WO2023030330A1 - 一种光控rna代谢调控系统 - Google Patents

一种光控rna代谢调控系统 Download PDF

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WO2023030330A1
WO2023030330A1 PCT/CN2022/115877 CN2022115877W WO2023030330A1 WO 2023030330 A1 WO2023030330 A1 WO 2023030330A1 CN 2022115877 W CN2022115877 W CN 2022115877W WO 2023030330 A1 WO2023030330 A1 WO 2023030330A1
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rna
light
controlled
polypeptide
protein
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French (fr)
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杨弋
陈显军
刘韧玫
杨菁
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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

Definitions

  • the present invention relates to multidisciplinary interdisciplinary fields such as genetic engineering, optogenetics, and synthetic biology, specifically to the field of RNA metabolism regulation, and more specifically to a light-controlled RNA metabolism regulation system and a method for regulating RNA metabolism in host cells using the system method.
  • Synthetic biology can allow people to reprogram living systems in an optimal way according to their understanding of the rules of operation, or create artificial laws that do not exist in nature. Synthetic biology can be used to construct biological components, modules, or even “artificial synthetic biological systems” with new functions. This can not only promote people's understanding of the essence of life, but also innovate the development model of biotechnology and cultivate new bioindustries.
  • Optogenetics is a combination of genetic engineering and optical technology. It allows people to precisely regulate the life activities of cells in time and space.
  • Traditional biological research methods have great advantages. So far, scientists have discovered a variety of light-sensitive proteins from bacteria, plants and even animal cells. These light-sensitive proteins are used in various life processes such as photosynthesis, vision, and biological rhythms. It has also been found that in bacteria and plants, light-sensitive proteins are also used to control gene transcription and expression (He, Q.
  • RNA binding proteins play a vital role in regulating the metabolism of RNA in different stages, such as Capping, splicing, polyadenylation, editing, transport, translation, degradation, etc. (Arrenberg, A.B. et al., Science, 2010, 330:971-974.).
  • RNA-binding proteins are widely involved in various cellular metabolisms of RNA, such as RNA splicing, transportation, localization, and degradation. The development of RNA-binding proteins that can regulate these metabolic activities will provide unique biological tools for the study of cellular functions.
  • RNA-binding proteins usually consist of an RNA-binding domain and a functional domain.
  • the former can specifically recognize and bind RNA sequences, and the latter exerts corresponding functions to regulate RNA metabolism.
  • people combined different functional domains with RNA-binding domains that recognize different specific RNA sequences, and synthesized some RNA-binding proteins with specific functions.
  • the most widely studied is the fusion of Pumilio/FBF repeat sequence (PUF) with different functional domains to synthesize engineered RNA-binding proteins that can regulate metabolic activities such as RNA stability, localization, splicing, and translation.
  • PEF Pumilio/FBF repeat sequence
  • the PUF domain itself can also act as a translation regulator, binding to the 5' end of mRNA, and affecting the initiation of translation through steric hindrance (Cao, J. et al., Nucleic acids research, 2015, 43:4353-4362 ; Cao, J. et al., Angewandte Chemie, 2014, 53:4900-4904.).
  • Zefeng Wang's research group fused the PUF domain with the PIN RNA nuclease domain of the SMG6 protein to obtain a site-specific RNA nuclease that regulates gene expression in bacteria and mammalian cells by controlling the stability of mRNA (Choudhury, R. et al., Nature communications, 2012, 3:1147).
  • RNA-binding protein could specifically control the polyadenylation of the 3' end of the mRNA, and affect gene expression by regulating the stability of the mRNA (Cooke, A. et at., Proceedings of the National Academy of Sciences of the United States of America, 2011, 108:15870-15875.). Recently, people have even combined RNA-binding proteins with the hot CRISPR/Cas9 technology to develop a system that can activate endogenous gene expression at high levels (Zalatan, J.G., et al., Cell, 2015, 160:339-350).
  • RNA-binding proteins Although they are naturally occurring or artificially synthesized RNA-binding proteins, their activities are difficult to regulate. As long as cells express these RNA-binding proteins, they function. However, most RNAs function in specific time and specific space (Wang, Y. et al., The FEBS journal, 2013280:3755-3767.), development can precisely regulate RNA in time and space Metabolic technology is crucial to the basic research of life sciences such as the study of RNA function and regulation of gene expression. The applicant believes that it is possible to synthesize light-controllable RNA-binding proteins by synthetic biology methods, use light to regulate its binding to RNA, and combine it with different functional domains to obtain RNA-binding proteins with different functions.
  • RNA-binding proteins will overcome the uncontrollable shortcomings of existing RNA-binding proteins, allowing people to regulate RNA transcription, translation, stability and other metabolic activities in time and space, and can be widely used in basic research in biological sciences and biotechnology especially in synthetic biology.
  • the applicant invented a light-controlled RNA metabolic regulation system, which consists of two parts: recombinant light-controlled RNA effector and its corresponding target regulatory unit, which has good ability to regulate RNA metabolic behavior, and can be used in time and time.
  • Various metabolic activities of RNA are spatially and precisely regulated.
  • the first object of the present invention is to provide a novel light-controlled RNA metabolic regulation system.
  • the second object of the present invention is to provide a prokaryotic or eukaryotic expression vector containing the light-controlled RNA metabolic regulation system.
  • the third object of the present invention is to provide a method for regulating target RNA metabolism in host cells by using the light-controlled RNA metabolism regulation system.
  • the fourth object of the present invention is to provide a kit containing each component of the light-controlled RNA metabolic regulation system.
  • the present invention relates to a light-controlled RNA metabolic regulation system, which includes two parts: a) recombinant light-controlled RNA effector, the recombinant light-controlled RNA effector includes the first polypeptide as the RNA binding domain, and the first polypeptide as the photosensitive domain The second polypeptide and the third polypeptide as the RNA effector domain; b) target regulatory unit: including at least one response element recognized/bound by the first polypeptide, the target RNA sequence regulated by the third polypeptide .
