WO2020186991A1 - 利用环形rna进行蛋白翻译及其应用 - Google Patents

利用环形rna进行蛋白翻译及其应用 Download PDF

Info

Publication number
WO2020186991A1
WO2020186991A1 PCT/CN2020/077026 CN2020077026W WO2020186991A1 WO 2020186991 A1 WO2020186991 A1 WO 2020186991A1 CN 2020077026 W CN2020077026 W CN 2020077026W WO 2020186991 A1 WO2020186991 A1 WO 2020186991A1
Authority
WO
WIPO (PCT)
Prior art keywords
translation initiation
construct
sequence
protein
translation
Prior art date
Application number
PCT/CN2020/077026
Other languages
English (en)
French (fr)
Inventor
王泽峰
杨赟
樊晓娟
Original Assignee
中国科学院上海营养与健康研究所
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 中国科学院上海营养与健康研究所 filed Critical 中国科学院上海营养与健康研究所
Priority to EP20773279.3A priority Critical patent/EP3943601A4/en
Priority to JP2021559486A priority patent/JP7297331B2/ja
Priority to CN202080036818.2A priority patent/CN113825837A/zh
Priority to US17/440,774 priority patent/US20220177898A1/en
Priority to CN202410093532.8A priority patent/CN117965533A/zh
Publication of WO2020186991A1 publication Critical patent/WO2020186991A1/zh
Priority to JP2023094068A priority patent/JP2023116620A/ja

