它作为手性中心使得甘氨酸以外的氨基酸均有L和D两种手性。L指的是左旋(levorotatory)或者left-handed)，D指的是右旋(dextrorotatory或者right-handed)。对于核酸来说，D型是自然界中存在的手性，核酸分子的手性中心位于它的骨架上。
生命体中的两种不同手性的化合物，生物活性可能会完全不同。生物个体中的酶和细胞表面的受体大多数都是手性的，两个对映体在生物体中往往以不同的途径被吸收、活化和降解。对于常见的手性药物而言，两种对映体可能有着等同的药理活性，也可能一个有活性另一个没有活性，甚至其对映体有毒。目前认为，生命体中存在的蛋白质、核酸这些分子，有着手性单一性的特点。如果在天然的蛋白质序列中掺入镜像的氨基酸，会破坏它本身的二级结构(Krause et al.，2000)，并对其蛋白功能产生严重影响。
在地球早期环境中，出现最原始的细胞之前应该会有氨基酸、核酸这些生物分子的存在，尽管RNA作为生命物质起源的理论比较充分，但是目前没有证据表明氨基酸和核酸是哪一种先在地球上出现。人们对1969年坠落在澳大利亚的Murchison的陨石研究发现，其中含有多种氨基酸，包括甘氨酸、丙氨酸和谷氨酸等，而且两种手性的氨基酸并不是1∶1的，L-氨基酸多于D氨基酸(Engel and Macko，2001)。Breslow和Levine的实验表明，即使是很小的手性差异，也可以在两次溶 液蒸发与结晶后，在溶液中保留90％以上的一种手性的氨基酸(Breslow and Levine，2006)，而这种过程在地球早期环境中是可以发生的。另外有其他假说认为，进化中的单一手性来源于方解石产生具有旋光性的晶体，选择性地吸附一种旋光性的氨基酸，使得另一种在溶液中的比例增加。除了以上两种解释，目前的RNA生命起源假说认为，在地球早期环境中由有机分子形成了RNA，RNA进化成有自我复制、自我剪切等活性的有生物功能的RNA——RNA核酶；在进一步进化中，氨基酸组成的蛋白酶或多肽开始参与催化RNA的复制、解旋等，可能是在这一阶段会经历手性选择的过程，这就导致了最后进化成的复杂生命体中手性的单一性。
发明内容
本研究中，我们用化学合成的方法构建了基于D-ASFV pol X镜像聚合酶的遗传信息转录和复制体系，实现了镜像中心法则中L-DNA的复制以及转录为L-RNA这两个步骤。我们证实了镜像DNA的复制和转录遵循碱基互补配对原则，并且有着良好的手性特异性。我们发现把天然和镜像的DNA复制体系放在同一个溶液体系中，两者可以分别工作而没有严重的相互干扰。镜像聚合酶体系扩增L-DNAzyme脱氧核酶序列，可以实现和天然脱氧核酶相对应的自我剪切活性。镜像遗传信息复制和转录的实现，说明了镜像生命分子的存在并具有生物活性的潜在可能，也为未来在实验室环境中构建镜像细胞奠定了基础。经过对系统的进一步优化，镜像复制系统还将用于生物方法高效筛选镜像核酸药物。
本发明提供了对镜像核酸进行复制的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得所述镜像核酸。
本发明提供了进行镜像PCR的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得镜像核酸。
本发明提供了筛选镜像核酸分子的方法，包括：在允许二者结合的条件下，使随机镜像核酸序列库与靶标分子接触；获得与靶标分子结合的镜像核酸分子；以及通过镜像PCR扩增所述与靶标分子结合的镜像核酸分子。
本发明还提供了D-型ASFV pol X，其序列如SEQ ID NO：17所示，其中除没有手性的甘氨酸以外，其余氨基酸均为D-型氨基酸。
附图说明
图1显示D-ASFV Pol X的化学合成路线。174个氨基酸的ASFV pol X分成肽段1：Met1-Lys85，肽段2：Cys86-Leu105和肽段3：Ala106-Leu174三段来合成 和连接。先将Cys86用Acm保护起来，合成Cys86-Leu105，再合成肽段3：Cys106-Glu107-Leu174，经过活化、连接得到肽段2和肽段3的连接产物，然后将Cys106脱硫形成Ala106。再合成肽段1，催化脱去肽段2的Acm保护基，进行活化和连接，获得全长ASFV pol X聚合酶。
图2显示D-ASFV pol X合成全长产物的检测。a.折叠后的D-ASFV pol X HPLC谱图。HPLC分析使用了214nm吸收波长，Vydac C18(4.6×250mm)液相色谱柱。b.ESI-MS谱图通过对于离子峰图的分析和计算，观察到主要合成产物的大小为20317.0Da，ASFV pol X理论值为20316.0Da。
图3显示L型和D型ASFV pol X检测。a.SDS-PAGE检测大肠杆菌表达的、化学合成L型和化学合成D型的ASFV pol X聚合酶在15％的SDS-PAGE胶上分离，并银染检测。M，蛋白分子量marker。b.CD检测L型和D型化学合成ASFV pol X在Applied Photophysics Pistar-180CD spectrometer上进行检测，吸收曲线是3次独立检测扣除背景后的平均值。
图4显示镜像DNA检测。a.HPLC检测(Chemgenes公司提供)分别对4种L-dNTP进行了HPLC的纯化和分析，L-dATP和L-dGTP的结果几乎没有杂峰，而L-dTTP和L-dCTP有明显的杂峰。b.primer12CD检测从公司获得的L型经过HPLC纯化和D型经过PAGE纯化的primer12，吸收曲线有对称关系(L-primer12的实际上样浓度小于D-primer)c.template18CD检测从公司获得的L型D型的template18，在CD检测中吸收曲线有对称关系(L-template18的样品实际上样浓度小于D-primer)。
图5显示镜像聚合酶催化DNA延伸。a.天然和镜像DNA复制体系示意图天然DNA复制系统包含L-聚合酶，D-DNA和D-dNTPs；镜像DNA复制系统包含D-聚合酶，L-DNA和L-dNTPs b.天然和镜像聚合酶催化DNA延伸(12nt引物，18nt模板)缓冲液条件为50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，在10ul天然或镜像反应体系中加入对应手性的0.7μg ASFV pol X，2.5μM primer12，2.5μM template18和4种0.4mM dNTPs。将反应体系置于37℃4小时。“*”表示5’FAM标记。c.L-DNA全长产物ESI-MS检测通过分析计算得到预期的全长产物分子量为5516.9(理论值5517.6)以及模板的分子量5481.9(理论值5481.6)d.天然和镜像聚合酶催化DNA延伸(15nt引物，21nt模板)反应体系在50mM Tris-HCl pH 7.5，20mM MgCl2，1mM DTT，50mM KCl中进行，加入了2.5μM 15-nt L-primer15(没有FAM修饰)，2.5μM 21-nt L-template21，0.2mM L-dNTPs(每种浓度)，以及1.4μg D-ASFV pol X.在37℃反应12小时后进行20％PAGE胶的分离，并且用Sybr Gold染色检测。
图6显示镜像DNA多循环扩增示意图。第一个循环不带FAM的reverse 11引物扩增得到双链的模板，第二个循环FAM标记的primer11只能与第一循环扩增出的模板(橘红色)进行互补，得到全长产物。第三个循环可以产生3倍于第二个循环的荧光全长产物。
图7显示镜像DNA复制体系多循环扩增。D-ASFV pol X多循环扩增L-DNA，Cycle0为对照组，在cycle1进行之前取样。Cycle1扩增得到reverse11的全长产物，可以被后续循环作为模板。样品在8M尿素变性的20％PAGE胶上分离，用Typhoon Trio+扫描荧光。检测到的所有产物均为带FAM标记的DNA。
图8显示镜像DNA延伸的碱基互补配对特异性。在天然或者镜像的系统中分别添加对应手性的0.2mM的dATP、dTTP、dCTP、dGTP中的一种，模板的下一个位点碱基分别为A、T、C、G(黑底白字方框标记)，缓冲液条件为50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，加入对应手性的0.7μg ASFV pol X，2.5μM primer，2.5μM template18。在37℃反应30分钟，在20％的PAGE胶上电泳并用Typhoon Trio+扫描仪扫描荧光信号。“*”表示5’FAM标记。“-”表示未添加D或L-dNTPs的对照组。
图9显示镜像DNA延伸的手性特异性。在缓冲液条件为50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，加入0.7μg ASFV pol X，2.5μM primer，2.5μM template18，蛋白、引物-模板、dNTPs的手性在图的下方列出，一共有8种组合。在37℃反应12小时，在20％的PAGE胶上电泳并用Typhoon Trio+扫描仪扫描荧光信号。“*”表示5’FAM标记。
图10显示天然和镜像系统在同一溶液中反应。在缓冲液条件为50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，加入两种手性的0.7μg ASFV pol X，2.5μM天然5’末端Cy5标记的primer20，2.5μM天然template26，2.5μM镜像5’末端FAM标记的primer12，2.5μM镜像template18，两种手性的dNTPs各四种，每种终浓度0.2mM，反应体系在37℃反应4小时，反应产物在8M尿素变性的20％PAGE胶上分离，用Typhoon Trio+的Cy5和FAM荧光模式扫描，并将图片整合到一起。
图11显示天然和镜像DNAzyme的酶合成与活性检测。a.Zn2+依赖的DNAzyme二级结构在44ntDNAzyme的5’末端添加了12nt的引物序列，全长为56nt。二级结构通过mfold服务器生成(Zuker，2003)。b.DNAzyme的酶合成天然和镜像的DNAzyme都在50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl缓冲液条件下反应，加入了66nt的DNAzyme template和12nt的引物，在37℃经过36小时获得全长产物。M，marker为化学合成的56nt序列作为分子量 标准。c.将延伸得到的全长DNAzyme从PAGE胶上切下，放在缓冲液里扩散过夜，用天恩泽PAGE胶回收试剂盒进行沉淀回收。将沉淀的DNA用buffer1∶50mM HEPES，pH 7.0和100mM NaCl溶解，并在90℃加热2min，再冰上降温5min。然后加入等体积的buffer 2∶50mM HEPES，pH 7.0，100mM NaCl，4mM ZnCl2或40mM MgCl2以开始反应(Zn2+与Mg2+终浓度为2mM与20mM)。在37℃反应36小时。最后用EDTA终止反应。样品在12％PAGE胶上分离并显影。
图12显示天然和镜像体系DNA模板依赖的RNA转录。a.天然和镜像ASFV pol X催化的RNA转录缓冲液条件为50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，在10ul反应体系中加入0.7μg ASFV pol X，2.5μM primer，2.5μM template和4种0.4mM rNTPs，天然体系中加入2unit RNase抑制剂。在37℃反应60小时后终止反应。b.加入rNTP的天然体系经过37℃36h的反应得到全长产物。加热到75℃10min使ASFV pol X和RNase抑制剂失活。在三个实验中分别加入1μg/μl，0.1μg/μl，0.01μg/μl的RNase A，然后在23℃孵育10min。加入20unit RNase抑制剂终止降解反应，并且加入上样缓冲液。反应产物在8M尿素变性的20％PAGE胶上分离，并且用Typhoon Trio+系统成像。Sample 1：对照组，延伸0h Sample 2：D-primer12经过36小时延伸的全长产物Sample 3：全长产物在75℃加热10min，失活RNase抑制剂以及ASFV pol X Sample 4-6：全长延伸产物，经75℃加热10min，加入0.01μg/μl，0.1μg/μl，1μg/μl的RNase A，23℃放置10min，加入20unit RNase抑制剂终止反应。在20％的PAGE胶上电泳并用Typhoon Trio+扫描仪扫描荧光信号。“*”表示5’FAM标记。
图13显示天然和镜像体系RNA转录的碱基互补配对特异性。天然或者镜像的系统中分别添加对应手性的0.2mM的rATP、rUTP、rCTP、rGTP中的一种，模板的下一个位点碱基分别为A、T、C、G(黑底白字方框标记)，缓冲液条件为50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，加入对应手性的0.7μg ASFV pol X，2.5μM primer，2.5μM template18。在37℃反应12小时，在20％的PAGE胶上电泳并用Typhoon Trio+扫描仪扫描荧光信号。“*”表示5’FAM标记。“-”表示未添加D或L-dNTPs的对照组。
具体实施方式
本发明提供了一种对镜像核酸进行复制的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得所述镜像核酸。
本文所用的术语“镜像”是指在手性上与天然物质呈镜像关系的异构体。
本文所用的术语“镜像核酸”是指L-型核酸，它与天然核酸(即D-型核酸)呈镜像关系。镜像核酸包括L-型DNA和L-型RNA。术语“镜像DNA”与“L-型DNA”或“L-DNA”可以互换使用。