  • the first polypeptide of the recombinant light-controlled RNA effector in the first part is an RNA binding domain, which can specifically recognize the response element.
  • the first polypeptide may be selected from the group consisting of an RNA recognition binding domain of an anti-transcription termination factor protein, an RNA attenuator RNA recognition binding domain, an RNA interferase RNA recognition binding domain, a small regulatory RNA binding protein RNA recognition binding domain, an RNA cleavage The gyrase RNA recognition binding domain, the ribozyme RNA recognition binding domain, the tRNA binding protein RNA recognition binding domain, and the rRNA binding protein RNA recognition binding domain.
  • the second polypeptide is a photosensitive domain, usually from a photosensitive protein with flavins as chromophores;
  • the third polypeptide is an RNA effector domain, including RNA splicing regulatory domain, RNA translation regulatory domain, RNA nuclease Domain, RNA epigenetic modification enzyme domain.
  • the first polypeptide, the second polypeptide and the third polypeptide may be connected directly, or may be operatively connected through a linker peptide.
  • the number of amino acids in the linker peptide is variable (eg 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).
  • the first polypeptide and the second polypeptide can constitute a light-controlled RNA-binding protein fusion protein (abbreviated as light-controlled RNA-binding protein), which can be used to study the RNA binding characteristics of recombinant light-controlled RNA effectors in vitro.
  • RNA-binding protein a light-controlled RNA-binding protein fusion protein
  • the response element in the second part of the target regulation unit can also be directly connected or operatively connected to the target RNA sequence.
  • the recombinant light-controlled RNA effector in the first part can further include additional polypeptides, such as the fourth polypeptide that promotes the transport of the recombinant light-controlled RNA effector fusion protein to different organelles (such as nuclear localization signal peptide).
  • additional polypeptides such as the fourth polypeptide that promotes the transport of the recombinant light-controlled RNA effector fusion protein to different organelles (such as nuclear localization signal peptide).
  • the fourth polypeptide and the first, second and third polypeptides can be connected directly or via linker peptides.
  • the present invention also relates to prokaryotic or eukaryotic expression vectors containing the light-controlled RNA metabolic regulation system of the present invention.
  • the expression vector may be a vector containing a gene encoding a recombinant light-controlled RNA effector alone, or a prokaryotic or eukaryotic expression vector containing a coding sequence of a target regulatory unit, which contains a response element but is to be regulated.
  • the target nucleic acid sequence is missing.
  • it can also be a prokaryotic or eukaryotic expression vector that simultaneously contains the gene encoding the recombinant light-controlled RNA effector and the response element in the target regulatory unit, but lacks the RNA coding sequence to be regulated.
  • the present invention also relates to a method for regulating RNA metabolism in host cells using the light-controlled RNA metabolism regulation system of the present invention, comprising the following steps:
  • the method for regulating the metabolism of target RNA in host cells of the present invention involves the illumination method, including the selection and control of the light source.
  • the light sources include LED lamps, incandescent lamps, fluorescent lamps, and lasers without limitation; the illumination method includes the selection of illumination amount, illumination time, illumination intensity and illumination frequency. It is also within the scope of the invention to spatially control the metabolism of the target RNA by means of scanning, projection, photomolding, and the like.
  • the present invention further relates to a kit, which is equipped with a prokaryotic or eukaryotic expression vector containing the light-controlled RNA metabolism regulation system of the present invention or/and a host introduced with the light-controlled RNA effector prokaryotic or eukaryotic expression vector cells, and corresponding instructions.
  • the kit of the present invention can also be equipped with a prokaryotic or eukaryotic expression vector of a target regulatory unit that contains a response element but lacks the target nucleic acid sequence to be regulated.
  • the invention provides a light-controlled RNA metabolic regulation system based on a photosensitive polypeptide, which is used to regulate various metabolic activities of target RNA in nuclear or eukaryotic host cells in time and space.
  • the light-controlled RNA metabolic regulation system of the present invention involves at least two parts: the first part is the coding nucleotide sequence of the recombinant light-controlled RNA effector fusion protein that can be expressed in host cells, and the fusion protein consists of three or four polypeptides Composition, wherein the first polypeptide is its RNA binding domain, the second polypeptide is the photosensitive domain, the third polypeptide is the RNA effector domain, and the fourth polypeptide is the organelle localization signal fragment; the second part is composed of the response element - The nucleotide sequence of the target regulatory unit composed of the target nucleic acid sequence to be regulated, wherein the response element is the RNA nucleotide motif recognized/bound by the first polypeptide of the recombinant light-controlled RNA
  • the three or four polypeptides of the first part preferably employ truncated functionally active fragments (ie domains) of the protein concerned.
  • the first part and the second part of the light-controlled RNA metabolic regulation system of the present invention can be constructed in one prokaryotic or eukaryotic expression vector or respectively constructed in two prokaryotic or eukaryotic expression vectors by genetic engineering technology. For specific host cell types, different conventional methods are used to introduce it into host cells to express the recombinant light-controlled RNA effector fusion protein of the present invention.
  • RNA effector domain of the third polypeptide of the light-controlled RNA effector regulates metabolic activities such as splicing, modification, transport, translation, and degradation of the target RNA.
  • the light-controlled RNA metabolism regulation system provided by the present invention can regulate the metabolism of target RNA in prokaryotic or eukaryotic host cells temporally and spatially by utilizing light irradiation that hardly damages cells or organisms.
  • Photo-controllable “photo-controllable”, “photo-controllable”, “photosensitive” and “photo-inducible” proteins have the same meaning herein and are used interchangeably, referring to proteins that are sensitive to light and can be irradiated with light of corresponding wavelengths at different intensities or frequencies Modulating the conformation or conformation of the protein to affect its activity, including activating, enhancing or repressing its activity.