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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to the field of biotechnology, in particular to the use of circular RNA for protein translation and its application.
  • Common protein replacement or expression therapies include ribonucleic acid (DNA) carrier-based delivery systems, deoxyribonucleic acid (RNA) carrier-based delivery systems, and protein delivery systems. These methods all need to produce protein through messenger RNA translation.
  • the common translation initiation method in eukaryotes is cap-dependent translation, which mainly uses the translation initiation factor to recognize the special cap structure at the 5′ end of the messenger RNA to initiate translation. This type of translation method only exists in linear messenger RNA. .
  • cap-independent translation initiation which mainly initiates translation through the interaction between specific protein factors and RNA elements. This type of translation can be initiated in linear or circular RNA.
  • Common cap-independent translation initiation elements are some elements with specific secondary structure in viral RNA. They can borrow the host cell's translation system to express their desired protein.
  • the internal ribosome entry site (IRES) element contained in RNA such as encephalomyocarditis virus or hepatitis C virus.
  • Circular RNA is a type of single-stranded closed-loop RNA form that is different from linear RNA. Due to its structural specificity, it is not easily degraded by exonuclease, which is more stable than linear RNA. Therefore, the expression of protein through circular RNA translation has the characteristics of more continuous and long-term effect and is an important means to replace linear RNA translation.
  • a common method is to use viral IRES to initiate the translation of circular RNA.
  • pathogenic viral RNA may have immune rejection in the host, the RNA elements derived from viruses basically contain complex RNA secondary structures and long sequences , Which limits the construction of viral IRES-based expression systems and the later application of gene therapy.
  • the purpose of the present invention is to provide a cap-independent translation initiation element of non-viral origin.
  • the first aspect of the present invention provides a circular RNA construct, which has the structure shown in formula I from the 5'-3' direction:
  • TI is the translation initiation element
  • Z1 is an expression cassette for expressing foreign protein
  • Z2 is none or other components
  • each "-" is a bond or a nucleotide connection sequence
  • the length of the TI element is 6-30 nt, preferably, 8-24 nt, more preferably, 10-20 nt;
  • the content of A is ⁇ 35%, preferably, ⁇ 45%, more preferably, ⁇ 60%;
  • the content of T is ⁇ 20%, preferably, ⁇ 30%, more preferably, ⁇ 50%;
  • the content of A+T is ⁇ 65%, preferably, ⁇ 80%, more preferably, ⁇ 90%;
  • the content of G is ⁇ 35%, preferably, ⁇ 25%, more preferably, ⁇ 10%.
  • the circular RNA construct is a circular messenger RNA construct.
  • the content of A in the TI element is 35-100%, preferably, 45-100%, more preferably, 60-100%.
  • the content of T in the TI element is 20-100%, preferably, 30-100%, more preferably, 50-100%.
  • the content of A+T in the TI element is 65-100%, preferably, 80-100%, more preferably, 90-100%.
  • the content of G in the TI element is 0-35%, preferably, 0-25%, more preferably, 0-10%.
  • the TI element contains one or more nucleotide sequences selected from the following group shown in Table 1:
  • the TI element is added with 1-24 (preferably 1-15, more preferably 1-10) at the 5'end and/or 3'end of the nucleotide sequence shown in Table 1.
  • 1-24 preferably 1-15, more preferably 1-10
  • One, more preferably, 1-6 nucleotides, and has the function of TI element.
  • the coding sequence of the TI element is selected from the following group;
  • the TI element has a sequence shown in SEQ ID NO.: 1-40.
  • the coding sequence of the TI element is shown in SEQ ID NO.: 1-40.
  • the Z1 element contains a stop codon.
  • the Z1 element does not contain a stop codon.
  • the coding sequence of the foreign protein is from a prokaryote or eukaryote.
  • the coding sequence of the foreign protein is derived from animals, plants, and pathogens.
  • the coding sequence of the foreign protein is derived from mammals, preferably primates, rodents, including humans, mice, and rats.
  • the encoding sequence of the foreign protein is selected from the group consisting of encoding luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthesis Enzymes, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, foreign DNA of variable regions of antibodies, DNA of luciferase mutants, or a combination thereof.
  • the foreign protein is selected from the following group: luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable regions of antibodies, luciferase mutations, ⁇ -amylase, enterobacteria A, hepatitis C virus E2 glycoprotein, insulin precursor , Interferon ⁇ A, interleukin-1 ⁇ , lysozyme, serum albumin, single-chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
  • luciferin or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydr
  • the Z2 element is selected from the group consisting of PolyA, multiple cloning site, aptamer, miRNA binding site, translation enhancement element, or a combination thereof.
  • one or more adenines (A) of the TI element are methylated.
  • sequence of the circular RNA construct is shown in SEQ ID NO.: 61.
  • the second aspect of the present invention provides a vector containing the expression cassette of the construct according to the first aspect of the present invention.
  • the expression cassette contains a first intron and a second intron.
  • first intron and the second intron are completely complementary or not completely complementary.
  • the vector has the sequence shown in SEQ ID NO.: 62.
  • sequence of the first intron is shown in SEQ ID NO.: 63.
  • sequence of the second intron is shown in SEQ ID NO.: 64.
  • the third aspect of the present invention provides a genetically engineered cell in which the nucleic acid construct of the first aspect of the present invention is integrated at one or more sites of the genome of the genetically engineered cell, or the genetically engineered cell contains the present invention
  • the carrier described in the second aspect of the invention is not limited to any particular order.
  • the genetically engineered cells include prokaryotic cells and eukaryotic cells.
  • the eukaryotic cells include higher eukaryotic cells.
  • the genetically engineered cells are selected from the group consisting of human-derived cells (such as Hela cells), Chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, yeast cells, or combinations thereof.
  • the genetically engineered cell is a yeast cell.
  • the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces yeast, or a combination thereof.
  • the Kluyveromyces yeast is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobriella, or a combination thereof.
  • the fourth aspect of the present invention provides a reaction system, including:
  • reaction system further includes YTHDF3, PABPC1, and/or hnRNPA1 protein.
  • the reaction system is an in vitro reaction system.
  • the fifth aspect of the present invention provides a method for synthesizing protein in vitro, including the steps:
  • step (ii) Under suitable conditions, incubate the synthesis system of step (i) for a period of T1 to synthesize the protein.
  • the method further includes: (iii) optionally separating or detecting the protein from the in vitro reaction system.
  • the reaction temperature is 25-42°C, preferably, 30-40°C, more preferably, 35-37°C.
  • the reaction time T1 is 1 hour to 20 hours, preferably, 2 hours to 12 hours, more preferably, 3 hours to 6 hours.
  • the sixth aspect of the present invention provides a kit for in vitro protein synthesis, including:
  • the second container, and other components required for the reaction in the second container, the other components are selected from the group consisting of spliceosome, ribosome, translation initiation factor EIF4G2, translation initiation factor EIF4A , The translation initiation factor EIF4B, or a combination thereof; and
  • first container and the second container are the same container or different containers.
  • the kit further includes one or more containers optionally selected from the following group:
  • the seventh aspect of the present invention provides a construct according to the first aspect of the present invention, a vector according to the second aspect of the present invention, a genetically engineered cell according to the third aspect of the present invention, and the fourth aspect of the present invention
  • the reaction system or the use of the kit according to the sixth aspect of the present invention is used for high-throughput in vitro protein synthesis.
  • Figure 1 shows the different cell populations screened by flow cytometry.
  • Figure 2 shows the activity of western blot to detect the characteristic sequence of the translation initiation element.