本文所用的术语“镜像核酸聚合酶”或“镜像聚合酶”是指D-型聚合酶，它与天然聚合酶(即L-型聚合酶)呈镜像关系。术语“镜像聚合酶”与“D-型聚合酶”或“D-聚合酶”可以互换使用。例如，“D-Dpo4”是指D-型Dpo4聚合酶，它与天然的L-型Dpo4聚合酶呈镜像关系。
特别适合于本发明的聚合酶包括D-ASFV pol X、D-Dpo4、D-Taq聚合酶以及D-Pfu聚合酶。
Dpo4(Sulfolobus solfataricus P2DNA polymerase IV，硫化叶菌P2DNA聚合酶IV)是一种热稳定的聚合酶，也能在37℃合成DNA。它的错配率在8×10-3-3×10-4之间。它是一种可以替代Taq做多循环PCR反应的聚合酶。它的氨基酸序列长度在目前的化学合成技术可实现范围内。
Taq聚合酶是1976年Chien及其同事在热泉微生物Thermus aquaticus中发现的热稳定聚合酶，它可以在DNA变性温度下保持活性，所以它替代E.coli聚合酶被用于PCR反应。Taq的最适温度在75℃-80℃，并且在92.5℃的半衰期约为2h。
Pfu聚合酶是在Pyrococcus furiosus中发现的，它在微生物中的功能是在细胞分裂期复制DNA。它优于Taq的地方在于它具有3‘-5’外切酶活性，可以在DNA合成过程中剪切掉延伸的链上错误添加的核苷酸。商业化的Pfu错配率在130万分之一左右。
在一些实施方案中，所述镜像核酸、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs为L-型，所述镜像核酸聚合酶为D型。在某些情况下，反应体系中可以混有不同类型的模板、引物或dNTPs/rNTPs(例如可以混有一部分D型模板引物或dNTPs/rNTPs)，但不会对反应产生严重干扰。
此处的核酸复制反应可以只进行一个循环，也可以进行多个循环。这可以由技术人员根据实际需要来确定。
此处所用的术语“多个”是指至少2个。例如，“多个循环”是指2个或更多个循环，例如3个、4个或10个循环。
此处所用的术语“复制”包括在DNA模板和dNTPs存在下获得目标DNA的一个或多个拷贝；也包括在DNA模板和rNTPs存在下获得目标RNA的一个或多个拷贝(此过程也可以称为RNA的“转录”)。
在核酸的复制过程中，模板和引物通常为DNA，如果目标核酸为DNA，则反应体系中应当添加dNTPs；如果目标核酸为RNA，则反应体系中应当添加rNTPs。
在一些实施方案中，所述镜像核酸为L型DNA，例如L-DNAzyme。在另一些实施方案中，所述镜像核酸为L型RNA。
在特别优选的实施方案中，所述反应为聚合酶链式反应。
本文所用的术语“PCR”具有本领域公知的含义，是指聚合酶链式反应((Polymerase Chain Reaction)。
在特别优选的实施方案中，所述反应在如下缓冲液中进行：50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。
本发明还提供了进行镜像PCR的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得镜像核酸。
本发明还提供了筛选镜像核酸分子的方法，包括：在允许二者结合的条件下，使随机镜像核酸序列库与靶标分子接触；获得与靶标分子结合的镜像核酸分子；以及通过镜像PCR扩增所述与靶标分子结合的镜像核酸分子。
优选地，所述靶标分子被固定在固相介质上，这样可能更有利于分离纯化。
例如，在随机镜像核酸序列库与靶标分子接触之后，可以通过洗涤去除不与靶标分子结合的镜像核酸序列，从而获得与靶标分子结合的镜像核酸分子。
所述镜像核酸分子可以为L型DNA或L型RNA。
优选地，所述镜像PCR中使用的镜像核酸聚合酶可以是D-ASFV pol X、D-Dpo4、D-Taq聚合酶或D-Pfu聚合酶。
在特别优选的实施方案中，镜像PCR在如下缓冲液中进行：50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。
本发明还提供了D-型ASFV pol X，其序列如SEQ ID NO：17所示，其中除没有手性的甘氨酸以外，其余氨基酸均为D-型氨基酸。
下面将参照附图和实施例对本发明进行进一步描述，附图和实施例只是出于解释本发明的目的，不应当被理解为是对本发明的限制。
实施例
哺乳动物中的D-氨基酸
D-氨基酸存在于哺乳动物的体内。1965年Hoeprich及同事在豚鼠和小鼠血液中检测到D-Ala，这是研究者首次在哺乳动物体内发现D-氨基酸(Corrigan，1969)。到现在为止，D-Ala被发现存在于多种哺乳动物的大脑、垂体，并且在尿液中发现D-Ala的排泄。D-Pro和D-Leu在小鼠大脑的7个区域被发现，说明了D-氨基酸在 垂体和松果体有着相对较高的浓度(Hamase et al.，2001)。另外，D-Ser和D-Ala也在人、小鼠等哺乳动物的脑和血液中被检测到(Hashimoto et al.，1993；Hashimoto et al.，1995)。D-氨基酸最早被认为是微生物、植物、无脊椎动物合成的，最近的研究表明D-Ser和D-Asp可以被哺乳动物组织合成。通过同位素标记实验发现带放射性的L-Ser在大鼠和小鼠的大脑可以被转化为D-Ser，1999年丝氨酸异构酶(Ser racemase)被克隆和纯化出来，该异构酶在大鼠脑部可以催化L-Ser向D-Ser的催化(Wolosker et al.，1999)。哺乳动物中另一大D-氨基酸的来源是外源的食物和微生物。
镜像蛋白质的化学合成
尽管镜像氨基酸在自然状态的生命体中被证实有着较为广泛的存在，镜像氨基酸自然存在状态以单体为主，也有存在于短肽片段中，例如革兰氏阳性菌的四肽侧链氨基酸d-丙-d-谷-r-l-赖-d-丙，目前尚未发现自然界中有功能性的镜像氨基酸组成的镜像蛋白，中心法则中的翻译是核糖体和tRNA参与的、按照mRNA遗传信息合成蛋白质的过程，镜像氨基酸没有在这一过程中当做底物被使用。一般认为，生命体的遗传信息转录、翻译和主要生物功能的实现，依赖于天然手性的L-氨基酸。
由于现有生物体的手性单一性特点，生物方法无法获得镜像蛋白质，目前对于镜像蛋白的研究均通过化学合成的方法来实现。多肽和小的蛋白可以通过固相多肽合成(SPSS，solid phase peptide synthesis)的方法来实现(Kent，1988)。这一方法通常可以得到60个氨基酸左右的片段，进而通过自然化学连接(native chemical ligation)在溶液中把脱保护的肽段逐一连接起来(Dawson and Kent，2000)。固相多肽合成以及自然化学连接的方法扩展了蛋白合成的范围，目前可以实现300个氨基酸以上蛋白的合成。通过这一技术也可以进行镜像蛋白质的合成研究。1992年Kent课题组合成了首个镜像蛋白酶，HIV-1蛋白酶(HIV-1protease)(Milton et al.，1992)。基于理论L和D对映异构体有互为镜像的结构，科学家通常猜测镜像蛋白有着和天然蛋白相对应的功能，这一猜想在镜像HIV-1蛋白酶项目上首次得到证实。L型和D型HIV-1蛋白酶有着相同的质谱分子量、相同的HPLC保留时间、相反的圆二色光谱曲线。在活性方面L型HIV-1蛋白酶可以切割L型肽底物，而D型蛋白酶可以切D型的底物。另一项研究中，两种手性的芽孢杆菌RNA酶(barnase)也显示出了类似的特性，天然L型barnase可以切割天然的RNA，而对于镜像RNA它的活性要弱4000倍左右。而对于镜像D型barnase，切割镜像RNA的活性则显著高于天然RNA(Vinogradov et al.，2015)。1995年4-草酰巴豆酯互变异构酶的L型和D型被化学合成，它的两种对映体在催化无手性的底物2-羟 粘康酸的异构反应中显示了相同的反应效率。同位素标记催化位点碳原子上的氢，表明两种手性的异构酶作用在碳原子的不同侧面(Fitzgerald et al.，1995)。上述研究一致地表明了镜像蛋白酶和天然手性蛋白酶有着相同的活性，但是作用于不同的异构位置。
2014年M.S.Kay课题组合成了最长的蛋白，312氨基酸的DapA。DapA是依赖分子伴侣GroEL/ES的蛋白，在表达后依赖分子伴侣的协助才能折叠成功能构象。GroEL/ES可以折叠D和L两种手性的DapA，但是对于天然L-DapA的折叠效率要高于镜像DapA(Weinstock et al.，2014)。
镜像核酸化学合成研究
关于生命起源早期假说的研究表明非酶催化的、模板指导的RNA扩增反应可以在单一手性的系统里进行。但是如果体系里有两种手性的RNA单体，那么延长反应会由于其镜像单体的加入而被阻止(Joyce et al.，1984)。这对于生命起源于自然产生的RNA的理论产生了严重的挑战。为了解释这个理论问题，Joyce及其同事通过体外筛选的方法得到了RNA聚合核酶(RNA polymerase ribozyme)，它由83个核糖核苷组成，可以催化对映手性的RNA聚合反应(Sczepanski and Joyce，2014)。在这项研究中的镜像RNA是通过化学合成的方法得到的。它可以通过连接11个寡聚核苷酸片段来产生全长的与其自身手性相反的RNA聚合核酶，在同一反应体系中对于两种RNA的扩增可以互不干扰地进行。这为生命起源早期，两种手性的RNA分子共存，并且通过RNA聚合核酶进行扩增提供了一种理论上的可能。
2013年Barciszewski课题组首次报道了有催化活性的镜像核苷酸酶，这种核苷酸酶是根据已有的天然核苷酸酶序列设计的，具有催化剪切镜像的L-核苷酸分子的功能(Wyszko et al.，2013)。镜像核苷酸酶可以在体内实现剪切功能。在实验中证实了镜像核苷酸酶在血清中不易降解，还有无毒性、不引起免疫反应的特点，这使它成为较为理想的药物分子。
镜像核酸适体研究
DNA/RNA最早被认为是遗传信息的携带工具，也被认为比蛋白质的结构要简单很多。但是实际上DNA/RNA也能够折叠成三级结构，也就有着一系列潜在的生理功能。最早在1990年，研究者就发现了RNA结构可以特异性地结合小分子底物。这些RNA结构像抗体一样，可以选择性地结合底物并且有很高的亲和性。这些能结合特定底物RNA结构就被称作核酸适体(aptamers)。后来，DNA的核酸适体也被研究者发现。
体外筛选技术利用随机DNA/RNA序列库寻找与特定靶标分子结合的核酸适体。体外筛选首先要有一个随机序列库，既可以是DNA也可以是RNA的。库一般含有30-80个核苷酸的随机序列，以及在两边有两段引物区，以方便PCR扩增。然后进行多个循环的筛选过程，将目标小分子底物固定在一个基质上面，再把随机序列库加到底物上面，在清洗之后，不结合的DNA或RNA分子流过被固定的底物，而被筛选的有结合能力的序列就会留在上面。然后将这些特殊的序列洗脱下来，进行PCR扩增，再经过多个循环的富集、筛选过程，就可以得到一个或者几个能和底物特异性结合的核苷酸序列。
与天然的核酸适体一样，镜像核酸序列根据其序列具有特定的二级、三级结构，可以紧密、高特异性地结合目标分子。如果应用体外筛选策略对于镜像的靶标分子进行筛选，再合成镜像的核苷酸序列，就可以得到能结合天然靶标的镜像核苷酸分子。研究者用体外筛选的方法得到了结合D-腺苷和L-精氨酸的镜像L-RNA适体(Klusmann et al.，1996；Nolte et al.，1996)，D.P.Bartel在1997年得到了结合抗利尿激素的L-DNA适体(Williams et al.，1997)。镜像核苷酸分子的优势在于在体内稳定不易降解、无毒性、不引起免疫反应，而且生产成本较为低廉，有着良好的作为药物分子的应用前景。
研究目的
镜像生物分子是否能够实现遗传信息的复制、转录，镜像分子在进化上是否有组成生命的理论可能，目前没有研究可以给出明确的论断。尽管已经有研究组合成并验证了镜像蛋白的活性以及比较了它们跟天然蛋白的特性，但是这些研究通常仅从化学合成的角度阐释了镜像蛋白的特性。
我们的研究旨在设计并合成基于聚合酶的镜像复制与转录体系，这一体系的意义有以下三个方面：
一、镜像复制与转录体系可以实现聚合酶对于镜像DNA、RNA的扩增，说明镜像聚合酶可以像天然手性聚合酶一样催化DNA、RNA的合成，证明镜像生物分子具有有效的生物活性；
二、镜像复制与转录体系实现了镜像中心法则中两个关键的步骤，为镜像原始细胞的合成工作奠定开创性的基础；
三、现阶段体外筛选技术得到的天然核酸分子，作为药物有着体内易水解的严重缺陷。为了避免这一问题，需要用特殊方法进行镜像核酸的筛选。现有的体外筛选技术，通过天然随机库对镜像靶标进行筛选，得到有效的核酸序列，再化学合成镜像核酸分子，这样得到的镜像核酸分子可以结合天然靶标，也就是潜在 的镜像药物。但是这一方法的局限在于生物体内的常见药物靶点有很多是大于300个氨基酸的蛋白，通过化学方法无法合成镜像靶标。镜像体外筛选如果能直接用天然的靶标分子和镜像的随机库进行，将极大地提高这一技术的普遍性，在更广泛的疾病领域筛选到药物分子。天然药物靶标、镜像随机库目前已经不存在技术难点，镜像药物筛选的瓶颈在于镜像PCR无法实现。而我们的镜像复制与转录体系可以实现镜像的PCR，尽管PCR效率有待进一步优化和提高，仍然为镜像药物筛选工作提供了理论和实践的基础。
实验材料与实验方法
实验药品与试剂
Figure PCTCN2016079006-appb-000001
甲基脲六氟磷酸酯
Figure PCTCN2016079006-appb-000002
Figure PCTCN2016079006-appb-000003
实验设备
Figure PCTCN2016079006-appb-000004
核酸序列
Figure PCTCN2016079006-appb-000005
Figure PCTCN2016079006-appb-000006
上述没有D-/L-标记的序列都指的是D-DNA.