  • “Host” refers to prokaryotes and eukaryotes
  • prokaryotes include various types of bacteria
  • eukaryotes include unicellular eukaryotes such as yeast
  • multicellular eukaryotes such as plants and animals, especially mammals, including humans .
  • “Host cells” in this patent refer to all prokaryotic and eukaryotic cells.
  • Prokaryotic cells include but not limited to Escherichia coli, Bacillus subtilis, lactic acid bacteria, actinomycetes, etc.
  • Eukaryotic cells include but not limited to yeast cells, fungal cells, plant Cells, nematode cells, Drosophila cells, insect cells, zebrafish cells, animal cells and mammalian cells, where mammalian cells can be original unmodified mammalian cells, such as HEK293, Hela, H1299 cells, etc., can also It can be a mammalian cell line obtained by further genome modification on the cell line, or other host cells compatible with the light-controlled RNA metabolic regulation system of the present invention.
  • RNA of interest refers to any functional RNA, including coding RNA and non-coding RNA, where non-coding RNA includes rRNA, tRNA, snRNA, snoRNA and microRNA, etc.
  • RNAs of known function also include RNAs of unknown function. The common feature of these RNAs is that they can all be transcribed from the genome, but they can perform their respective biological functions at the RNA level without being translated into proteins.
  • reporter RNA is a kind of target RNA, and refers to a useful RNA whose expression can be easily detected.
  • the following known reporter RNA can be selected: Pepper fluorescent RNA, which is based on Pepper RNA aptamers that specifically recognize and bind HBC series dyes and significantly activate The principle of fluorescence, Gaussian luciferase (Gluc) mRNA, green fluorescent protein (GFP) mRNA, red fluorescent protein (mCherry) mRNA.
  • the light-controlled RNA metabolism regulation system of the present invention is not limited to regulating the metabolism of reporter RNA, but can be used to regulate the metabolism of any functional RNA.
  • reporter protein is a protein produced by reporter RNA translation, and generally refers to a protein whose activity can be easily detected.
  • the following widely used reporter protein of reporter RNA coding can be selected: Gaussian luciferase (Gluc), green fluorescent protein (GFP), red fluorescent protein ( mCherry) and so on.
  • Transcription herein specifically refers to the process of transcribing a DNA sequence into a corresponding RNA sequence by RNA polymerase in a prokaryotic or eukaryotic host cell.
  • the transcription of eukaryotic genes is much more complex than that of prokaryotic organisms.
  • the three types of RNA polymerases I, II and III of eukaryotes transcribe the three types of eukaryotic gene DNA respectively to produce three types of RNA (rRNA, mRNA, tRNA) and antisense RNA.
  • the transcription process regulated by the transcription factors herein is the transcription initiated by RNA polymerase II, that is, the transcription of DNA into mRNA.
  • Transcription regulation refers herein to regulation of eukaryotic gene transcription, including initiation or repression of transcription, enhancement or repression of transcription, upregulation or downregulation of transcription.
  • RNA metabolism RNA metabolic activity
  • RNA metabolic behavior have the same meaning and are used interchangeably herein, referring to a series of metabolic processes that RNA undergoes after transcription, including but not limited to splicing, apparent Genetic modification, transportation, positioning, translation, degradation, etc., wherein epigenetic modification includes but not limited to methylation modification and pseudouracil modification.
  • RNA metabolism regulatory effect in this paper refers to the difference between recombinant light-controlled RNA effectors regulating target RNA metabolism under blue light light and dark conditions, which can be direct or indirect. It can be directly responded by detecting the content of mRNA, and can also be indirectly reflected by the level of protein produced by its translation. In general, the greater the metabolic difference of the target RNA under light and dark conditions, the better the regulation effect of the recombinant light-controlled RNA effector. In practical applications, as long as there is a statistically significant difference in the metabolism of the target RNA under light and dark conditions, it can be considered that the recombinant light-controlled RNA effector has the ability to regulate the metabolism of the target RNA.
  • the degradation of the target RNA by the recombinant light-controlled RNA nuclease factor under light conditions is 7.4 times that under dark conditions; in another specific embodiment of the present invention, the recombinant light-controlled RNA nucleic acid
  • the degradation of target RNA by enzyme factors under light conditions is reflected in the level of its encoded protein which is 13.1 times that under dark conditions.
  • the degradation of the target RNA by the recombinant light-controlled RNA nuclease factor under light conditions is reflected in the level of its encoded protein being 65% of that under dark conditions.
  • the level of recombinant light-controlled RNA translation initiation factor activating target RNA translation under light conditions is 8.5 times that under dark conditions; in another specific embodiment of the present invention, recombinant light
  • the splicing factors controlling RNA splicing promote target RNA exon-containing splicing 2.6-fold under light conditions compared with dark conditions.
  • “Expression”, “target protein gene expression”, and “gene expression” have the same meaning and can be used interchangeably herein, referring to the transcription of the DNA sequence of the target gene to produce RNA (mRNA or antisense RNA) carrying the gene information and the RNA carrying the gene information.
  • RNA mRNA or antisense RNA
  • the information in the ribosome is translated to produce the target protein, that is, the transcription to produce the information RNA and the translation to produce the target protein are called expression.
  • This article includes these two meanings, mainly referring to the production of the target protein.
  • RNA effector refers to a protein that can regulate RNA metabolism in prokaryotes or eukaryotes.
  • RNA effector protein can regulate various metabolic activities of the target RNA by binding and interacting with the response elements in the target regulatory unit alone or recruiting other RNA effector polypeptides.
  • Light-controlled RNA-binding protein in the present invention refers to a fusion protein composed of a first polypeptide and a second polypeptide. The binding capacity of the element changes.
  • the first polypeptide LicT CAT is operatively linked to the second polypeptide VIVID (N56K+C71V+I85V) to obtain the recombinant light-controlled RNA binding protein LicV, whose amino acid sequence is the sequence SEQ ID NO: 1.