  • Figure 3 shows the western blot detection of the activity of translation initiation elements produced by anti-learning.
  • the translation initiation element has high translation activity.
  • the translation enlightening element of the present invention was inserted into the circular RNA. Expression vectors can significantly enhance translation efficiency in vitro and in vivo. On this basis, the inventor completed the present invention.
  • the 3'end of the first intron contains a splice acceptor site, which contains a cis element (50bp-300bp in length) that is paired with the second intron.
  • the 5'end of the second intron contains a splice donor site, which contains a cis element (50bp-300bp in length) paired with the first intron.
  • the first aspect of the present invention provides a circular RNA construct, which has the structure shown in formula I from the 5'-3' direction:
  • TI is the translation initiation element
  • Z1 is an expression cassette for expressing foreign protein
  • Z2 is none or other components
  • each "-" is a bond or a nucleotide connection sequence
  • the length of the TI element is 6-30 nt, preferably, 8-24 nt, more preferably, 10-20 nt;
  • the content of A is ⁇ 35%, preferably, ⁇ 45%, more preferably, ⁇ 60%;
  • the content of T is ⁇ 20%, preferably, ⁇ 30%, more preferably, ⁇ 50%;
  • the content of A+T is ⁇ 65%, preferably, ⁇ 80%, more preferably, ⁇ 90%;
  • the content of G is ⁇ 35%, preferably, ⁇ 25%, more preferably, ⁇ 10%.
  • the content of A in the TI element is 35-100%, preferably, 45-100%, more preferably, 60-100%.
  • the content of T in the TI element is 20-100%, preferably, 30-100%, more preferably, 50-100%.
  • the content of A+T in the TI element is 65-100%, preferably, 80-100%, more preferably, 90-100%.
  • the content of G in the TI element is 0-35%, preferably, 0-25%, more preferably, 0-10%.
  • the selection of the encoding sequence of the foreign protein is not particularly limited.
  • the encoding sequence of the foreign protein is selected from the following group: encoding luciferin, or luciferase (such as firefly luciferase), green Fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody variable region foreign DNA, luciferase mutant DNA , Or a combination thereof.
  • the coding sequence of the foreign protein can also encode a protein selected from the group consisting of: ⁇ -amylase, enterobacteria A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon ⁇ A, interleukin-1 ⁇ , bacteriolysis Enzyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
  • ⁇ -amylase enterobacteria A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon ⁇ A interleukin-1 ⁇
  • bacteriolysis Enzyme bacteriolysis Enzyme
  • serum albumin serum albumin
  • scFV single chain antibody fragment
  • transthyretin tyrosinase
  • xylanase xylanase
  • nucleic acid construct of the present invention is circular.
  • nucleic acid construct of the present invention is single-stranded.
  • nucleic acid construct of the present invention is RNA.
  • sequence of the circular RNA construct of the present invention is shown in SEQ ID NO.: 61.
  • Circular RNA sequence using GFP as an example is an example.
  • sequence of the circular RNA precursor (containing the first intron and the second intron) taking GFP as an example is as follows:
  • the TI element of the present invention contains the nucleotide sequence shown in Table 1 selected from the following group:
  • the coding sequence of the TI element of the present invention is shown in SEQ ID NO.: 1-40.
  • the circular RNA construct of the present invention has high translation activity and can significantly enhance the translation efficiency in vivo or in vitro.
  • the present invention provides a reaction system, including:
  • reaction system further includes YTHDF3, PABPC1, and/or hnRNPA1 protein.
  • the reaction system may be in vitro or in vivo.
  • the present invention provides a kit for in vitro protein synthesis, including:
  • the second container, and other components required for the reaction in the second container, the other components are selected from the group consisting of spliceosome, ribosome, translation initiation factor EIF4G2, translation initiation factor EIF4A , The translation initiation factor EIF4B, or a combination thereof; and
  • first container and the second container are the same container or different containers.
  • coding sequence of foreign protein and “foreign DNA” are used interchangeably, and both refer to exogenous DNA molecules used to direct protein synthesis.
  • the DNA molecule is linear or circular.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include (but not limited to): genomic sequence, cDNA sequence.
  • the sequence encoding the foreign protein also contains a promoter sequence, a 5'untranslated sequence, and a 3'untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the following group: encoding luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein , Aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA in the variable region of an antibody, luciferase mutant DNA, or a combination thereof.
  • the exogenous DNA can also be selected from the following group: encoding ⁇ -amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon ⁇ A, interleukin-1 ⁇ , lysozyme, serum white Exogenous DNA of protein, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.
  • the foreign DNA encodes a protein selected from the group consisting of green fluorescent protein (enhanced GFP, eGFP), yellow fluorescent protein (YFP), and E. coli ⁇ -galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase (Lysine-tRNA synthetase), human leucine-tRNA synthetase (Leucine-tRNA synthetase), Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate) dehydrogenase), mouse catalase, or a combination thereof.
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • YFP yellow fluorescent protein
  • E. coli ⁇ -galactosidase ⁇ -galactosidase, LacZ
  • human lysine-tRNA synthetase Lysine-t
  • the present invention provides an in vitro protein synthesis method, including the steps:
  • step (ii) Under suitable conditions, incubate the synthesis system of step (i) for a period of T1 to synthesize the protein.
  • the method further includes: (iii) optionally separating or detecting the protein from the reaction system.
  • the present invention has developed a set of methods for designing and synthesizing new artificially synthesized eukaryotic translation initiation elements of non-viral origin with high translation activity and controllable sequence structure and length. Drive the translation of circular RNA.
  • the present invention screens for the first time a specific translation initiation element, which is very short, only 6-30 nt, but has high translation activity, and inserts the translation initiation element into circular RNA expression
  • the carrier can significantly enhance translation efficiency both in vivo and in vitro.
  • the circular RNA reporter gene can be expressed under the drive of the translation initiation element to produce green fluorescent protein) to construct a library containing millions of different sequences, through Cell transfection and flow cytometry screen different cell populations (negative: no green fluorescence, positive: green fluorescence with different intensities). Perform amplicon sequencing on the collected different cell populations and analyze the sequence information contained in negative and different positive cells in combination with computational biology analysis, and extract sequence features of different lengths from the sequence information;
  • a high-throughput screening system based on circular RNA separates cell populations with different green fluorescence intensities (positive) and cell populations without fluorescence (negative).
  • results are shown in Table 1 and Figure 1.
  • the results show that the circular RNA system can express green fluorescent protein and be used for screening. At the same time, the system can isolate cell populations with different fluorescence intensities, indicating that different inserted sequences in the library have a differential effect on the translation initiation of circular RNA.
  • Table 1 Sequence features of translation initiation elements contained in cell populations with high green fluorescence signals (the first 100 sequence features displayed in the form of 6 bases)
  • Translation initiation elements (take 12 bases as an example) produced by anti-learning based on the characteristic sequences of positive and negative cell populations. List the top 20 sequences with different translational activity strengths.
  • the highly active translation initiation elements are basically AT-enriched sequences, as shown in Table 2.
  • the results show that the anti-learning method of the present invention can effectively infer the activity of translation initiation elements, and the method can be used to generate translation initiation elements of different lengths and strengths, and the translation initiation elements of the present invention have high translation activity and can Significantly enhance translation efficiency.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种环形RNA构建物及其应用，所述环形RNA构建物从5'-3'方向具有式I所示的结构：TI-Z1-Z2(I)，式中，TI为翻译起始元件；Z1为表达外源蛋白的表达盒；Z2为无或其他元件。