ASFV pol X的蛋白和DNA序列
蛋白序列
1 mltliqgkki vnhlrsrlaf eyngqlikil sknivavgsl rreekmlndv dlliivpekk
61 llkhvlpnir ikglsfsvkv cgerkcvlfi ewekktyqld lftalaeekp yaifhftgpv
121 syliriraal kkknyklnqy glfknqtlvp lkittekeli kelgftyrip kkrl(SEQ ID NO：17)
DNA序列
Figure PCTCN2016079006-appb-000007
实验方法
ASFV pol X的化学合成
我们将D-ASFV pol X的氨基酸序列分为三片段，采取从C端到N端顺序自 然链接法。每一个多肽片段的合成采用的是基于9-芴甲氧羰基(Fmoc)为保护基策略的固相多肽合成法(Fmoc-SPPS)。合成片段1采用的是2-Cl-trityl-Cl树脂(2CTC，取代度为0.5mmol/g)，而合成片段2以及片段6则使用肼取代的2CTC树脂。多肽片段的合成首先将树脂在二氯甲烷(DCM)以及N，N-二甲基甲酰胺(DMF)的混合液中溶胀半小时，然后除去溶剂。随后，对于片段1，将溶有4倍当量的氨基酸和8倍当量的N，N-二异丙基乙基胺的5ml DMF溶液加入到反应管中，在30℃的恒温摇床内反应12小时，之后加入200μL甲醇，封闭未反应的活性氯。对于片段2以及片段6，将溶有4倍当量的氨基酸、3.8倍当量的2-(7-偶氮苯并三氮唑)-N，N，N′，N′-四甲基脲六氟磷酸酯(HATU)、3.8倍当量的苯并三氮唑(HOAT)和8倍当量的N，N-二异丙基乙基胺的5ml DMF溶液加入到反应管中，在30℃的恒温摇床内反应1小时。多肽片段合成过程中，脱去Fmoc保护基的方法是使用含有20％哌啶的DMF溶液，浸泡两次，一次5分钟，另一次10分钟。从第二个氨基酸开始直到最后，缩合体系都使用的是HATU/HOAT/DIEA。多肽片段合成完毕后，使用切割试剂K(三氟乙酸/苯酚/水/茴香硫醚/乙二硫醇＝82.5/5/5/5/2.5)浸泡3小时，然后用高纯氮气除去三氟乙酸将体系浓缩，随后加入乙醚，使多肽沉淀出来，最后离心，收集固体沉淀，使用半制备级别的反相高效液相色谱(RP-HPLC)分离纯化目标多肽片段。
按照以下方法进行多肽片段的自然化学连接。首先将含有酰肼基团的多肽片段溶于缓冲液中(6M盐酸胍，200mM磷酸氢二钠，pH 3.0)。在冰盐浴中，往反应液中加入15倍当量的NaNO2溶液，反应20分钟。随后加入混有40倍当量4-巯基苯乙酸(MPAA)，等当量的N端为半胱氨酸的多肽片段，pH值为7.0的缓冲溶液。搅拌均匀后，将体系的pH值调至7.0，反应12小时。反应结束后，加入80mM磷酸三氯乙酯(TCEP)缓冲液，将体系浓度稀释一倍。最后使用半制备级别的RP-HPLC分离目标产物。
按照以下方法进行多肽的脱硫反应。首先将1μmol多肽片段3溶于2.5ml200mM TCEP缓冲溶液中(6M盐酸胍，0.2M磷酸氢二钠，pH＝6.9)，然后加入50μmol VA-044和100μmol还原型谷胱甘肽，溶液在37℃下反应12小时。最后脱硫产物4用半制备级别的RP-HPLC分离纯化。
按照以下方法脱除Cys86侧链的乙酰胺甲基(Acm)保护基。0.5μmol多肽片段4溶于1ml 50％醋酸水溶液中。然后加入5mg醋酸银，在30℃下搅拌过夜。随后加入2.5mmol巯基乙醇，并且用6M盐酸胍水溶液将体系稀释2倍。通过离心，除去沉淀，用RP-HPLC分离上清液，获得目标产物5。
ASFV pol X的折叠复性
按照以下方法对D-ASFV pol X进行折叠复性。将5mg D-ASFV pol X溶于10ml 6M盐酸胍溶液中，将溶液置于3K Da的透析袋。然后把透析袋放在含有4M盐酸胍的缓冲液体系内(50mM Tris-HCl，40mM KCl，6mM醋酸镁，0.01M EDTA和16％甘油)浸泡10小时，随后将盐酸胍浓度逐渐降低为2M，1M和0M。透析袋在每种浓度盐酸胍溶液中浸泡时间均为10小时。使用圆二色谱和质谱可以证明，D-ASFV pol X折叠正确并且D-Cys81和D-Cys86之间通过空气氧化形成二硫键。
天然和镜像DNA、RNA聚合酶反应方法
DNA聚合反应方法：配置聚合酶反应缓冲液，50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。在10ul反应体系中加入0.7μg ASFV pol X，2.5μM primer，2.5μM template和4种0.4mM dNTPs。将反应体系置于37℃4小时，加入0.5M EDTA 1μl终止反应。反应得到与模板互补的DNA片段。
RNA聚合反应方法：配置聚合酶反应缓冲液，50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。在10ul反应体系中加入0.7μg ASFV pol X，2.5μM primer，2.5μM template和4种0.4mM rNTPs。将反应体系置于37℃60小时，加入0.5M EDTA 1μl终止反应。反应得到primer DNA和RNA的复合体。
PAGE胶回收DNA/RNA片段方法
我们采用PAGE胶回收的方法分离纯化扩增产物，首先在终止了反应的体系中加入含有二甲苯青、溴酚蓝的上样缓冲液，在合适浓度的变性丙烯酰胺凝胶上进行电泳，在电泳结束后将胶取下。用溴化乙锭进行DNA/RNA染色，在UV光下把DNA/RNA的目的大小片段切下，弃去杂带和空白的区域，切割的胶块应该尽量小。然后将胶块放在TE缓冲液中，颠倒混合过夜。小心吸取上清，加入1/10体积的乙酸钠(3mol/L，PH＝5.2)于DNA溶液中充分混匀，使其最终浓度为0.3mol/L。加入2倍体积用冰预冷的乙醇混合后再次充分混匀置于-20℃中30分钟。12,000g离心10分钟，小心移出上清液，吸去管壁上所有的液滴。于室温下将开盖的EP管的置于实验桌上以使残留的液体挥发至干。加适量的ddH2O溶解DNA/RNA，可以获得高纯度的酶扩增L-DNA/RNA片段。
镜像DNAzyme反应方法
我们用100μl的反应体系扩增了一段DNAzyme的序列，这种DNAzyme是单 链具有自我剪切活性的DNA短链。反应体系为：50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，and 50mM KCl，28.9μg D-ASFV pol X，5μM primer12引物，5μM DNAzymeTemplate模板和1.6mM dNTPs。在37℃反应36小时。下一步将反应产物加上上样缓冲液，在12％PAGE胶上用300V 3个小时将条带分开。为了方便跑胶分离和切胶回收全长反应产物——单链的DNAzyme，DNAzymeTemplate模板在设计时比全长产物多10个核苷酸，拆下胶板后显影并切下全长产物序列。将胶块用上面描述的方法处理，扩散过夜并乙醇沉淀回收DNA。沉淀下来的产物用buffer 1∶50mM HEPES，pH 7.0和100mM NaCl溶解，并在90℃加热2min，再冰上降温5min。然后加入等体积的buffer 2∶50mM HEPES，pH 7.0，100mM NaCl，4mM ZnCl2or 40mM MgCl2以开始反应。在37℃反应36小时。最后用EDTA终止反应。样品在12％PAGE胶上分离并显影。
镜像多循环聚合酶反应方法
配置聚合酶反应缓冲液，50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。在反应体系中加入2.672μg D-ASFV pol X，2.5μM L-FAM-primer，2.5μM L-template和4种0.4mM L-dNTPs。将反应体系置于37℃4小时，加入0.5M EDTA 1μl终止反应。由于ASFV pol X有着很强的结合DNA的能力，即使在95℃加热变性的条件下也无法解离，我们用酚氯仿抽提的方法去除前一轮反应中的聚合酶。第二轮反应中，反应体系加热到95℃30s，然后降至室温。在反应体系中再加入0.334μg D-ASFV pol X，反应体系在37℃放置20h。第三个循环用同样的方法处理。反应产物在8M尿素变性的20％PAGE胶上分离，并且用Typhoon Trio+系统成像。电泳条带定量使用ImageQuant软件进行。
RNase A消化天然RNA聚合产物
L-ASFV pol X RNA反应体系(见2.2.3)，经过37℃36h的反应，加热到75℃10min使ASFV pol X和RNase抑制剂失活。在三个实验中分别加入1μg/μl，0.1μg/μl，0.01μg/μl的RNase A，在23℃孵育10min。加入20unit RNase抑制剂终止反应，并且加入上样缓冲液。反应产物在8M尿素变性的20％PAGE胶上分离，并且用Typhoon Trio+系统成像。
镜像手性的遗传信息复制和转录体系的设计
概述
在地球上的生命体中，组成蛋白质的氨基酸除了没有手性的甘氨酸，几乎都为L型，核酸中的核糖都为D型。蛋白质和核酸有着手性单一性的特点，在天然的蛋白质中错误地加入一个或几个镜像的氨基酸，可能会使得蛋白质二级结构发生改变，甚至失去生物活性(Krause et al.，2000)。生物体具有严格的手性单一性特点。虽然在进化中研究者尚无法找到关于镜像手性为何丢失的明确证据，我们仍然可以通过化学合成的方法来研究镜像的蛋白质和核酸的特性，尝试构建镜像细胞生命体所需要的生物原件。作为镜像生命体的核心，尝试构建镜像中心法则中的关键步骤，DNA的复制和转录RNA是重点研究的方向。
镜像DNA复制体系的设计
在天然生命体中，DNA的复制需要作为模板的DNA长链、作为引物的DNA短链、DNA聚合酶、dATP、dCTP、dTTP、dGTP四种分子，以及适宜的溶液条件，例如合适的pH和Mg2+离子。我们参照天然生命体系，设计镜像DNA复制体系的成分，50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl，再加入镜像的蛋白和核酸分子，D-聚合酶、L-DNA模板、L-DNA引物和四种L-dNTPs。镜像蛋白和核酸分子在天然的生命体中不存在，而对于化学合成的过程来说，投入特定的原料，合成一种手性的分子与对映手性的分子所需要的化学过程并没有区别。所以我们确定用化学的方法获得镜像的D-聚合酶和L-DNA。
蛋白质的化学合成主要通过60氨基酸左右的多肽固相合成，以及自然化学连接在溶液中把脱保护的肽段逐一连接起来，目前能够合成的蛋白上限根据序列的不同大约是350个氨基酸。大肠杆菌聚合酶I的大小是928个氨基酸，我们常用的taq聚合酶是832个氨基酸，比通常认为聚合酶功能更简单的T4DNA连接酶也有487个氨基酸。这些酶超出了我们的合成范围。通过文献检索我们选定了一个174氨基酸的聚合酶ASFV pol X，非洲猪瘟病毒聚合酶X。
ASFV编码两种DNA聚合酶，一种是真核类型的family B的DNA聚合酶，用于病毒基因组的复制；还有一种是family X的DNA聚合酶，被命名为ASFV pol X(Oliveros et al.，1997)。ASFV Pol X是现在发现的最小的DNA聚合酶(Showalter and Tsai，2001)，由174个氨基酸组成，大小为20kDa。ASFV Pol X是模板依赖的聚合酶，保真度很低，缺少3’-5’外切酶活性，对于双脱氧核苷酸的识别能力非常差(Oliveros et al.，1997)。
将ASFV Pol X的基因序列和其他代表性的family X的聚合酶进行比对，可以 看到ASFV Pol X和人、牛、鼠的TdT、人、鼠polβ等在结构和功能上有一定的联系。ASFV Pol X的三维结构已经用NMR的方法解出。Pol X和其它聚合酶不一样，它只具有一个palm结构和一个C端子域(Macieiewski et al.，2001)。在真核的Polβ中，一般还有一个N端结构域负责DNA结合。Pol X没有这个关键的结构域，结合DNA的能力却强于Polβ。
另外，ASFV Pol X可以结合单个核苷酸剪切修复(BER)的中间产物，并且可以高效地修复单个核苷酸缺口(Showalter et al.，2001)。BER的步骤中，Pol X修复缺口并且很容易引入突变。在最后一步再由一种ASFV编码的一种容错连接酶把带有错配的新合成链连入基因组。Pol X在修复基因组时将新的突变引入，有助于病毒变异的产生，以便在有生存压力的环境中存活下来。
ASFV pol X聚合酶的化学合成
D-ASFV PolX的化学全合成方法采用的是由Merrifield于1963年提出的固相多肽合成技术(SPPS)，该技术是目前最有效的多肽合成方法。根据保护策略的不同，主要有叔丁氧羰基(Boc)和芴甲氧羰酰基(Fmoc)两种方法。在本项目中，我们拟使用Fmoc-固相合成技术将D型氨基酸连接成多肽。
尽管理论上SPPS的每一步缩合反应其产率能达到99％，但是在实践中Fmoc-固相合成技术通常只能合成少于50个氨基酸的肽链。由于这个原因，合成蛋白质往往需要把多肽片段连接起来，即将目标蛋白质分子分割成几个片段，使得每一片段的氨基酸都少于50个，然后通过高效的化学反应将各个片段连接起来获得目标蛋白质分子。因此，在本项目中我们拟将目标D-ASFV蛋白质分子的多肽链分为4段，采用收敛法两两组合，通过改良后的一锅法自然化学连接(Native chemical ligation，NCl)---蛋白酰肼连接法，得到目标蛋白质的全长氨基酸序列。