  • Recombinant light-controlled RNA localization factor in the present invention means that the light-controlled RNA-binding protein of the present invention is directly or operatively linked (that is, separated by several amino acids) to the fourth polypeptide.
  • the light-controlled RNA localization factor can regulate the localization of target RNA in living cells.
  • the light-controlled RNA-binding protein is operatively linked to the intracellular membrane localization signal; in another specific embodiment of the present invention, the light-controlled RNA-binding protein is operatively linked to the cell nucleus localization signal.
  • Target regulatory unit refers to an artificial RNA sequence (not a protein) consisting of a response element and a target RNA sequence, wherein the response element can be located at the 5' end of the target RNA sequence, at the 3' end of the target RNA sequence, or at the In the middle of the target RNA sequence, the two can be connected directly or operationally (that is, separated by several nucleotides).
  • Nucleotide sequence encoding target regulatory unit in this patent refers to the DNA sequence that can be transcribed to produce the target regulatory unit described in this patent.
  • RNA-encoding nucleotide sequence to be regulated has the same meaning and can be used interchangeably herein, which means that the nucleotide sequence to be regulated described in this patent can be produced by transcription.
  • DNA sequence of RNA or RNA of interest or target RNA This DNA sequence can be included in the chromosomal DNA sequence of the host cell or in an artificially constructed expression vector.
  • RNA effector element refers to one or more RNA motifs that are specifically recognized/bound by RNA effectors. Different RNA effectors have different response elements corresponding to them. RNA effectors contain RNA that can bind to such RNA motifs. binding domain. When the RNA effector specifically binds to its corresponding response element, the RNA effector itself or its recruited cofactors act synergistically on the target RNA sequence to regulate the metabolic activity of RNA.
  • the response element refers to the RNA motif that can specifically recognize/bind with the first polypeptide of the recombinant light-controlled RNA effector, for example, the response element of LicT is a 25bp RNA motif (Sequence 2).
  • Promoters refers to a DNA sequence that initiates and causes the transcription of a downstream gene to produce RNA. Promoters include prokaryotic promoters and eukaryotic promoters, and can be natural gene promoters or artificially modified promoters. Different promoters can direct gene transcription in different types of tissues or cells at different developmental stages, or gene expression in response to different environmental or physiological conditions. Promoters can generally be classified as “constitutive promoters”, “inducible promoters” or “regulatable promoters”; divided into “cell-specific promoters” and “tissue-specific promoters” by tissue and cell , “development specific promoter” or “cell differentiation specific promoter”.
  • the upstream of the expressible natural cell structural protein gene has a matching promoter, and different gene DNA fragments can have the same promoter.
  • Non-limiting examples of commonly used constitutive promoters that can be used to express the recombinant light-sensitive transcription factors of the invention include promoters derived from polyoma virus, adenovirus 2, cytomegalovirus CMV, and simian virus 40 (SV40).
  • SV40 simian virus 40
  • nucleotide motifs necessary for transcription upstream of the TATA box that is, the response element specifically recognized/bound by the transcription factor described herein, which is bound by its corresponding transcription factor to convey a response to the minimal promoter Activation of the minimal promoter under the cooperative action of cofactors recruited by transcription factors leads to the transcription of downstream genes to produce the corresponding target RNA.
  • Vector expression vector
  • gene expression vector recombinant gene expression vector
  • plasmid plasmid
  • Transfection means that host cells are treated by physical or chemical methods, such as electroporation, calcium phosphate co-precipitation, lipofectamine or DEAE-dextran-mediated transfection, DNA particle bombardment and microinjection, etc., so that Cells take up exogenously added gene-carrying expression vectors, or deliver gene-carrying expression vectors into host cells through biological media, such as retroviral vectors, adenoviral vectors, receptor-mediated DNA uptake, etc. After entering the host cell, these vectors can exist in the cytoplasm in the form of episomes, or be integrated into the chromosome of the cell. Under appropriate conditions, the cell can transiently express or long-term express the protein or functional RNA carried by the gene encoded by the vector.
  • Such host cells are called vector-transfected cells.
  • Methods for transfecting host cells with expression vectors can be found in Sambrooka et al. (A Laboratory Manual of Molecular Cloning, Second Edition, Cold Spring Harbor Press (1989)), and other related textbooks.
  • the recombinant light-controlled RNA effector of the first part of the photosensitive polypeptide-based light-controlled RNA metabolic regulation system of the present invention is a fusion protein formed by connecting three or four functional polypeptide fragments directly through peptide bonds in series or through linker peptides in series. Under the irradiation of light of appropriate wavelength, the fusion protein can bind to the response element in the target regulatory unit of the second part of the present invention, and cooperate with the target in the target regulatory unit by itself or recruiting other cofactors of the host cell itself RNA sequence, thereby regulating the metabolism of the target RNA.
  • RNA effector fusion protein and “recombinant RNA effector” have the same meaning and can be used interchangeably.
  • the recombinant light-controlled RNA effector of the present invention contains a first polypeptide, which can specifically recognize and bind to the response element RNA sequence in the target regulatory unit; the first polypeptide is selected from: RNA recognition and binding of anti-transcription termination factor protein domain, RNA attenuator RNA recognition binding domain, RNA interferase RNA recognition binding domain, small regulatory RNA binding protein RNA recognition binding domain, RNA helicase RNA recognition binding domain, ribozyme RNA recognition binding domain, tRNA binding protein RNA recognition binding domain, rRNA binding protein RNA recognition binding domain.
  • the first polypeptide that can be used as the present invention preferably includes but is not limited to: the RNA recognition binding domain of Bacillus crassa LicT protein, the RNA recognition binding domain of Escherichia coli BglG protein, the RNA of Bacillus subtilis SacY protein Recognition binding domain, RNA recognition binding domain of Bacillus subtilis GlcT protein, RNA recognition binding domain of PyrR protein, RNA recognition binding domain of RapZ protein, RNA recognition binding domain of EndoA protein, RNA recognition binding structure of protein domain, more preferably the RNA recognition binding domain of LicT protein, the RNA recognition binding domain of BglG protein, the RNA recognition binding domain of SacY protein and the RNA recognition binding domain of GlcT protein.