Description

利用环形RNA进行蛋白翻译及其应用 技术领域
本发明涉及生物技术领域，具体地，涉及利用环形RNA进行蛋白翻译及其应用。
背景技术
蛋白质是生物体内最重要的生物大分子，其突变或者表达异常都会导致疾病发生。因而，通过蛋白质替代或者表达可以治疗相应的疾病。常见的蛋白质替代或者表达疗法包括基于核糖核酸(DNA)载体的递送系统、基于脱氧核糖核酸(RNA)载体的递送系统以及蛋白质递送系统。这些方法都需要通过信使RNA翻译产生蛋白质。在真核生物体内常见的翻译起始方式是帽依赖性翻译，其主要通过翻译起始因子识别信使RNA 5′端的特殊帽结构来起始翻译，该类翻译方式只存在于线性信使RNA中存在。此外，还存在着一类非帽依赖性的翻译起始，其主要通过特定蛋白因子与RNA元件间的互作启动翻译。这类翻译可以在线性或者环形RNA中起始翻译。常见的非帽依赖性翻译起始元件是病毒RNA中一些具有特定二级结构的元件，它们可以借用宿主细胞的翻译系统表达自己所需的蛋白质。例如，脑心肌炎病毒或者丙型肝炎病毒等RNA中含有的内部核糖体进入位点(Internal Ribosome Entry Site：IRES)元件。
环形RNA是一类不同于线性RNA的单链闭环RNA形式，因其结构特异性导致其不易被核酸外切酶降解，顾其相较于线性RNA更稳定。所以，通过环形RNA翻译表达蛋白具有更加持续长效的特点，是替代线性RNA翻译的一种重要手段。但因环状RNA的翻译只能利用非帽依赖性翻译的反式，如何设计并选择合适的非帽翻译起始元件是该应用的关键技术。一种常见的手段是利用病毒IRES启动环形RNA的翻译，但由于致病病毒RNA在宿主体内可能存在免疫排斥反应，同时病毒来源的RNA元件基本都含有复杂的RNA二级结构而且较长的序列，限制了基于病毒IRES的表达系统构建及后期的基因治疗应用。
因此，本领域迫切需要开发一种非病毒来源的非帽依赖性翻译起始元件。
发明内容
本发明的目的在于提供一种非病毒来源的非帽依赖性翻译起始元件。
本发明第一方面提供了一种环形RNA构建物，所述环形RNA构建物从5’-3’方向具有式I所示的结构：
TI-Z1-Z2   (I)
式中，
TI为翻译起始元件；
Z1为表达外源蛋白的表达盒；
Z2为无或其他元件；
并且，各“-”为键或核苷酸连接序列；
其中，TI元件的长度为6-30nt，较佳地，8-24nt，更佳地，10-20nt；
在TI元件中，A的含量≥35％，较佳地，≥45％，更佳地，≥60％；
在TI元件中，T的含量≥20％，较佳地，≥30％，更佳地，≥50％；
在TI元件中，A+T的含量≥65％，较佳地，≥80％，更佳地，≥90％；
在TI元件中，G的含量≤35％，较佳地，≤25％，更佳地，≤10％。
在另一优选例中，所述环形RNA构建物为环形信使RNA构建物。
在另一优选例中，所述TI元件中，A的含量为35-100％，较佳地，45-100％，更佳地，60-100％。
在另一优选例中，所述TI元件中，T的含量为20-100％，较佳地，30-100％，更佳地，50-100％。
在另一优选例中，所述TI元件中，A+T的含量为65-100％，较佳地，80-100％，更佳地，90-100％。
在另一优选例中，所述TI元件中，G的含量为0-35％，较佳地，0-25％，更佳地，0-10％。
在另一优选例中，所述TI元件含有表1所示的选自下组的一种或多种核苷酸序列：
表1
1 AATATA 11 ATAAAT 21 AAAATT 31 TAAATA 41 AATAAG 51 TATACT
2 AAAATA 12 ATATTA 22 AAATAC 32 TTATAA 42 AAATTC 52 AGATAT
3 AAATAT 13 AATAAT 23 TAATAT 33 AGAAGA 43 GAGATA 53 TCAAGC
4 AAATAA 14 TATATA 24 CATATA 34 ATTATA 44 CAAAAA 54 AAGAAT
5 AATAAA 15 ATAATA 25 ATTAAT 35 TATAAA 45 AATTTA 55 AAACAT
6 ATATAA 16 AATTAA 26 ACATAT 36 AACATA 46 AAGATA 56 ATTATT
7 AAAAAA 17 ATAAAA 27 AATACA 37 TTAATA 47 AACATT 57 ACAAAA
8 AAATTA 18 TATAAT 28 ATACAA 38 TATATT 48 ATTAGG 58 AAAAGA
9 ATATAT 19 ATAAGA 29 AATTAT 39 AATACT 49 ACATAA 59 AATCAA
10 AAAAAT 20 AATATT 30 ATATAG 40 ATATAC 50 GAAGAA 60 AAAGAC
61 TAAGAA 71 GGAGAT 81 TAAACA 91 TATACA
62 ATAAAC 72 TAATCT 82 CGAAAC 92 GACATA
63 TAGATT 73 TAAAAA 83 TATTAA 93 TGAATA
64 ATAAAG 74 AAATCC 84 AATAGA 94 TAAGAC
65 AATATC 75 ATCAAG 85 AATTCA 95 AACTGA
66 TAATAA 76 ATACTG 86 ATAAGT 96 TTATAT
67 ATTCGA 77 CATTAG 87 AAACAA 97 TTTAAA
68 TATTTT 78 TGACAT 88 ATACTA 98 TAATAG
69 TAATTA 79 ATTTAA 89 ATATCT 99 AAATAG
70 TATATG 80 AGATTA 90 AAGAAG 100 AACAAA。
在另一优选例中，所述TI元件在表1所示的核苷酸序列的5'端和/或3'端添加1-24个(较佳地1-15，更佳地1-10个，更佳地，1－6个)核苷酸，且具有TI元件的功能。
在另一优选例中，所述TI元件的编码序列选自下组；
(a)序列如SEQ ID NO.:1－40所示的多核苷酸；
(b)核苷酸序列与SEQ ID NO.:1－40所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％或≥98％或≥99％)的多核苷酸；
(c)在SEQ ID NO.:1-40所示多核苷酸的5'端和/或3'端截短或添加1-18个(较佳地1-10，更佳地1-6个)核苷酸的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述TI元件具有如SEQ ID NO.:1-40所示的序列。
在另一优选例中，所述TI元件的编码序列如SEQ ID NO.:1-40所示。
在另一优选例中，所述Z1元件含有终止密码子。
在另一优选例中，所述Z1元件不含有终止密码子。
在另一优选例中，所述外源蛋白的编码序列来自原核生物、真核生物。
在另一优选例中，所述外源蛋白的编码序列来自动物、植物、病原体。
在另一优选例中，所述外源蛋白的编码序列来自哺乳动物，较佳地灵长动物，啮齿动物，包括人、小鼠、大鼠。
在另一优选例中，所述的外源蛋白的编码序列选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
在另一优选例中，所述外源蛋白选自下组：荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域、萤光素酶突变、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
在另一优选例中，所述Z2元件选自下组：PolyA、多克隆位点、适配体、miRNA结合位点、翻译增强元件、或其组合。
在另一优选例中，所述TI元件的一个或多个腺嘌呤(A)被甲基化。
在另一优选例中，所述环形RNA的构建物的序列如SEQ ID NO.:61所示。
本发明第二方面提供了一种载体，所述载体含有本发明第一方面所述的构建物的表达盒。
在另一优选例中，所述表达盒含有第一内含子和第二内含子。
在另一优选例中，所述第一内含子和第二内含子完全互补或不完全互补。
在另一优选例中，所述载体具有SEQ ID NO.:62所示的序列。
在另一优选例中，所述第一内含子的序列如SEQ ID NO.:63所示。
在另一优选例中，所述第二内含子的序列如SEQ ID NO.:64所示。