由于酰肼链接的位点必须利用半胱氨酸，因此，我们拟将36位以及129位的丙氨酸突变为半胱氨酸，在连接反应完成后，进行脱硫反应，将半胱氨酸还原为丙氨酸(Qian and Danishefsky，2007)。与此同时，在连接反应过程中，为了避免副反应的发生，需要对81位以及86位的半胱氨酸进行侧链巯基保护，在连接反应结束后，再进行脱除。我们拟使用乙酰胺甲基(Acm)作为反应过程中的巯基保护基团(Liu et al.，2012)。合成D-ASFV pol X的具体合成路线和步骤参照图1。
为了验证合成途径的可行性，同时处于节约成本的考虑，我们先进行了L-ASFV pol X的合成。此后用相同的方法合成了D-ASFV pol X。
化学ASFV pol X聚合酶的复性和分析检测
在得到全长的L和D型ASFV pol X之后，我们对它进行了反相色谱(RP-HPLC)纯化，使用了Vydac C18(4.6×250mm)液相色谱柱，加入TFA并用20％-70％的乙腈梯度进行分离(图2)。得到强吸收的主峰和微弱的杂峰。电喷雾电离质谱(ESI-MS)得到的D-ASFV pol X合成全长产物分子量为20317.0Da，理论值为20316.0Da.
我们通过变性-透析的方法对合成的D-ASFV pol X进行复性，首先把冻干的聚合酶干粉加入含有6M盐酸胍的10ml透析液，透析液中含有50mM Tris-HCl，pH 7.4，40mM KCl，6mM(AcO)2Mg，0.01M EDTA和16％甘油。透析过程不断降低透析液中的盐酸胍浓度从4M、2M到0，每次都在4℃搅拌透析10小时。D-Cys81和D-Cys86之间通过空气氧化形成二硫键。我们又通过SDS-PAGE比较了大肠杆菌表达、合成的L型和合成的D型ASFV pol X，它们的条带位置保持一致(图3)。我们同时观察到少量未完全连接的肽段。我们通过圆二色光谱(CD)的方法对比了L和D型ASFV pol X。由于D-ASFV pol X不能被常用的蛋白酶降解(如胰蛋白酶和胃蛋白酶，实验结果未列出)无法进行测序，我们只用质谱测序分析了L-ASFV pol X的序列，在检测结果中覆盖到了100％的pol X序列(表3.1)。
表3.1化学合成的L-ASFV pol X质谱测序肽段序列
Figure PCTCN2016079006-appb-000008
镜像DNA的检测
在D-ASFV pol X聚合酶成功合成之后，镜像遗传信息复制体系中还缺少镜像核酸。我们在美国Chemgenes公司购买了L-primer12、L-template18等镜像DNA片段(序列见2.1.3)和四种L-dNTP。
小结
我们设计了镜像DNA复制的体系，包括镜像聚合酶、镜像DNA、四种镜像dNTP以及适宜的缓冲液条件。我们通过Chemegenes获得了镜像DNA和四种dNTP。由于镜像蛋白质不能通过生物方法获得，我们找到了174个氨基酸的ASFV pol X聚合酶，并且设计了合成路线并合成了L型和D型的ASFV pol X聚合酶。通过盐酸胍变性-透析复性的方法将蛋白折叠至正确的构象。经过质谱、SDS-PAGE检测，验证化学合成蛋白的大小和天然蛋白一致。又通过CD检测，看到天然和镜像聚合酶的吸收谱对称。通过对合成L型ASFV pol X的测序，验证了化学合成蛋白序列的正确性。至此我们得到了镜像DNA复制系统的各个组成部分，具备了进行后续 对于镜像聚合酶反应研究的条件。
镜像手性遗传信息复制和转录体系的功能研究
概述
目前为止前人没有关于镜像DNA复制和转录的研究，我们合成的镜像聚合酶，首先要验证镜像蛋白质和DNA相互作用的可能性，镜像聚合酶的手性特异性，以及复制出的DNA是否具有生物活性。我们同时尝试实现镜像DNA的多循环扩增，未来可以在研究中用于获得更多镜像核酸分子。由于ASFV pol X所在的X聚合酶家族中有一些酶可以利用NTP，模板互补扩增得到RNA分子，我们也尝试基于镜像DNA复制体系尝试依赖模板的转录。
镜像聚合酶催化的DNA延伸
我们已经设计了镜像DNA复制体系，并且通过化学合成的方法获得了体系中的主要组成部分(图5a)。下面我们在非手性的pH 7.5Tris-HCl缓冲液中，加入20mM Mg2+，在天然的体系中加入0.7μg L-ASFV pol X聚合酶，2.5μM D-template18模版，2.5μM D-primer12引物以及0.2mM D-dNTPs；在镜像体系中加入相同浓度的D-ASFV pol X聚合酶以及D-引物、模板和dNTPs。将反应体系置于37℃静置4h。我们在L和D型的引物5’末端都添加了荧光素氨基磷酸酯(FAM)标记，而模板未加修饰。这样在未经染色的条件下用488nm光激发，可以看到primer条带。在两个体系中，天然和镜像的12nt引物都延伸到18nt(图5b)。在8M尿素变性的20％PAGE胶上，可以清晰分辨从12-18nt每个碱基的位置。0h作为对照组，引物长度为12nt，而4h是完全延伸的终产物，长度为18nt。根据模板序列推测延伸的6个碱基为ACTGAG，说明在镜像体系中4种dNTP均可以被用作底物合成DNA链，这一结论仍然需要后续的互补配对验证。我们进一步希望验证镜像延伸产物是否添加了正确的镜像核苷。由于目前的一代测序方法仍然依赖聚合酶和天然的dNTP衍生物，这一体系无法用于L-DNA的测序。我们尝试通过质谱的方法来验证18nt延伸产物的质量是否正确。我们通过ESI-MS检测得到镜像全长延伸产物分子量为5516.9Da(理论值5517.6Da)，在误差范围内认为18nt的全长产物质量正确。另外，我们希望了解镜像扩增体系是否有局限性，是否可以应用到不同的引物、模板序列，在另一组实验中使用了15nt引物、21nt模板(15nt引物不带FAM标记，PAGE胶经过Sybr Gold染色)，仍然可以观察到引物减少、 中间条带出现以及全长DNA的量增大这些现象，说明镜像DNA延伸可以用于不同的DNA序列(图5d)。
镜像DNA复制系统多循环扩增DNA
我们尝试镜像DNA复制系统尝试进行多循环的PCR扩增。尽管ASFV pol X不是一个热稳定的酶，我们仍然可以通过每个循环加酶的方式进行多个循环的扩增。ASFV pol X结合DNA会阻碍下一个循环的扩增，所以我们在每个循环结束后进行酚氯仿抽提去除蛋白，再加入新的ASFV pol X。每个循环的抽提会带来较大的DNA损失(回收效率约为40％)，我们只进行3个循环的扩增。由于循环较少难以检测到DNA产物的增加，我们使用了FAM标记的引物，而且它只能使用第一个循环产生的全长延伸产物才能进行第二轮的扩增(图6)。在反应中使用了FAM标记的primer11，不带标记的reversel1，以及template27，全长产物为22nt(序列见2.1.3)。我们通过FAM标记的全长产物定量，检测到第2到第3循环发生了2.3倍的扩增，理论扩增值为3(图7)。结果在理论上证实了镜像DNA复制体系多循环扩增DNA的可行性。
镜像DNA复制系统的碱基互补配对特异性
我们进一步需要检测镜像聚合酶催化的DNA扩增是否遵守碱基互补配对的规则。我们将4种dNTP分别加入反应体系，在模板的下一个碱基分别为A、T、C、G时检测延伸情况(图8)。对于天然的系统，我们使用了primer12和四种不同的template，在其3’端第13个碱基的位置分别为A、T、C、G序列。而对于镜像系统，我们为了减少购买镜像DNA的成本，分别用primer12进行了1、2、3nt的延伸，并进行PAGE胶回收得到primer13、primer14、primer15，它们与template18互补，下一个模板上的碱基分别为T、G、A、C。我们观察到只有A∶T、T∶A、C∶G、G∶C4种正确的配对条件下dNTP可以高效率地添加到引物序列的3’末端，没有明显的错配发生，在天然和镜像的系统中保持一致。所以镜像DNA复制系统也遵循互补配对原则，并且有着一定的保真度。
镜像DNA复制系统的手性特异性
关于聚合酶在添加dNTP时的手性特异性，在天然的聚合酶上已经有了一些研究。之前的研究表明哺乳动物聚合酶γ、大肠杆菌聚合酶I、HIV-1逆转录酶催化的DNA延伸会受到L-dTTP的抑制，而对于哺乳动物聚合酶α，L-dTTP不会抑制 其催化的DNA聚合反应，对于哺乳动物β，抑制的效果比较微弱(Yamaguchi et al.，1994)。L-dTTP会抑制人的DNA聚合酶γ、δ、牛胸腺末端转移酶，但是不会抑制人DNA聚合酶β，但是DNA聚合酶β、γ、δ不能使用L-dTTP作为底物。在3’末端添加的L-核苷会让DNA难以被3’-5’外切酶降解(Focher et al.，1995)。所以有的聚合酶在添加两种底物时会受到镜像dNTP的抑制而有的不会。我们的研究希望阐释对于镜像ASFV pol X是否会有手性特异性，以及它的聚合酶活性是否会受到天然手性dNTP的抑制。我们的研究中首先尝试单独添加不同手性的聚合酶、引物-模板、dNTP的组合，再尝试将天然和镜像两个体系放在同一溶液中进行反应。
我们在与之前相同的缓冲液条件下，分别改变ASFV pol X、引物-模板和dNTP的手性，每个成分L型和D型两种手性，体系共有8种组合(图9)。将体系置于37℃孵育12小时，在20％PAGE胶上观察结果。我们发现L-ASFV pol X、D-引物-模板、D-dNTPs的天然体系可以延伸6个碱基到全长，D-ASFV pol X、L-引物模板、L-dNTPs的镜像体系可以延伸到全长，而其他的组合均不可以延伸。例如L-ASFV pol X、D-引物-模板、L-dNTPs，天然的聚合酶不能将镜像的dNTPs添加到天然引物的3’末端；对于L-ASFV pol X、L-引物-模板、D-dNTPs，天然的聚合酶不能将天然的dNTPs添加到镜像引物的3’末端；D-ASFV pol X、L-引物-模板、D-dNTPs，镜像的聚合酶不能将天然的dNTPs添加到镜像引物的3’末端；对于D-ASFV pol X、D-引物-模板、L-dNTPs，镜像的聚合酶不能将镜像的dNTPs添加到天然引物的3’末端。所以在镜像和天然的体系中，错误手性的引物-模板、dNTP均不能被作为底物参加延伸反应，天然和镜像的DNA复制体系有着良好的手性特异性。
我们进而希望探究天然和镜像的系统在同一个溶液体系之中，是否会发生相互干扰。前面所述的文献表明，对于某些聚合酶镜像的dNTP会阻止DNA延伸反应的继续进行。我们的实验使用了与之前相同的缓冲液体系，并同时加入了两种手性，相同浓度的ASFV pol X和dNTPs。为了分别观察引物的延伸，我们设计了长度不同的引物和模板序列，并且使用了不同的荧光修饰，天然反应使用Cy5标记的primer20以及template26，镜像反应是FAM标记的primer12和template18。我们将相同浓度的两种手性引物和模版都加入同一个溶液体系，在37℃反应4小时以后在20％PAGE胶上分离检测反应产物(图10)。红色的条带为天然反应，在21nt和26nt之间，而绿色的条带为镜像反应，在12nt和18nt之间。左边的样品是只有天然体系的对照组，中间的是镜像体系单独反应的对照组，右侧是天然和镜像相同浓度在同一溶液体系中反应的实验组。我们可以看到在实验组中两个体系都可以分别延伸到18nt和26nt的全长，混合体系反应速率与单独反应相差不 多，说明天然和镜像体系在同一个溶液之中不存在严重的相互干扰。
镜像DNAzyme的酶合成以及功能检测
我们已经验证了镜像DNA复制系统可以依赖模板合成DNA，而且具有一定的碱基互补配对特异性。进而我们希望验证镜像聚合酶合成的镜像DNA序列具有生物活性。
上世纪九十年代，体外筛选技术(in vitro selection)创立并得到应用。这项技术可以通过复杂度高的随机DNA或RNA序列库进行设计筛选，得到功能性的DNA或RNA分子，主要有可以结合特定靶标的核酸适体(aptamer)和有催化活性的核酶(ribozyme)和脱氧核酶(DNAzyme)。以核酸适体为例，体外筛首先要合成单链核酸序列库，其中含有20-80个随机序列，文库复杂度达到1014。文库中不同核酸的一级序列不同，它们在溶液中会形成不同的空间构型。在适宜的条件下将随机库与靶标分子相互作用，捕获能够与靶标结合的DNA或RNA分子，再将这些分子进行PCR扩增。得到扩增的这些分子相对于原始随机库对于靶标的亲和力得到了增强，将它作为次级库，再对于靶标分子进行第二轮的筛选。这样经过反复的扩增就能筛选到高亲和力的核酸适体序列。
我们选择用D-ASFV pol X合成一段L-DNAzyme序列，再验证它的脱氧核酶活性。R.Breaker及其同事在2013年的研究中发现并验证了多个DNAzyme序列的活性(Gu et al.，2013)，我们选取了其中一个44nt的Zn2+依赖的DNAzyme作为合成的目标。我们设计出的全长DNAzyme包括5’末端12nt的引物序列和完整的44ntDNAzyme序列(图11a)：
Figure PCTCN2016079006-appb-000009
我们将它的反向互补序列作为模板(为了方便胶回收分离单双链，我们在模板的3’末端额外添加了10nt)，加入primer12经过36小时的延伸获得了全长的DNAzyme序列(图11b)。与marker的比对发现通过聚合酶合成的DNAzyme大小是正确的。天然的聚合酶催化合成DNAzyme全长序列的速率高于镜像系统，这可能由于镜像引物、模板和dNTP中含有杂质。此实验进一步说明镜像DNA复制系统的延伸长度在44nt以上，并且没有明显的序列选择性。我们进一步将反应到全长的DNAzyme胶染色并切下，与66nt的template小心分开，放在缓冲液中扩散过夜，并且用试剂盒沉淀DNAzyme序列。溶解DNAzyme之后先在90℃加热再降至室温使56nt的DNAzyme序列折叠成正确的结构。然后再反应体系中加入buffer2以开始反应。对照组buffer2含有20mM Mg2+，实验组含有2mM Zn2+，在37℃反 应36h。天然和镜像酶合成的DNAzyme，均可以在2mM Zn2+离子存在的条件下实现高效率的自我剪切，在缺少Zn2+或者加入Mg2+的对照实验中都不能自我剪切(图11c)。所以说明通过镜像ASFV pol X合成的DNAzyme序列具有自我剪切的活性。