  • the first polypeptide is the RNA recognition and binding domain of LicT protein. In another preferred embodiment of the present invention, the first polypeptide is the RNA recognition and binding domain of BglG protein. In another preferred embodiment of the present invention, the first polypeptide is the RNA recognition and binding domain of SacY protein. In another preferred embodiment of the present invention, the first polypeptide is the RNA recognition and binding domain of GlcT protein.
  • the first polypeptide can also be selected from the RNA recognition binding domain of other transcription termination factor proteins, including but not limited to the RNA recognition of Bacillus subtilis SacT protein Binding domain, RNA recognition binding domain of Erwinia chrysanthemi Arbg protein, RNA recognition binding domain of Lactococcus lactis BglR protein, RNA recognition binding domain of Lactobacillus casei LacT protein , the RNA recognition binding domain of Staphylococcus carnosus (Staphylococcus carnosus) GlcT protein.
  • the first polypeptide can also be selected from the group consisting of RNA attenuator RNA recognition binding domain, RNA interferase RNA recognition binding domain, small regulatory RNA binding protein RNA recognition Binding domain, RNA helicase RNA recognition binding domain, ribozyme RNA recognition binding domain, tRNA binding protein RNA recognition binding domain, rRNA binding protein RNA recognition binding domain.
  • the second polypeptide in the recombinant light-controlled RNA effector fusion protein of the present invention is a light-sensitive polypeptide, and the polypeptide comes from a light-sensitive structural domain with flavins (FMN or FAD) as chromophores.
  • FMN or FAD flavins
  • LOV light-sensitive structural domain with flavins
  • photolyase-like cryptochromes photolyase-like cryptochromes
  • blue light using FAD blue light using FAD, BLUF.
  • the photosensitive protein containing the LOV domain is preferred.
  • the dimerization ability of the second polypeptide After being irradiated with light of an appropriate wavelength, the dimerization ability of the second polypeptide will change, so that the dimerization ability of the recombinant light-controlled RNA effector will change, and the dimerized recombinant light-controlled RNA effectors bind to corresponding response elements to regulate the metabolic activity of the target RNA.
  • the present invention includes but not limited to the following preferred photosensitive protein or its functionally active truncated body: VIVID LOV domain of Neurospora crassa, EL222 LOV domain of bacteria Erythrobacter litoralis, LOV domain AsLOV2 of oat phytochrome 1 gene , LOV domain AuLOV of Aureus aureoprotein 1, PpLOV LOV domain of Pseudomonas putida.
  • the first preferred second polypeptide of the present invention is the photosensitive domain of the VIVID protein of Neurospora crassa and its mutants.
  • VIVID is a light-sensitive protein that exists in Neurospora crassa cells and participates in blue light regulation of cell signal transduction pathways. Under blue light irradiation, it can react with flavin adenine dinucleotide (FAD) to form a dimer.
  • FAD flavin adenine dinucleotide
  • the full-length VIVID protein contains 186 amino acids and contains only one light-sensitive LOV domain.
  • VIVID-36 truncated protein with 36 amino acids missing from the N-terminus of the VIVID protein is more stable than the full-length protein, and the VIVID-36 dimer formed after blue light irradiation returns to the monomeric form under dark conditions , VIVID-36 containing point mutations N56K and C71V had stronger dimerization ability.
  • the second polypeptide is VIVID (N56K+C71V) containing two point mutations and deleting the first 1-36 amino acids.
  • a second preferred second polypeptide of the invention is the LOV domain of the bacterial Erythrobacter litoralis EL222 protein.
  • the LOV domain of EL222 protein is located at the 1-182 amino acids of its N-terminus, which can combine with flavin mononucleotide (FMN) to generate an addition product under blue light irradiation, and then form a homodimer.
  • FMN flavin mononucleotide
  • the LOV domain of the EL222 protein is connected to the first polypeptide, which successfully leads to the ability of the first polypeptide of the recombinant light-controlled RNA effector to be regulated by light to bind to the corresponding response element.
  • the LOV domain of the EL222 protein is the light-controlled RNA effector LicEB of the second polypeptide, which can be combined with its corresponding response element under light conditions to promote the degradation of target RNA.
  • the third preferred second polypeptide of the present invention is the LOV2 domain (AsLOV2) of the phytochrome 1 gene of oat (Avena sativa).
  • the N-terminal of oat cell phytochrome 1 is LOV1 and LOV2 photo-oxygen voltage (LOV) domains, both of which can combine with flavin mononucleotide (FMN) to generate an addition product under blue light irradiation.
  • the LOV2 domain of oat phytochrome 1 is connected to the first polypeptide, which successfully leads to the ability of the first polypeptide of the recombinant light-controlled RNA effector to be regulated by light to bind to the corresponding response element.
  • the invention contains the light-controlled RNA effector LicAsB containing AsLOV2 as the second polypeptide, which can combine with its corresponding response element under light conditions to promote the degradation of target RNA.
  • the fourth preferred second polypeptide of the present invention is the LOV domain (abbreviated as AuLOV) at the C-terminus of the aureochrome 1 protein of Stramenopile algae Vaucheria frigida.
  • the LOV domain can combine with flavin mononucleotide (FMN) to generate an addition product under blue light irradiation.
  • the LOV2 domain of Aurea aureus 1 is connected to the first polypeptide, which successfully leads to the ability of the first polypeptide of the recombinant light-controlled RNA effector to be regulated by light to bind to the corresponding response element.
  • the invention contains the light-controlled RNA effector LicAuB containing AuLOV as the second polypeptide, which can be combined with its corresponding response element under light conditions to promote the degradation of target RNA.
  • a fifth preferred second polypeptide of the present invention is the LOV domain of Pseudomonas putida (abbreviated as PpLOV).