本发明第三方面提供了一种基因工程细胞，所述基因工程细胞的基因组的一个或多个位点整合有本发明第一方面所述的核酸构建物，或者所述基因工程细胞中含有本发明第二方面所述的载体。
在另一优选例中，所述基因工程细胞包括原核细胞、真核细胞。
在另一优选例中，所述真核细胞包括高等真核细胞。
在另一优选例中，所述基因工程细胞选自下组：人源细胞(如Hela细胞)、 中国仓鼠卵巢细胞、昆虫细胞、麦胚细胞、兔网织红细胞、酵母细胞、或其组合。
在另一优选例中，所述基因工程细胞为酵母细胞。
在另一优选例中，所述酵母细胞选自下组：酿酒酵母、克鲁维酵母属酵母、或其组合。
在另一优选例中，所述克鲁维酵母属酵母选自下组：乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母、或其组合。
本发明第四方面提供了一种反应体系，包括：
(a)本发明第一方面所述的构建物；
(b)反应所需的其他组分，所述其他组分选自下组：剪接小体、核糖体、翻译起始因子EIF4G2、翻译起始因子EIF4A、翻译起始因子EIF4B、或其组合。
在另一优选例中，所述反应体系还包括YTHDF3、PABPC1、和/或hnRNPA1蛋白。
在另一优选例中，所述反应体系为体外反应体系。
本发明第五方面提供了一种体外合成蛋白质的方法，包括步骤：
(i)提供本发明第四方面所述的反应体系；
(ii)在适合的条件下，孵育步骤(i)的合成体系一段时间T1，从而合成所述的蛋白质。
另一优选例中，所述的方法还包括：(iii)任选地从所述体外反应体系中，分离或检测所述的蛋白质。
在另一优选例中，所述步骤(ii)中，反应温度为25－42℃，较佳地，30－40℃，更佳地，35－37℃。
在另一优选例中，所述步骤(ii)中，反应时间T1为1小时－20小时，较佳地，2小时－12小时，更佳地，3小时－6小时。
本发明第六方面提供了一种用于体外蛋白合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的本发明第一方面所述的构建物；
(k2)第二容器，以及位于第二容器内的反应所需的其他组分，所述其他组分选自下组：剪接小体、核糖体、翻译起始因子EIF4G2、翻译起始因子EIF4A、翻译起始因子EIF4B、或其组合；和
(kt)标签或说明书。
在另一优选例中，所述的第一容器、第二容器是同一容器或不同容器。
在另一优选例中，所述试剂盒还包括任选的选自下组的一个或多个容器：
(k3)第三容器，以及位于第三容器内的YTHDF3、PABPC1、和/或hnRNPA1蛋白。
本发明第七方面提供了一种本发明第一方面所述的构建物、本发明第二方面所述的载体、本发明第三方面所述的基因工程细胞、本发明第四方面所述的反应体系、或本发明第六方面所述的试剂盒的用途，用于进行高通量的体外蛋白合成。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了流式细胞仪筛选获得的不同细胞群体。
图2显示了western blot检测翻译起始元件特征序列的活性。
图3显示了western blot检测对抗学习产生的翻译起始元件的活性。
具体实施方式
经过广泛而深入的研究，通过大量筛选和摸索，首次意外地筛到一种特定的翻译起始元件，该翻译起始元件具有很高的翻译活性，将本发明的该翻译启示元件插入环形RNA表达载体，在体内体外均可显著增强翻译效率。在此基础上，本发明人完成了本发明。
第一内含子
在本发明中，第一内含子3'端含有一个剪接受体位点，内部包含一段与第二内含子配对的顺式元件(长度50bp-300bp)。
序列如下所示：
Figure PCTCN2020077026-appb-000001
Figure PCTCN2020077026-appb-000002
第二内含子
在本发明中，第二内含子5'端含有一个剪接供体位点，内部包含一段与第一内含子配对的顺式元件(长度50bp-300bp)。
序列如下所示：
Figure PCTCN2020077026-appb-000003
环形RNA构建物
本发明第一方面提供了一种环形RNA构建物，所述环形RNA构建物从5’-3’方向具有式I所示的结构：
TI-Z1-Z2  (I)
式中，
TI为翻译起始元件；
Z1为表达外源蛋白的表达盒；
Z2为无或其他元件；
并且，各“-”为键或核苷酸连接序列；
其中，TI元件的长度为6-30nt，较佳地，8-24nt，更佳地，10-20nt；
在TI元件中，A的含量≥35％，较佳地，≥45％，更佳地，≥60％；
在TI元件中，T的含量≥20％，较佳地，≥30％，更佳地，≥50％；
在TI元件中，A+T的含量≥65％，较佳地，≥80％，更佳地，≥90％；
在TI元件中，G的含量≤35％，较佳地，≤25％，更佳地，≤10％。
在一优选实施方式中，所述TI元件中，A的含量为35-100％，较佳地，45-100％，更佳地，60-100％。
在一优选实施方式中，所述TI元件中，T的含量为20-100％，较佳地，30-100％，更佳地，50-100％。
在一优选实施方式中，所述TI元件中，A+T的含量为65-100％，较佳地，80-100％，更佳地，90-100％。
在一优选实施方式中，所述TI元件中，G的含量为0-35％，较佳地，0-25％，更佳地，0-10％。
在本发明中，所述外源蛋白的编码序列的选择没有特别限制，通常，外源蛋白的编码序列选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源蛋白的编码序列还可以编码选自下组的蛋白：α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、或其组合。
此外，本发明的所述核酸构建物是环状的。本发明的所述核酸构建物是单链的。本发明的所述核酸构建物是RNA。
在一优选实施方式中，本发明的环形RNA构建物的序列如SEQ ID NO.:61所示。
以GFP为例的环形RNA序列：
Figure PCTCN2020077026-appb-000004
Figure PCTCN2020077026-appb-000005
在一优选实施方式中，以GFP为例的环形RNA前体(含有第一内含子、第二内含子)的序列如下所示：
Figure PCTCN2020077026-appb-000006
Figure PCTCN2020077026-appb-000007
在一优选实施方式中，本发明的TI元件含有表1所示的选自下组的核苷酸序列：
表1
1 AATATA 11 ATAAAT 21 AAAATT 31 TAAATA 41 AATAAG 51 TATACT
2 AAAATA 12 ATATTA 22 AAATAC 32 TTATAA 42 AAATTC 52 AGATAT
3 AAATAT 13 AATAAT 23 TAATAT 33 AGAAGA 43 GAGATA 53 TCAAGC
4 AAATAA 14 TATATA 24 CATATA 34 ATTATA 44 CAAAAA 54 AAGAAT
5 AATAAA 15 ATAATA 25 ATTAAT 35 TATAAA 45 AATTTA 55 AAACAT
6 ATATAA 16 AATTAA 26 ACATAT 36 AACATA 46 AAGATA 56 ATTATT
7 AAAAAA 17 ATAAAA 27 AATACA 37 TTAATA 47 AACATT 57 ACAAAA
8 AAATTA 18 TATAAT 28 ATACAA 38 TATATT 48 ATTAGG 58 AAAAGA
9 ATATAT 19 ATAAGA 29 AATTAT 39 AATACT 49 ACATAA 59 AATCAA
10 AAAAAT 20 AATATT 30 ATATAG 40 ATATAC 50 GAAGAA 60 AAAGAC
61 TAAGAA 71 GGAGAT 81 TAAACA 91 TATACA
62 ATAAAC 72 TAATCT 82 CGAAAC 92 GACATA
63 TAGATT 73 TAAAAA 83 TATTAA 93 TGAATA
64 ATAAAG 74 AAATCC 84 AATAGA 94 TAAGAC
65 AATATC 75 ATCAAG 85 AATTCA 95 AACTGA
66 TAATAA 76 ATACTG 86 ATAAGT 96 TTATAT