DNA模板依赖的镜像转录
我们已经验证了镜像的DNA聚合酶可以依照DNA模板进行引物的延伸，进而希望研究是否可以将镜像DNA转录为镜像RNA。已知的X家族DNA聚合酶对于dNTP和rNTP的选择性很差，polμ可以依照模板添加dNTP或者rNTP到DNA链上，定量来看，它对于rNTP的选择特异性低于polβ1000倍(Sa and Ramsden，2003)。ASFV pol X没有被验证过是否可以利用rNTP。我们首先用天然手性的ASFV pol X在primer12和template18的体系中加入rNTP。我们发现尽管延伸的效率降低了很多，仍然可以在36小时后得到全长的延伸产物(图12a)。
RNase A特异性地催化RNA的核糖在C和U残基的下一个核苷酸5′磷酸二酯键断裂，它可以识别3’，5’磷酸二酯键而不能切断2’，5’键，形成具有2′，3′-环磷酸衍生物3′C或3′U寡聚核苷酸。我们将36小时反应后的延伸产物加热到75℃10min时间，失活RNase抑制剂以及ASFV pol X，然后再加入不同浓度的RNase A进行消化(图12b)。我们可以看到第一步的加热反应没有使RNA发生降解，但是RNase A的加入使得全长的18nt产物降解为13nt，并且随着RNase A的浓度越高降解越明显。这一实验证明了RNase A消化RNA合成产物验证了添加的是rNTP，并且rNTP的添加方式为3’，5’连接。
在镜像的ASFV pol X和DNA的体系中，也可以将L-rNTP添加到引物链上，尽管速率比天然的体系要慢(图12a)。我们同样认为这可能是镜像模板、引物和rNTP的纯度不够高引起的(在HPLC中可以观察到杂峰，在CD中发现模板、引物的量小于标称值)。
镜像转录RNA系统的碱基互补配对特异性
最后我们需要验证RNA转录体系是否依照模板遵循碱基互补配对规则。将4种rNTP分别加入反应体系，使用了5’末端FAM标记的primer12作为引物，在模板的下一个碱基分别为A、T、C、G时，观察是否可以延伸到13nt。对于天然的系统，我们使用了primer12和四种不同的template，在其3’端第13个碱基的位置分别为A、T、C、G序列。而对于镜像系统，分别用primer12添加A、AC、ACT进行了延伸反应，并进行PAGE胶回收得到primer13、primer14、primer15，它们 与template18互补时下一个模板上的碱基分别为T、G、A、C。我们观察到A∶U、U∶A、C∶G、G∶C4种正确的配对条件下rNTP可以高效率地添加到引物序列的3’末端。镜像体系中出现了一些较为明显的错配，比如T∶rG，A∶rC等等，但是错误核苷添加的效率显著低于正确配对的(图13)。所以镜像RNA转录系统遵循互补配对原则，有着一定的保真度。
小结
我们设计构建的镜像手性遗传信息复制和转录体系，包含核心成分为D-ASFV pol X、L-引物、L-模板、L-dNTPs/rNTPs，可以依照模板实现DNA的扩增和转录RNA，说明镜像手性的聚合酶和DNA有相互作用。利用ASFV pol X聚合酶，我们实现了理论上的多循环扩增DNA，在第二至第三循环实现了2.3倍的DNA扩增。另外，我们验证了在利用天然和镜像体系添加dNTP或rNTP延伸引物链，遵循碱基互补配对原则，保真度良好。聚合酶、引物模板和dNTP在反应体系中存在手性特异性，只有三者全是天然或者全是镜像分子时引物链可以延伸，其他的各种组合均不能实现扩增。在同一溶液体系中加入相同浓度的两种手性的DNA复制体系，两个体系可以分别进行延伸反应，没有严重的相互阻碍。我们用镜像ASFV pol X延伸了44nt的Zn2+依赖的DNAzyme序列，在含有2mM Zn2+的缓冲液环境中，经过纯化分离的单链L-DNAzyme有着自我剪切的生物活性。
总结与展望
总结
起源于对镜像生物分子的存在与生物活性的探究，我们的工作构建了基于D-ASFV pol X的镜像遗传信息复制和转录体系。主要结论如下：
一.我们设计了镜像DNA复制的体系，包括镜像聚合酶、镜像DNA、四种镜像dNTP，以及适宜的缓冲液条件(50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl)。通过全化学合成的方法得到了D-ASFV pol X的全长序列，并且通过盐酸胍变性-透析的方法成功对其进行折叠复性。通过HPLC、SDS-PAGE、ESI-MS、CD等方法对其进行了分析检测。
二.在镜像DNA复制体系中，在上述缓冲液条件下加入0.7μg D-ASFV pol X聚合酶，2.5μM L-模版，2.5μM L-引物以及0.2mM L-dNTPs(每种浓度)，我们证实在多个模板引物组合中(primer12-template18，primer15-template21， primer12-DNAzymeTemplate)镜像DNA的复制延伸均可以进行。并且在DNA复制过程中新合成的DNA与模板链互补，镜像复制过程遵循碱基互补配对法则，有着较好的保真性。
三.在反应体系中加入合适的模板和双链引物，使用95℃变性-室温退火-37℃延伸的PCR循环，每次退火后重新加入D-ASFV pol X，可以实现对于DNA模板的多循环扩增。
四.在实验中变换ASFV pol X、引物-模板、dNTP的手性组合，一共有8种情况，在适宜的反应体系中尝试进行延伸反应，只有L-ASFV pol X、D-引物-模板、D-dNTPs的全天然组合，以及D-ASFV pol X、L-引物-模板、L-dNTPs的全镜像组合可以反应，其余六种条件在反应时间内均未检测到延伸。说明基于ASFV pol X的天然和镜像DNA复制系统可以识别错误手性的底物，在延伸反应中有着良好的手性特异性。
五.在混合两种手性的实验中，同一个反应体系中加入两种手性的ASFV pol X、引物、模板和dNTP，两个体系可以正确地识别本体系的蛋白或核酸分子，分别完成DNA链的延伸，没有严重的相互干扰。
六.用镜像DNA复制体系扩增一段44nt的Zn2+依赖DNAzyme序列，在经过36小时的延伸后获得全长DNA链。通过PAGE胶回收分离得到单链DNAzyme，在含有2mM Zn2+的缓冲液环境中，L-DNAzyme有着自我剪切的生物活性，而对照组20mM Mg2+条件下不能剪切。镜像DNA复制系统可以扩增长链的、有活性的L-DNA序列。
七.镜像转录体系含有D-ASFV pol X、L-模板、L-引物和L-rNTP，在50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl条件下可以将L-rNTP添加到模板的3’末端，合成一段RNA序列。D-ASFV pol X模板依赖的转录RNA反应速率低于复制DNA，转录同样遵循碱基互补配对原则，有一些错配产生。
我们的工作实现了构建镜像DNA复制和转录体系，并且证实了镜像聚合酶可以依照模板复制镜像DNA或者生成RNA链。虽然生命起源早期的进化证据已经无从查找，我们仍然不知道为何镜像的分子在高等细胞生命的进化过程中被舍弃了，但是我们在实验上证实了镜像生命分子可以实现相应的生物功能，提供了镜像分子存在的可能性依据。对于镜像中心法则中两个重要环节的实现，为未来在实验室环境中构建完整的镜像生命奠定了基础。
应用及展望
我们的工作延续了对于镜像手性生物分子的研究，不仅证实了镜像分子如研 究者预期的那样具有和天然分子相对应的活性和手性特异性，而且作为理论和实践应用的开端，完成了镜像中心法则中的DNA复制和转录RNA两个环节。本发明有两个主要的应用领域：一是将镜像DNA复制体系用于体外筛选获得核酸药物，二是尝试构建完整的镜像原始细胞。
一.镜像体外筛选获得核酸药物是未来很有应用前景的方向。天然和镜像的核酸序列都可以由于其多样的序列折叠成复杂多样的结构，有一些可以结合特定靶标分子DNA或RNA被称为核酸适体(aptamer)。通常获得核酸适体的方式是体外筛选技术，体外筛选使用含有通常30-80个随机序列的DNA或RNA，一般可以达到1014以上的复杂度，两端加上固定的引物互补区方便PCR扩增。在筛选的过程中通常要用不同的方法固定靶标分子，将随机序列库与靶标分子放在一起结合，然后用洗涤液分离去除不与靶标分子结合的核酸序列，进而再获得亲和力较强的核酸分子，通过PCR扩增富集高亲和强度的序列。经过多循环的筛选，即可得到与靶标分子较强结合的一个或几个核酸序列。
如果针对疾病相关的某种蛋白或小分子进行体外筛选，例如CDK、GPCR、Bcl-2等，可能可以获得与之高亲和力的核酸适体分子。然而将这些分子应用于临床的效果并不理想，在体内会快速被降解。进而有研究者希望以在体内更稳定的镜像核酸分子作为药物。目前已有的研究通过合成镜像的靶标分子，再用天然的随机序列库进行体外筛选，获得可以结合镜像靶标的天然核酸序列。由于手性镜像的关系，使用同样的序列合成镜像核酸适体，即可结合天然靶标(Williams et al.，1997)。这种筛选方法可以有效的获得能结合天然分子的镜像核酸适体，但是问题在于蛋白质化学合成的技术难度很高，很少有学术或商业机构可以合成较为复杂的蛋白质，而且生命体中的大多数的靶点都超出了目前蛋白合成的技术范围，例如PD-L1蛋白大小为40kDa。
镜像体外筛选的难度在于目前没有办法在筛选的每一轮进行镜像PCR扩增。我们的镜像PCR技术将可以实现这一点。通过DNA合成仪合成镜像随机序列库，直接将其用于天然药物靶点的结合，再通过漂洗、洗脱、扩增高亲和序列，经多个循环直接得到镜像核酸适体序列，可以经优化和临床试验作为潜在药物分子。对于镜像PCR技术，本文用ASFV pol X每个循环加酶的方式实现了镜像PCR。ASFV pol X的反应速率很低(在病毒中主要用于基因组缺口的修复)，以及它不是耐热聚合酶，在3.5M脯氨酸保护的条件下50℃1分钟即失去活性。未来可以选用已经发现的高效、耐热聚合酶，在合成技术范围内的比如352个氨基酸的Dpo4(Sulfolobus solfataricus P2DNA polymerase IV)，文献报道Dpo4可以用于PCR扩增，未来可以使用镜像Dpo4蛋白进行PCR和镜像的体外筛选。
二.构建镜像生命的重要步骤是将镜像中心法则中的另一个重要步骤——翻译过程在实验室实现。镜像核糖体和tRNA是实现翻译的主要生物原件，目前的技术水平仍然倾向于使用化学合成的方法来构建。细菌的核糖体包含rRNA，通常还有50-80个核糖体蛋白组成(Wilson et al.，2009)，大多数的都短于240个氨基酸，目前的蛋白合成技术可以实现，还有一个557个氨基酸的rpsA蛋白需要未来改进的蛋白合成技术来实现。各组成部分别合成之后在体外进行复性和组装，成为有功能的镜像核糖体。通过化学合成DNA和PCR扩增更长的L-DNA模板，转录出长的L-mRNA，再用镜像的核糖体和tRNA可以得到实现镜像细胞功能的蛋白分子，例如DNA连接酶、解旋酶、丙酮酸脱氢酶等等。如果能尽量多地合成出镜像细胞所需的蛋白和核酸组成成分，未来通过进一步的技术进展将会有可能构建简单的镜像细胞，也可以使用镜像细胞生产镜像的药物或镜像生物材料。
主要符号对照表
ASFV pol X   非洲猪瘟病毒聚合酶X
DNAzyme      脱氧核酶
SDS-PAGE     十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate 
             polyacrylamide gel electropheresis)
Tris         三羟甲基氨基甲烷
NMDA         N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid)
HPLC         高效液相色谱(high performance liquid chromatography)
ESI-MS       电喷雾电离质谱(electrospray ionization mass spectrometry)
EDTA         乙二胺四乙酸(ethylene diamine tetraacetic acid)
FAM          荧光素氨基磷酸酯(fluorescein amidite)
参考文献
Breslow，R.，and Levine，M.(2006).Amplification of enantiomeric concentrations under credible prebiotic conditions.Proceedings of the National Academy of Sciences of the United States of America 103，12979-12980.
Corrigan，J.(1969).D-Amino Acids in Animals.Science 164，142-149.
Dawson，P.E.，and Kent，S.B.H.(2000).Synthesis of Native Proteins by Chemical Ligation.Annual Review of Biochemistry 69，923-960.
Dawson,P.E.,Muir,T.W.,Clark-Lewis,I.,.,and Kent,S.B.(1994).Synthesis of proteins by native chemical ligation.Science 266,776-779.
Engel,M.,and Macko,S.(2001).The stereochemistry of amino acids in the Murchison meteorite.Precambrian Research 106,35-45.
Fields,G.(2002).Introduction to Peptide Synthesis,Vol Chapter 11.