  • the LOV domain can combine with flavin mononucleotide (FMN) to generate an addition product under blue light irradiation.
  • FMN flavin mononucleotide
  • the PpLOV structural domain is connected to the first polypeptide, which successfully leads to the ability of the first polypeptide of the recombinant light-controlled RNA effector to be regulated by light to bind to the corresponding response element.
  • the invention contains the light-controlled RNA effector LicPB with PpLOV as the second polypeptide, which can combine with its corresponding response elements under light conditions to promote the degradation of target RNA.
  • the recombinant light-controlled RNA effector of the present invention contains a third polypeptide, which is an RNA effector domain, which can be a protein that regulates any metabolic activity of RNA, including RNA splicing, epigenetic modification, transport, Localization, translation, degradation and other metabolic activities.
  • the third polypeptide includes but not limited to RNA splicing regulatory domain, RNA translation initiation factor domain, RNA translation repressor domain, RNA nuclease domain, RNA exonuclease domain, RNA epigenetic modification Structural domains, wherein the RNA epigenetic modification domains include but are not limited to RNA methylase domains, RNA demethylase domains, and RNA pseudouracil synthetase domains.
  • the effector domain of RNA can regulate various metabolic activities of the target RNA by binding and interacting with the response elements in the target regulatory unit alone or by recruiting other RNA effector polypeptides.
  • the third polypeptide is hnRNP A1 RNA splicing regulatory domain, which can regulate the alternative splicing process of RNA; in another embodiment of the present invention, the third polypeptide is serine-arginine (SR) rich in arginine-threonine (RS) RNA splicing regulatory domain of protein, it can regulate the alternative splicing process of RNA; In another embodiment of the present invention, the 3rd polypeptide is eIF4E RNA The translation initiation factor domain, which can specifically recognize the cap structure at the 5' end of the mRNA, forms the eIF4F complex by recruiting eIF4A and eIF4G to participate in translation initiation; in another embodiment of the present invention, the third polypeptide For barnase RNA nuclease domain in Bacillus amyloliquefaciens, it can catalyze and hydrolyze RNA, cause its degradation to lose function; In another embodiment of the present invention, the 3rd polypeptide
  • the recombinant light-controlled RNA effector fusion protein of the present invention may also include a fourth polypeptide, which is a positioning signal peptide to promote the transport of the fusion protein to different organelles.
  • the fourth polypeptide is linked directly or via a linker peptide to the first, second and third polypeptides.
  • the fourth polypeptide can be selected from a nucleus localization signal peptide, a mitochondrial localization signal peptide, a Golgi apparatus localization signal peptide, an endoplasmic reticulum localization signal peptide, a cytoplasmic localization signal peptide, a mitochondrial outer membrane localization signal peptide, and a cell membrane inner membrane localization signal.
  • the fourth polypeptide is a cell nucleus localization signal peptide, which can mediate the localization of the recombinant light-controlled RNA effector of the present invention to the nucleus.
  • the fourth polypeptide can be one or more, preferably more if necessary or more effective.
  • the three or four polypeptides contained in the recombinant light-controlled RNA effector of the present invention can have multiple choices, and the three or four polypeptides can be linked into a fusion protein and can have multiple combinations.
  • the functional domain fragments of each polypeptide with good activity are preferred to prepare the recombinant light-controlled RNA effector fusion protein, and the preferred RNA metabolism regulation ability is expressed in the host cell, that is, the induction and non-induction lead to a large difference in the metabolism of the target RNA
  • the recombinant light-controlled RNA effector is used to regulate the metabolism of the target RNA, but regardless of the selection and combination, as long as the recombinant light-controlled RNA effector that utilizes light to regulate the metabolism of the target RNA in the host cell as envisioned in the present invention can be realized, various Combinations are within the scope of the present invention.
  • the second part of the light-controlled RNA metabolism regulation system based on the recombinant light-controlled RNA effector of the present invention is a target regulatory unit (nucleotide) composed of a response element-the RNA sequence to be regulated, which is specifically recognized/bound by the recombinant light-controlled RNA effector sequence), specifically, the nucleotide motif of the response element is different depending on the first polypeptide of the recombinant light-controlled RNA effector fusion protein selected in different embodiments of the present invention.
  • the response element is the specific response element of the first polypeptide, and the corresponding response element must be selected according to the selected first polypeptide.
  • the corresponding response element should be the "sequence 2, 3, 4, 5" motif.
  • the number of response elements is 1, 2, 3, 4 or 5, preferably multiple if necessary or more effective.
  • the target RNA sequence to be regulated is operably linked to the response element, which may be any functional RNA nucleotide sequence.
  • the response element which may be any functional RNA nucleotide sequence.
  • exemplary reporter RNAs are used: Pepper fluorescent RNA, Gaussian luciferase (Gluc) mRNA, green fluorescent protein (GFP) mRNA, red fluorescent protein (mCherry) mRNA, but the target RNA of the present invention is not limited to these reporter RNAs.
  • operably linked means that the response element and the target RNA sequence or between multiple response elements are not directly connected but can be separated by several nucleotides, as long as they can still act synergistically.
  • Standard recombinant DNA technology can be used to construct the first part and the second part of the light-controlled RNA metabolic regulation system of the present invention in one prokaryotic or eukaryotic expression vector or respectively in two prokaryotic or eukaryotic expression vectors. Standard techniques can be used to introduce this expression vector into various host cell groups to regulate various metabolic activities of the target RNA, and further select to produce useful transgenic organisms, such as transgenic mice.
  • the light-controlled RNA metabolism regulation system of the present invention can be used for regulation of endogenous RNA or exogenous RNA metabolism in host cells.
  • amino acid codon nucleotides have degeneracy (that is, certain amino acids may have two, or three, or four codons, which are called degenerate codons for the amino acid), and the present invention
  • the coding nucleic acids of various recombinant light-controlled RNA effectors mentioned above, the present invention includes all their respective degenerate nucleotide sequences.