67 ATTCGA 77 CATTAG 87 AAACAA 97 TTTAAA
68 TATTTT 78 TGACAT 88 ATACTA 98 TAATAG
69 TAATTA 79 ATTTAA 89 ATATCT 99 AAATAG
70 TATATG 80 AGATTA 90 AAGAAG 100 AACAAA
在一优选实施方式中，本发明的TI元件的编码序列如SEQ ID NO.:1-40所示。
在本发明中，应用本发明的环形RNA构建物具有很高的翻译活性，可显著增强体内或体外的翻译效率。
反应体系
本发明提供了一种反应体系，包括：
(a)本发明第一方面所述的构建物；
(b)反应所需的其他组分，所述其他组分选自下组：剪接小体、核糖体、翻译起始因子EIF4G2、翻译起始因子EIF4A、翻译起始因子EIF4B、或其组合。
在另一优选例中，所述反应体系还包括YTHDF3、PABPC1、和/或hnRNPA1蛋白。
在本发明中，反应体系可以在体外也可以在体内。
试剂盒
本发明提供了用于体外蛋白合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的权利要求1所述的核酸构建物；
(k2)第二容器，以及位于第二容器内的反应所需的其他组分，所述其他组分选自下组：剪接小体、核糖体、翻译起始因子EIF4G2、翻译起始因子EIF4A、翻译起始因子EIF4B、或其组合；和
(kt)标签或说明书。
在一优选实施方式中，所述的第一容器、第二容器是同一容器或不同容器。
外源蛋白的编码序列(外源DNA)
如本文所用，术语“外源蛋白的编码序列”与“外源DNA”可互换使用，均指外源的用于指导蛋白质合成的DNA分子。通常，所述的DNA分子为线性的或环状的。所述的DNA分子含有编码外源蛋白的序列。
在本发明中，所述的编码外源蛋白的序列的例子包括(但并不限于)：基因组序列、cDNA序列。所述的编码外源蛋白的序列还含有启动子序列、5’非翻译序列、 3’非翻译序列。
在本发明中，所述外源DNA的选择没有特别限制，通常，外源DNA选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源DNA还可以选自下组：编码α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶的外源DNA、或其组合。
在一优选实施方式中，所述外源DNA编码选自下组的蛋白：绿色荧光蛋白(enhanced GFP,eGFP)、黄色荧光蛋白(YFP)、大肠杆菌β-半乳糖苷酶(β-galactosidase,LacZ)、人赖氨酸-tRNA合成酶(Lysine-tRNA synthetase)、人亮氨酸-tRNA合成酶(Leucine-tRNA synthetase)、拟南芥甘油醛3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase)、鼠过氧化氢酶(Catalase)、或其组合。
体外蛋白合成方法
本发明提供了一种体外蛋白合成方法，包括步骤：
(i)提供本发明第二方面所述的反应体系；
(ii)在适合的条件下，孵育步骤(i)的合成体系一段时间T1，从而合成所述的蛋白质。
另一优选例中，所述的方法还包括：(iii)任选地从所述反应体系中，分离或检测所述的蛋白质。
本发明的主要优点包括：
(1)本发明首次开发了一套用以设计合成具有高翻译活性同时序列结构和长度可控的非病毒来源的新型人工合成的真核生物翻译起始元件的方法，并以此翻译起始元件驱动环形RNA的翻译。
(2)本发明首次筛选到一种特定的翻译起始元件，该翻译起始元件非常短，只有6-30nt，但是具有很高的翻译活性，并且将该翻译起始元件插入到环形RNA表达载体，在体内体外均可显著增强翻译效率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。
外源蛋白以GFP为例。
实验方法
1.基于本发明特有的一种环状RNA报告基因系统(该环状RNA报告基因可以在翻译起始元件的驱动下表达产生绿色荧光蛋白)构建一套含有百万个不同序列的文库，通过细胞转染及流式细胞仪筛选不同的细胞群体(阴性：没有绿色荧光，阳性：具有不同强度的绿色荧光)。将收集的不同细胞群体进行扩增子测序并结合计算生物学分析解析阴性和不同阳性细胞内包含的序列信息，根据这些序列信息从中提取不同长度的序列特征；
2.以这些阴性和阳性序列特征为基础，通过对抗神经网络学习生成非病毒来源的翻译起始元件模块；
3.将生成的翻译起始元件模块及模块组合元件插入环状RNA表达载体，通过细胞转染和western blot检测翻译起始元件的活性。
实验结果
1.基于环状RNA的高通量筛选系统分离出含有不同绿色荧光强度的细胞群体(阳性)及没有荧光的细胞群体(阴性)。
结果如表1和图1所示。结果表明，环形RNA系统可以表达绿色荧光蛋白并用于筛选。同时，该系统可以分离出具有不同荧光强度的细胞群体，说明文库中不同的插入序列对环形RNA的翻译起始具有差异化的效果。
2.通过筛选获得的不同细胞群体的扩增子测序数据的计算生物学分析挖掘获得的高绿色荧光细胞群体包含的序列特征，这些序列通常都含有大量AU碱基较少包含易形成RNA二级结构的GC碱基，表明该类翻译起始元件的核心模块不依赖于结构同时也表明其与病毒IRES具有明显差异，是一类新的可促进环形RNA的非帽依赖翻译的元件，如表1所示。
表1具有高绿色荧光信号的细胞群体所含翻译起始元件的序列特征(以6个碱基形式展示的前100个序列特征)
1 AATATA 11 ATAAAT 21 AAAATT 31 TAAATA 41 AATAAG 51 TATACT
2 AAAATA 12 ATATTA 22 AAATAC 32 TTATAA 42 AAATTC 52 AGATAT
3 AAATAT 13 AATAAT 23 TAATAT 33 AGAAGA 43 GAGATA 53 TCAAGC
4 AAATAA 14 TATATA 24 CATATA 34 ATTATA 44 CAAAAA 54 AAGAAT
5 AATAAA 15 ATAATA 25 ATTAAT 35 TATAAA 45 AATTTA 55 AAACAT
6 ATATAA 16 AATTAA 26 ACATAT 36 AACATA 46 AAGATA 56 ATTATT
7 AAAAAA 17 ATAAAA 27 AATACA 37 TTAATA 47 AACATT 57 ACAAAA
8 AAATTA 18 TATAAT 28 ATACAA 38 TATATT 48 ATTAGG 58 AAAAGA
9 ATATAT 19 ATAAGA 29 AATTAT 39 AATACT 49 ACATAA 59 AATCAA
10 AAAAAT 20 AATATT 30 ATATAG 40 ATATAC 50 GAAGAA 60 AAAGAC
61 TAAGAA 71 GGAGAT 81 TAAACA 91 TATACA
62 ATAAAC 72 TAATCT 82 CGAAAC 92 GACATA
63 TAGATT 73 TAAAAA 83 TATTAA 93 TGAATA
64 ATAAAG 74 AAATCC 84 AATAGA 94 TAAGAC
65 AATATC 75 ATCAAG 85 AATTCA 95 AACTGA
66 TAATAA 76 ATACTG 86 ATAAGT 96 TTATAT
67 ATTCGA 77 CATTAG 87 AAACAA 97 TTTAAA
68 TATTTT 78 TGACAT 88 ATACTA 98 TAATAG
69 TAATTA 79 ATTTAA 89 ATATCT 99 AAATAG
70 TATATG 80 AGATTA 90 AAGAAG 100 AACAAA
3.通过细胞转染和western blot检测翻译起始元件基础特征序列的翻译活性(6个碱基的序列特征)，结果如图2所示。结果表明，这些翻译元件均具有翻译起始功能，但不同序列具有的翻译起始能力有差异。这表明基于这些不同的序列特征组合将可能可以得到具有不同活性的翻译起始元件。
4.基于阳性和阴性细胞群体特征序列的对抗学习产生的翻译起始元件(以12个碱基为例)。分别列出具有不同翻译活性强度的前20个序列。高活性的翻译起 始元件基本为AT富集序列，如表2所示。