Fitzgerald,M.C.,Chernushevich,I.,Standing,K.G.,Kent,S.B.H.,and Whitman,C.P.(1995).Total Chemical Synthesis and Catalytic Properties of the Enzyme Enantiomers L-and D-4-Oxalocrotonate Tautomerase.Journal of the American Chemical Society 117,11075-11080.
Focher,F.,Maga,G.,Bendiscioli,A.,Capobianco,M.,Colonna,F.,Garbesi,A.,and Spadari,S.(1995).Stereospecificity of human DNA polymerases alpha,beta,gamma,delta and epsilon,HIV-reverse transcriptase,HSV-1 DNA polymerase,calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D-and L-thymidine 5'-triphosphate as substrate.Nucleic Acids Research 23,2840-2847.
Gu,H.,Furukawa,K.,Weinberg,Z.,Berenson,D.F.,and Breaker,R.R.(2013).Small,Highly Active DNAs That Hydrolyze DNA.Journal of the American Chemical Society 135,9121-9129.
Hamase,K.,Inoue,T.,Morikawa,A.,Konno,R.,and Zaitsu,K.(2001).Determination of Free[formula omitted]-Proline and[formula omitted]-Leucine in the Brains of Mutant Mice Lacking[formula omitted]-Amino Acid Oxidase Activity.Analytical Biochemistry 298,253-258.
Hashimoto,A.,Kumashiro,S.,Nishikawa,T.,Oka,T.,Takahashi,K.,Mito,T.,Takashima,S.,Doi,N.,Mizutani,Y.,Yamazaki,T.,et al.(1993).Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex.Journal of Neurochemistry 61,348-351.
Hashimoto,A.,Oka,T.,and Nishikawa,T.(1995).Anatomical Distribution and Postnatal Changes in Endogenous Free D-Aspartate and D-Serine in Rat Brain and Periphery.European Journal of Neuroscience 7,1657-1663.
Joyce,G.,Visser,G.,Van Boeckel,C.,Van Boom,J.,Orgel,L.,and Van Westrenen,J.(1984).Chiral selection in poly(C)-directed synthesis of oligo(G).Nature 310,602.Kent,S.B.(1988).Chemical synthesis of peptides and proteins.Annual Review of Biochemistry 57,957-989.
Klusmann,S.,Nolte,A.,Bald,R.,Erdmann,V.,and Furste,J.(1996).Mirror-image RNA that binds D-adenosine.Nature Biotechnology 14,1112.
Krause,E.,Bienert,M.,Schmieder,P.,and Wenschuh,H.(2000).The Helix-Destabilizing Propensity Scale of d -Amino Acids:The Influence of Side Chain Steric Effects.
Liu,S.,Pentelute,B.L.,and Kent,P.S.B.H.(2012).Convergent Chemical Synthesis of [Lysine 24,&thinsp；38,&thinsp；83]Human Erythropoietin &dagger.Angewandte Chemie International Edition 51,993&ndash；999.
Maciejewski,M.,Shin,R.,Pan,B.,Marintchev,A.,Denninger,A.,Mullen,M.,Chen, K.,Gryk,M.,and Mullen,G.(2001).Solution structure of a viral DNA repair polymerase.Nature Structural &Molecular Biology 8,936.
Milton,R.,Milton,S.,and Kent,S.(1992).Total chemical synthesis of a D-enzyme:the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity[corrected].Science 256,1445-1448.
Mothet,J.,Parent,A.,Wolosker,H.,Brady,R.,Linden,D.,Ferris,C.,Rogawski,M.,and Snyder,S.(2000).d-Serine is an endogenous ligand for the glycine site of the N-methyl-d-aspartate receptor.Proceedings of the National Academy of Sciences of the United States of America 97,4926.
Nagata,Y.,Homma,H.,Lee,J.,and Imai,K.(1999).d-Aspartate stimulation o ftestosterone synthesis in rat Leydig cells.FEBS Letters 444,160-164.
Nolte,A.,Klusmann,S.,Bald,R.,Erdmann,V.,and Furste,J.(1996).Mirror-design of L-oligonucleotide ligands binding to L-arginine.Nature Biotechnology 14,1116.
Ohide,H.,Miyoshi,Y.,Maruyama,R.,Hamase,K.,and Konno,R.(2011).d-Amino acid metabolism in mammals:Biosynthesis, degradation and analytical aspects of the metabolic study.Journal of Chromatography B 879,3162-3168.
Oliveros,M.,Yanez,R.,Salas,M.,Salas,J.,Vinuela,E.,and Blanco,L.(1997).
Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair.Journal of Biological Chemistry 272,30899-30910.
Qian,W.,and Danishefsky,S.J.(2007).Free-radical-based,specific desulfurization of cysteine:a powerful advance in the synthesis of polypeptides and glycopolypeptides.
Angewandte Chemie International Edition 46,9248-9252.
Sa,N.,and Ramsden,D.(2003).Polymerase mu is a DNA-directed DNA/RNA polymerase.Molecular and Cellular Biology 23,2309-2315.
Schell,M.,Molliver,M.,and Snyder,S.(1995).D-serine,an endogenous synaptic modulator:localization to astrocytes and glutamate-stimulated release.Proceedings of the National Academy of Sciences of the United States of America 92,3948-3952.
Sczepanski,J.,and Joyce,G.(2014).A cross-chiral RNA polymerase ribozyme.Nature 515,440.
Showalter,A.,Byeon,I.,Su,M.,and Tsai,M.(2001).Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.Nature Structural &Molecular Biology 8,942.
Showalter,A.,and Tsai,M.(2001).A DNA Polymerase with Specificity for Five Base Pairs.
Vinogradov,A.,Evans,E.,and Pentelute,B.(2015).Total synthesis and biochemical characterization of mirror image barnase.Chemical Science 6,2997-3002.
Weinstock,M.,Jacobsen,M.,and Kay,M.(2014).Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity.Proceedings of the National Academy of Sciences of the United States of America 111,11679-11684.
Williams,K.,Liu,X.,Schumacher,T.,Lin,H.,Ausiello,D.,Kim,P.,and Bartel,D.(1997).Bioactive and nuclease-resistant l-DNA ligand of vasopressin.Proceedings of  the National Academy of Sciences of the United States of America 94,11285-11290.
Wilson,D.,Gupta,R.,Mikolajka,A.,and Nierhaus,K.(2009).Ribosomal Proteins:Role in Ribosomal Functions,Vol 2009.
Wolosker,H.,Blackshaw,S.,and Snyder,S.(1999).Serine racemase:A glial enzyme synthesizing d-serine to regulate glutamate-N-methyl-d-aspartate neurotransmission.
Proceedings of the National Academy of Sciences of the United States of America 96,13409-13414.
Wyszko,E.,Szymanski,M.,Zeichhardt,H.,Muller,F.,Barciszewski,J.,and Erdmann,V.(2013).Spiegelzymes:Sequence Specific Hydrolysis of L-RNA with Mirror Image Hammerhead Ribozymes and DNAzymes.PloS one 8,e54741.
Yamaguchi,T.,Iwanami,N.,Shudo,K.,and Saneyoshi,M.(1994).Chiral Discrimination of Enantiomeric 2′-Deoxythymidine 5′-Triphosphate by HIV-1 Reverse Transcriptase and Eukaryotic DNA Polymerases.Biochemical and Biophysical Research Communications 200,1023-1027.
Zuker,M.(2003).Mfold web server for nucleic acid folding and hybridization prediction.Nucleic Acids Research 31,3406-3415.