  • the present invention includes all their respective amino acid sequence analogs that contain conservative deletions, additions, and substitutions but still retain their original functional activities.
  • the present invention provides a prokaryotic or eukaryotic expression vector containing a target regulatory unit with a vacant target RNA-encoded nucleotide sequence to be regulated.
  • the vacancy of the target RNA-encoded nucleotide sequence to be regulated allows users to select the required nucleotide sequence to be regulated.
  • the target RNA coding nucleotide sequence such as the coding nucleotide sequence of the target RNA, is inserted into the expression vector of the present invention by standard recombinant DNA technology, and the transcription is regulated by the above-mentioned recombinant light-controlled RNA effector Metabolic activity of the target RNA.
  • the present invention also provides host cell expression vectors transformed with various recombinant light-controlled RNA effector genes or bacterial strains or cell strains with various recombinant light-controlled RNA effector expression cassettes integrated on the genome, and simultaneously provides response elements-to-be-regulated
  • the target RNA encodes an expression vector with a nucleotide sequence gap.
  • RNAst strains or cell strains that have integrated the expression cassettes of various recombinant light-controlled RNA effectors, and cultivate such strains or cell lines to express recombinant light-controlled RNA effectors and response elements-target RNAs, and use light to regulate the metabolic activities of target RNAs to explore their biological functions.
  • kits for host cells equipped with various expression vectors or transformed with such vectors or integrated with expression cassettes of various recombinant light-controlled RNA effectors in the genome are respectively equipped with expression vectors containing one or more recombinant light-controlled RNA effector genes.
  • some containers in the kit are respectively equipped with expression vectors containing one or more recombinant light-controlled RNA effector genes, and other containers are respectively equipped with target regulatory units (wherein the response element-to-be An expression vector that regulates the target RNA coding nucleotide sequence vacancy).
  • kits in the kit contain host cells that have been transformed with expression vectors containing recombinant light-controlled RNA effector genes or have integrated expression cassettes for various recombinant light-controlled RNA effectors in the genome, Other containers are equipped with response elements-host cell expression vectors lacking in the nucleotide sequence encoding the target RNA to be regulated.
  • the kit of the present invention may also include corresponding lighting control equipment, such as LED lights and their regulating devices. All kits are equipped with corresponding instructions to explain the components in the kit, purpose and method of use, and provide relevant reference lists.
  • the present invention also includes a method for regulating target RNA metabolism in a host cell by a light-controlled RNA metabolism regulation system, comprising the steps of:
  • the method for inducing the host cells by light includes the selection of light source and the use of light source.
  • Light sources include, without limitation, LED lights, incandescent lights, fluorescent lights, lasers.
  • the light source is blue LED (460-470nm).
  • Illumination methods including illumination amount, illumination intensity, illumination time, illumination frequency, and methods such as scanning, projection, and photomold to spatially control the metabolism of target RNA are also included in the scope of the present invention.
  • the light intensity is 0-1.8W/m 2 etc; Metabolism;
  • neutral gray patches are used as photomasks to spatially modulate the metabolic levels of target RNAs in cells at different locations.
  • Fig. 1 Schematic diagram of recombinant light-controlled RNA effector plasmids containing different first polypeptides.
  • FIG. 2 Schematic diagram of recombinant light-controlled RNA effector plasmids containing different second polypeptides.
  • Fig. 3 is a schematic diagram of recombinant light-controlled RNA effector plasmids containing different third polypeptides.
  • Fig. 4 Degradation effect of recombinant light-controlled RNA nuclease factors containing different first polypeptides on target Gluc mRNA in living cells.
  • the test results were analyzed by Student’s t-test under light and dark conditions, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, N.S., no significant difference.
  • Fig. 5 Degradation effect of recombinant light-controlled RNA nuclease factors containing different second polypeptides on target Gluc mRNA in living cells.
  • the test results were analyzed by Student’s t-test under light and dark conditions, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, N.S., no significant difference.
  • Fig. 6 Degradation effect of recombinant light-controlled RNA nuclease factor LicVB on target mCherry mRNA in living cells. Scale bar, 100 ⁇ m.
  • Fig. 7 Degradation effect of recombinant light-controlled RNA nuclease factor LicVB on Pepper target RNA in living cells. Scale bar, 50 ⁇ m.
  • Fig. 8 Degradation effect of recombinant light-controlled RNA nuclease factor LicVPIN on Pepper target RNA in living cells. Scale bar, 50 ⁇ m.
  • Fig. 9 Degradation effect of recombinant light-controlled RNA nuclease factor LicVB on target mCherry mRNA in Escherichia coli and yeast cells.
  • the test results were analyzed by Student’s t-test under light and dark conditions, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, N.S., no significant difference.
  • FIG. 10 Regulatory effects of recombinant light-controlled RNA splicing factors LicVA1 and LicVRS on target RNA splicing in living cells.
  • A Gel running results of spliced products after reverse transcription and amplification;
  • B Statistical results of (A) electrophoresis gel bands. The test results were analyzed by Student’s t-test under light and dark conditions, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, N.S., no significant difference.
  • Figure 11 Regulatory effect of recombinant light-controlled RNA translation initiation factor LicV4E on target RNA translation in living cells. Scale bar, 200 ⁇ m.
  • Figure 13 The recombinant light-controlled RNA translation initiation factor LicV4E precisely regulates the translation effect of target RNA in space. Ruler, 3mm.
  • Fig. 14 Regulatory effect of recombinant light-controlled RNA localization factor on target RNA localization.
  • A Imaging effect of LicV-mKalama1-CAAX on Pepper-RAT LicT target RNA localization regulation;
  • B Statistical results of target RNA imaging in (A);
  • C LicV-mKalama1-3xNLS on Pepper-RAT LicT target Imaging effect of RNA localization regulation;
  • D Statistical results of imaging target RNA in (A). Scale bar, 25 ⁇ m.