表2对抗学习产生的翻译起始元件(12个碱基为例)
Figure PCTCN2020077026-appb-000008
5.将对抗学习得到的不同强度翻译起始元件插入环形RNA表达载体，利 用细胞转染和western blot检测其翻译活性。结果如图3所示。结果显示，表2中的强活性元件能够翻译产生更多的GFP蛋白，中等和弱活性元件也有一定的翻译效率，但是比强活性元件的翻译效率低。
结果表明，本发明的对抗学习方法可以有效推测翻译起始元件的活性，可以利用该方法产生不同长度和强度的翻译起始元件，并且本发明的翻译起始元件具有很高的翻译活性，能够显著增强翻译效率。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种环形RNA构建物，其特征在于，所述环形RNA构建物从5’-3’方向具有式I所示的结构：
    TI-Z1-Z2    (I)
    式中，
    TI为翻译起始元件；
    Z1为表达外源蛋白的表达盒；
    Z2为无或其他元件；
    并且，各“-”为键或核苷酸连接序列；
    其中，TI元件的长度为6-30nt，较佳地，8-24nt，更佳地，10-20nt；
    在TI元件中，A的含量≥35％，较佳地，≥45％，更佳地，≥60％；
    在TI元件中，T的含量≥20％，较佳地，≥30％，更佳地，≥50％；
    在TI元件中，A+T的含量≥65％，较佳地，≥80％，更佳地，≥90％；
    在TI元件中，G的含量≤35％，较佳地，≤25％，更佳地，≤10％。
  2. 如权利要求1所述的构建物，其特征在于，所述TI元件含有表1所示的选自下组的一种或多种核苷酸序列：
    表1
    1 AATATA 11 ATAAAT 21 AAAATT 31 TAAATA 41 AATAAG 51 TATACT 2 AAAATA 12 ATATTA 22 AAATAC 32 TTATAA 42 AAATTC 52 AGATAT 3 AAATAT 13 AATAAT 23 TAATAT 33 AGAAGA 43 GAGATA 53 TCAAGC 4 AAATAA 14 TATATA 24 CATATA 34 ATTATA 44 CAAAAA 54 AAGAAT 5 AATAAA 15 ATAATA 25 ATTAAT 35 TATAAA 45 AATTTA 55 AAACAT 6 ATATAA 16 AATTAA 26 ACATAT 36 AACATA 46 AAGATA 56 ATTATT 7 AAAAAA 17 ATAAAA 27 AATACA 37 TTAATA 47 AACATT 57 ACAAAA 8 AAATTA 18 TATAAT 28 ATACAA 38 TATATT 48 ATTAGG 58 AAAAGA 9 ATATAT 19 ATAAGA 29 AATTAT 39 AATACT 49 ACATAA 59 AATCAA 10 AAAAAT 20 AATATT 30 ATATAG 40 ATATAC 50 GAAGAA 60 AAAGAC
    61 TAAGAA 71 GGAGAT 81 TAAACA 91 TATACA 62 ATAAAC 72 TAATCT 82 CGAAAC 92 GACATA
    63 TAGATT 73 TAAAAA 83 TATTAA 93 TGAATA 64 ATAAAG 74 AAATCC 84 AATAGA 94 TAAGAC 65 AATATC 75 ATCAAG 85 AATTCA 95 AACTGA 66 TAATAA 76 ATACTG 86 ATAAGT 96 TTATAT 67 ATTCGA 77 CATTAG 87 AAACAA 97 TTTAAA 68 TATTTT 78 TGACAT 88 ATACTA 98 TAATAG 69 TAATTA 79 ATTTAA 89 ATATCT 99 AAATAG 70 TATATG 80 AGATTA 90 AAGAAG 100 AACAAA。
  3. 如权利要求1所述的构建物，其特征在于，所述TI元件的编码序列选自下组；
    (a)序列如SEQ ID NO.:1－40所示的多核苷酸；
    (b)核苷酸序列与SEQ ID NO.:1－40所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％或≥98％或≥99％)的多核苷酸；
    (c)在SEQ ID NO.:1-40所示多核苷酸的5'端和/或3'端截短或添加1-18个(较佳地1-10，更佳地1-6个)核苷酸的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
  4. 如权利要求1所述的构建物，其特征在于，所述TI元件的一个或多个腺嘌呤(A)被甲基化。
  5. 一种载体，其特征在于，所述载体含有权利要求1所述的构建物的表达盒。
  6. 一种基因工程细胞，其特征在于，所述基因工程细胞的基因组的一个或多个位点整合有权利要求1所述的构建物，或者所述基因工程细胞中含有权利要求5所述的载体。
  7. 一种反应体系，其特征在于，包括：
    (a)权利要求1所述的构建物；
    (b)反应所需的其他组分，所述其他组分选自下组：剪接小体、核糖体、翻译起始因子EIF4G2、翻译起始因子EIF4A、翻译起始因子EIF4B、或其组合。
  8. 一种体外合成蛋白质的方法，其特征在于，包括步骤：
    (i)提供权利要求7所述的反应体系；
    (ii)在适合的条件下，孵育步骤(i)的合成体系一段时间T1，从而合成所述的蛋白质。
  9. 一种用于体外蛋白合成的试剂盒，其特征在于，包括：
    (k1)第一容器，以及位于第一容器内的权利要求1所述的构建物；
    (k2)第二容器，以及位于第二容器内的反应所需的其他组分，所述其他组分选自下组：剪接小体、核糖体、翻译起始因子EIF4G2、翻译起始因子EIF4A、翻译起始因子EIF4B、或其组合；和
    (kt)标签或说明书。
  10. 一种权利要求1所述的构建物、权利要求5所述的载体、权利要求6所述的基因工程细胞、权利要求7所述的反应体系、或权利要求9所述的试剂盒的用途，其特征在于，用于进行高通量的体外蛋白合成。
PCT/CN2020/077026 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用 WO2020186991A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP20773279.3A EP3943601A4 (en) 2019-03-20 2020-02-27 PROTEIN TRANSLATION USING CIRCULAR RNA AND ITS APPLICATION
JP2021559486A JP7297331B2 (ja) 2019-03-20 2020-02-27 環状ｒｎａを使用したタンパク質翻訳およびその応用
CN202080036818.2A CN113825837A (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用
US17/440,774 US20220177898A1 (en) 2019-03-20 2020-02-27 Protein translation using circular rna and application thereof
CN202410093532.8A CN117965533A (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用
JP2023094068A JP2023116620A (ja) 2019-03-20 2023-06-07 環状ｒｎａを使用したタンパク質翻訳およびその応用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910214234.9A CN111718929B (zh) 2019-03-20 2019-03-20 利用环形rna进行蛋白翻译及其应用
CN201910214234.9 2019-03-20