Claims (25)

  1. 对镜像核酸进行复制的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得所述镜像核酸。
  2. 权利要求1的方法，其中所述反应重复多个循环。
  3. 权利要求1或2的方法，其中所述镜像核酸、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs为L-型，所述镜像核酸聚合酶为D型。
  4. 权利要求1-3之任一项的方法，其中所述镜像核酸聚合酶选自由D-ASFV pol X、D-Dpo4、D-Taq聚合酶以及D-Pfu聚合酶构成的组。
  5. 权利要求1-4之任一项的方法，其中所述镜像核酸为L-DNAzyme。
  6. 权利要求1-5之任一项的方法，其中所述镜像核酸为L型DNA。
  7. 权利要求1-5之任一项的方法，其中所述镜像核酸为L型RNA。
  8. 权利要求1-7之任一项的方法，其中所述反应为聚合酶链式反应。
  9. 权利要求1-8之任一项的方法，其中所述反应在如下缓冲液中进行：50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。
  10. 进行镜像PCR的方法，包括：在镜像核酸聚合酶、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs的存在下进行反应，以获得镜像核酸。
  11. 权利要求10的方法，其中所述反应重复多个循环。
  12. 权利要求10或11的方法，其中所述镜像核酸、镜像核酸模板、镜像核酸引物以及镜像dNTPs/rNTPs为L-型，所述镜像核酸聚合酶为D型。
  13. 权利要求10-12之任一项的方法，其中所述镜像核酸聚合酶选自由D-ASFV pol X、D-Dpo4、D-Taq聚合酶以及D-Pfu聚合酶构成的组。
  14. 权利要求10-13之任一项的方法，其中所述镜像核酸为L型DNA。
  15. 权利要求10-13之任一项的方法，其中所述镜像核酸为L型RNA。
  16. 权利要求10-13之任一项的方法，其中所述镜像核酸为L-DNAzyme。
  17. 权利要求10-16之任一项的方法，其中所述反应在如下缓冲液中进行：50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。
  18. 筛选镜像核酸分子的方法，包括：
    在允许二者结合的条件下，使随机镜像核酸序列库与靶标分子接触；
    获得与靶标分子结合的镜像核酸分子；以及
    通过镜像PCR扩增所述与靶标分子结合的镜像核酸分子。
  19. 权利要求18的方法，其中所述靶标分子被固定在固相介质上。
  20. 权利要求18-19之任一项的方法，其中在随机镜像核酸序列库与靶标分子 接触之后，通过洗涤去除不与靶标分子结合的镜像核酸序列，从而获得与靶标分子结合的镜像核酸分子。
  21. 权利要求18-20之任一项的方法，其中所述镜像核酸分子为L型DNA。
  22. 权利要求18-20之任一项的方法，其中所述镜像核酸分子为L型RNA。
  23. 权利要求18-22之任一项的方法，其中所述镜像PCR中使用的镜像核酸聚合酶选自由D-ASFV pol X、D-Dpo4、D-Taq聚合酶以及D-Pfu聚合酶构成的组。
  24. 权利要求18-23之任一项的方法，其中所述镜像PCR在如下缓冲液中进行：50mM Tris-HCl，pH 7.5，20mM MgCl2，1mM DTT，50mM KCl。
  25. D-型ASFV pol X，其序列如SEQ ID NO：17所示，其中除没有手性的甘氨酸以外，其余氨基酸均为D-型氨基酸。
PCT/CN2016/079006 2015-08-18 2016-04-11 镜像核酸复制体系 WO2017028548A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US16/092,757 US11371027B2 (en) 2015-08-18 2017-02-15 Mirror nucleic acid replication system
EP17781735.0A EP3444341A4 (en) 2015-08-18 2017-02-15 MIRROR NUCLEIC ACID REPLICATION SYSTEM
CN201780026142.7A CN109072203B (zh) 2015-08-18 2017-02-15 镜像核酸复制体系
PCT/CN2017/073573 WO2017177759A1 (zh) 2015-08-18 2017-02-15 镜像核酸复制体系