Publications (1)

Publication Number Publication Date
WO2020186991A1 true WO2020186991A1 (zh) 2020-09-24

Family

ID=72519626

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/077026 WO2020186991A1 (zh) 2019-03-20 2020-02-27 利用环形rna进行蛋白翻译及其应用

Country Status (5)

Country Link
US (1) US20220177898A1 (zh)
EP (1) EP3943601A4 (zh)
JP (2) JP7297331B2 (zh)
CN (4) CN115806976A (zh)
WO (1) WO2020186991A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112608946B (zh) * 2021-01-17 2023-07-28 楷拓生物科技(苏州)有限公司 一种环状rna载体及其在疫苗中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012177639A2 (en) * 2011-06-22 2012-12-27 Alnylam Pharmaceuticals, Inc. Bioprocessing and bioproduction using avian cell lines
CN105985978A (zh) * 2015-03-06 2016-10-05 中国科学院上海生命科学研究院 一种新型rna环化表达载体的构建及其应用
CN109423496A (zh) * 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 一种细胞中内源性表达rna聚合酶的核酸构建物
CN109423497A (zh) * 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 增强蛋白质合成效率的rna元件
CN109971775A (zh) * 2017-12-27 2019-07-05 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT1282697E (pt) * 2000-03-28 2008-02-01 Univ Queensland Construção capaz de libertação na forma circular fechada a partir de uma grande sequência nucleotídica permitindo a expressão específica do local e/ou a expressão regulada durante o desenvolvimento de sequências genéticas seleccionadas
CN111819185A (zh) * 2017-12-15 2020-10-23 旗舰创业创新第六有限责任公司 包含环状多核糖核苷酸的组合物及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012177639A2 (en) * 2011-06-22 2012-12-27 Alnylam Pharmaceuticals, Inc. Bioprocessing and bioproduction using avian cell lines
CN105985978A (zh) * 2015-03-06 2016-10-05 中国科学院上海生命科学研究院 一种新型rna环化表达载体的构建及其应用
CN109423496A (zh) * 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 一种细胞中内源性表达rna聚合酶的核酸构建物
CN109423497A (zh) * 2017-08-31 2019-03-05 康码（上海）生物科技有限公司 增强蛋白质合成效率的rna元件
CN109971775A (zh) * 2017-12-27 2019-07-05 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
XU HAI-DONG;LENG QI-YING;PATRICIA ADU-ASIAMAH;WANG ZHANG;LI TING;ZHANG LI: "Circular RNAs: Research Progress and Its Significance in Birds and Livestock", BIOTECHNOLOGY BULLETIN, vol. 34, no. 11, 30 November 2018 (2018-11-30), pages 56 - 69, XP055856153, ISSN: 1002-5464, DOI: 10.13560/j.cnki.biotech.bull.1985.2018-0403 *

Also Published As

Publication number Publication date
JP7297331B2 (ja) 2023-06-26
CN111718929B (zh) 2022-10-18
JP2023116620A (ja) 2023-08-22
CN115806976A (zh) 2023-03-17
US20220177898A1 (en) 2022-06-09
CN117965533A (zh) 2024-05-03
CN111718929A (zh) 2020-09-29
CN113825837A (zh) 2021-12-21
EP3943601A4 (en) 2022-12-14
EP3943601A1 (en) 2022-01-26
JP2022528252A (ja) 2022-06-09

Similar Documents

Publication Publication Date Title
Cao et al. High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies
Pogson et al. Immunogenomic engineering of a plug-and-(dis) play hybridoma platform
Öztürk et al. scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution
KR102695752B1 (ko) Aav 벡터 및 프로모터의 단일 세포 전사체 기반 개발을 위한 방법 및 재료
Sasso et al. A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells
Rajendra et al. Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system
Guerin et al. A novel next-generation sequencing and analysis platform to assess the identity of recombinant adeno-associated viral preparations from viral DNA extracts
Meyer et al. Library generation by gene shuffling
WO2020186991A1 (zh) 利用环形rna进行蛋白翻译及其应用
Luo et al. High efficiency CHO cell display-based antibody maturation
Han et al. Development of a glycerol-inducible expression system for high-yield heterologous protein production in Bacillus subtilis
Yu et al. Transcriptome analyses of insect cells to facilitate baculovirus-insect expression
CN109988777A (zh) 谷氨酰胺合成酶基因以及应用
CN114901828A (zh) 一种哺乳动物表达系统使用的高表达载体
CN118834867A (zh) 利用环形rna进行蛋白翻译及其应用
Royle et al. A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris
US20230340460A1 (en) Method for identifying regulatory elements
Liu et al. Leaderless bicistronic design for precise and reliable control of gene expression in Corynebacterium glutamicum
Crone et al. gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells
CN118834868A (zh) 利用环形rna进行蛋白翻译及其应用
Nguyen et al. A Baculovirus Expression Vector Derived Entirely from Non-Templated, Chemically Synthesized DNA Parts
Öztürk et al. scAAVengr: Single-cell transcriptome-based quantification of engineered AAVs in non-human primate retina
Kondratov et al. Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System
Sharma et al. Identification of optimal flow rate for culture media, cell density, and oxygen toward maximization of virus production in a fed‐batch baculovirus‐insect cell system
Bannach et al. Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

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: 20773279

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021559486

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020773279

Country of ref document: EP

Effective date: 20211020