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510507780.3A CN106467910A (zh) 2015-08-18 2015-08-18 L-dna/l-rna聚合酶及其应用
CN201510507780.3 2015-08-18

Publications (1)

Publication Number Publication Date
WO2017028548A1 true WO2017028548A1 (zh) 2017-02-23

Family

ID=58050673

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2016/079006 WO2017028548A1 (zh) 2015-08-18 2016-04-11 镜像核酸复制体系
PCT/CN2017/073573 WO2017177759A1 (zh) 2015-08-18 2017-02-15 镜像核酸复制体系

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/073573 WO2017177759A1 (zh) 2015-08-18 2017-02-15 镜像核酸复制体系

Country Status (4)

Country Link
US (2) US11371027B2 (zh)
EP (1) EP3444341A4 (zh)
CN (2) CN106467910A (zh)
WO (2) WO2017028548A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109022387A (zh) * 2018-08-29 2018-12-18 华南理工大学 一种突变型Pfu DNA聚合酶及其制备方法和应用
US11371027B2 (en) 2015-08-18 2022-06-28 Tsinghua University Mirror nucleic acid replication system
WO2023148646A1 (en) * 2022-02-03 2023-08-10 Tsinghua University Mirror-image selection of l-nucleic acid aptamers

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3775264A4 (en) * 2018-04-05 2022-03-23 Tsinghua University METHODS FOR SEQUENCING AND PRODUCING NUCLEIC ACID SEQUENCES
CN111217892A (zh) * 2019-07-26 2020-06-02 翔宇药业股份有限公司 卡贝缩宫素的合成方法
CN114891868A (zh) * 2022-05-31 2022-08-12 广州市金圻睿生物科技有限责任公司 一种基于ngs平台的微生物定量方法及试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101818142A (zh) * 2009-01-09 2010-09-01 富士胶片株式会社 用于复制核酸序列的方法
CN102517241A (zh) * 2011-12-19 2012-06-27 中国药科大学 一种含有d型氨基酸的双羰基还原酶的突变体的制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102203292B (zh) 2008-10-29 2014-06-25 南克森制药公司 通过质谱分析法测序核酸分子
JP6138684B2 (ja) * 2010-08-31 2017-05-31 キヤノン ユー．エス． ライフ サイエンシズ， インコーポレイテッドＣａｎｏｎ Ｕ．Ｓ． Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ， Ｉｎｃ． マイクロ流体デバイスのインシステムプライミングのための組成物及び方法
JP6522496B2 (ja) * 2012-05-16 2019-05-29 ノクソン ファーマ エージー Ｌ−核酸の酵素性合成
CN105916992A (zh) * 2013-11-20 2016-08-31 诺松制药股份公司 通过酶合成l-核酸
CN106467910A (zh) 2015-08-18 2017-03-01 清华大学 L-dna/l-rna聚合酶及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101818142A (zh) * 2009-01-09 2010-09-01 富士胶片株式会社 用于复制核酸序列的方法
CN102517241A (zh) * 2011-12-19 2012-06-27 中国药科大学 一种含有d型氨基酸的双羰基还原酶的突变体的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE DATABASE Genbank 19 November 2014 (2014-11-19), YANEZ, R.J. ET AL.: "DNA polymerase beta-like protein [African swine fever virus", XP055367467, Database accession no. NP_042790 *
WANG, YUHAI ET AL.: "Synthesis of New Chiral Compounds of Purine and Pyridine", SCIENCE IN CHINA (SERIES B, vol. 28, no. 6, 31 December 1998 (1998-12-31), pages 531 - 539, ISSN: 1006-9240 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11371027B2 (en) 2015-08-18 2022-06-28 Tsinghua University Mirror nucleic acid replication system
CN109022387A (zh) * 2018-08-29 2018-12-18 华南理工大学 一种突变型Pfu DNA聚合酶及其制备方法和应用
CN109022387B (zh) * 2018-08-29 2020-12-22 华南理工大学 一种突变型Pfu DNA聚合酶及其制备方法和应用
WO2023148646A1 (en) * 2022-02-03 2023-08-10 Tsinghua University Mirror-image selection of l-nucleic acid aptamers

Also Published As

Publication number Publication date
WO2017177759A1 (zh) 2017-10-19
EP3444341A4 (en) 2019-09-04
CN106467910A (zh) 2017-03-01
EP3444341A1 (en) 2019-02-20
CN109072203B (zh) 2023-04-04
US20190376050A1 (en) 2019-12-12
US20220325260A1 (en) 2022-10-13
US11371027B2 (en) 2022-06-28
CN109072203A (zh) 2018-12-21

Similar Documents

Publication Publication Date Title
WO2017028548A1 (zh) 镜像核酸复制体系
Halpin et al. DNA display III. Solid-phase organic synthesis on unprotected DNA
JP2021003134A (ja) 改善された発現のための、細菌抽出物中の選択タンパク質のタンパク質分解不活性化
US11905541B2 (en) Efficient product cleavage in template-free enzymatic synthesis of polynucleotides
Weidmann et al. Copying life: synthesis of an enzymatically active mirror-image DNA-ligase made of D-amino acids
AU2015248673A1 (en) Method for synthesising nucleic acids, in particular long nucleic acids, use of said method and kit for implementing said method
CN116199733A (zh) 融合蛋白合成的方法和产品
WO2016154675A1 (en) Platform for non-natural amino acid incorporation into proteins
CN110637086A (zh) Rna分子与肽的复合体的制造方法及其利用
EP2952582A1 (en) Flexible display method
CN110997922B (zh) 使用双链多联体dna的无细胞蛋白质表达
Lander et al. D‐peptide and d‐protein technology: recent advances, challenges, and opportunities
CN108795893B (zh) 一种氨基酸脱氢酶突变体及其制备方法和应用
JP2022543569A (ja) ポリ（ａ）およびポリ（ｕ）ポリメラーゼを使用するポリヌクレオチドの鋳型なしの酵素による合成
WO2013065827A1 (ja) 核酸リンカー
US20130189757A1 (en) Affinity purification of rna under native conditions based on the lambda boxb/n peptide interaction
WO2003062417A1 (fr) Produit de ligation arn-adn, et utilisation correspondante
Sachdeva et al. DNA-catalyzed reactivity of a phosphoramidate functional group and formation of an unusual pyrophosphoramidate linkage
CN116547380A (zh) 大型及镜像蛋白质的化学合成及其用途
CN116547382A (zh) 从动物细胞系和/或组织外植体生产、分离和/或提取胶原蛋白和/或明胶
WO2023143123A1 (zh) 可控合成单链dna的末端转移酶变体及应用
US20210380967A1 (en) Methods of Identifying Adenosine-to-Inosine Edited RNA
WO2006052016A1 (ja) タンパク質合成法、固相固定化ｍＲＮＡ及びタンパク質合成装置
CN117070514A (zh) 非天然rna的制备方法及产品

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16836413

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16836413

Country of ref document: EP

Kind code of ref document: A1