  • All the primers used for PCR in the examples were synthesized, purified and identified by mass spectrometry by Shanghai Jereh Bioengineering Technology Co., Ltd.
  • the expression plasmids constructed in the examples have all been sequenced, and the sequence determination was completed by Jerry Sequencing Company.
  • the Taq DNA polymerase used in each example was purchased from Dongsheng Bio, the pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the PrimeSTAR DNA polymerase was purchased from TaKaRa Company. Polymerase buffer and dNTPs.
  • T4 ligase and T4 phosphorylase were purchased from Fermentas Company, and corresponding buffers and the like were included with the purchase.
  • the one-step rapid cloning kit (containing homologous recombinase) used in the examples was purchased from Yisheng Biotechnology Co., Ltd. Unless otherwise stated, inorganic salt chemical reagents were purchased from Sinopharm Shanghai Chemical Reagent Company. Kanamycin (Kanamycin) was purchased from Ameresco Company; ampicillin (Amp) was purchased from Ameresco Company; streptomycin was purchased from Ameresco Company; 384-well luminescence detection white plate and 384-well fluorescence detection black plate were purchased from Grenier Company.
  • the gel recovery kit used in the examples was purchased from Sangon Company, and the ordinary plasmid mini-prep kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • the cloned strain MachI was purchased from Beijing Quanshijin Company, and the BL21 (DE3) strain was purchased from Beijing Quanshijin Company.
  • the cell lines HEK293T and HEK293 were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.
  • Plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, it is necessary to perform phosphate group addition reaction on the 5' terminal base of PCR products. Only DNA molecules with phosphate groups at the ends can occur. Link reaction.
  • the self-circularization ligation reaction refers to the ligation reaction of the 3' end and the 5' end of the linearized vector.
  • T4 PNK is the abbreviation of T4 polynucleotide kinase, which is used for the addition reaction of the phosphate group at the 5' end of the DNA molecule. Reaction system for self-circularization of 5' phosphorylated DNA fragment products:
  • Overlap PCR is a commonly used method to connect two different or identical genes. For example, as shown in Figure 1, to connect gene AD and gene BC, first design two pairs of primers A, D, C, and B to amplify genes AD and BC respectively. The 5' ends of primers D and C contain a certain length of complementary sequence. The amplified products AD and BC obtained in the first round of PCR were recovered and used as templates for the second round of PCR.
  • the second round was amplified for 10 rounds according to the conventional PCR process, and the PCR system was:
  • Inverse PCR is a technique used in the following examples for site-directed mutagenesis, truncation mutagenesis, and insertion mutagenesis.
  • the basic principle refers to the experimental process of the MutaBEST kit of Takara Company.
  • Reverse PCR primers were designed at the corresponding variation site, and the 5' end of one primer contained the variation nucleotide sequence. After gel recovery and purification, the amplified product undergoes 5' end phosphorylation reaction and self-cyclization reaction, and is transformed into competent cells.
  • the 5' and 3' end sequences of the PCR target fragment used for homologous recombination are completely consistent with the end sequences of the linearized vector, and the one-step cloning kit is used for homologous recombination reaction.
  • the reaction system is:
  • the experiment of detecting the relative expression level of the reporter gene luciferase Gluc was carried out according to the instructions provided by the kit of NEB Company.
  • draw 10 ⁇ l from the cell culture medium and add it to a 384-well white plate from Grenier Company and prepare fresh Gluc detection solution (1 ⁇ M coelenterazine, 0.1M Tris-HCl buffer, 0.3M sodium ascorbate, pH 7.4), use Eppendolf's 12-channel electric dispenser to draw 10 ⁇ l/channel of the detection solution and add it to the cell culture medium in a 384-well white plate, and immediately use a multi-functional microplate reader to read the relative luminescence intensity (RLU).
  • RLU relative luminescence intensity
  • HEK293 and HEK293T were cultured with DMEM (HyClone, catalog number: SH302431) at an appropriate temperature and gas mixture (usually 37° C., 5% CO 2 ).
  • DMEM contained 4 mM glutamine, 4.5 g/l glucose, 10% fetal bovine serum (FBS), sodium pyruvate, and final concentrations of 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • FBS fetal bovine serum
  • streptomycin sodium pyruvate
  • a Leica SP8 confocal microscope was used to image mCherry fluorescence with a 561nm laser, EGFP fluorescence with a 488nm laser, and Pepper485 and mKalama1 fluorescence with a 458nm laser.
  • Example 1 Construction of expression vectors containing genes encoding different recombinant light-controlled RNA effectors.
  • RNA recognition binding domain encoding DNA of LicT, BglG, SacY and GlcT protein was synthesized genetically
  • the above DNA fragments were amplified by P1 and P2, P3 and P4, P5 and P6, P7 and P8 respectively to obtain LicT CAT , BglG CAT , SacY CAT and GlcT CAT gene fragments.
  • the DNA fragment encoding the third polypeptide barnase (K27A+N58D+R59A+E73A) (barnase M4 for short) was synthesized from the whole gene, and the fragment was amplified by P9 and P10 to obtain the barnase M4 gene fragment.
  • the primers for amplifying the LicT CAT gene fragment are:
  • Upstream primer (P1) 5'-agatccgctagcgctatgaaaattgcgaaggtgat-3'
  • Downstream primer (P2) 5'-agcgtagagcgtatgtcctgcggctttgctaattcttgctgatacatccttgttatcga-3'
  • the primers for amplifying the BglG CAT gene fragment are:
  • the primers for amplifying the SacY CAT gene fragment are:
  • the primers for amplifying the GlcT CAT gene fragment are:
  • the primers for amplifying the barnase M4 gene fragment are:
  • the primers for amplifying the VVD (N56K/C71V) gene fragment are:
  • the primers for amplifying the pEGFP-N1-FLAG vector to linearize it are:
  • DNA fragments encoding the LOV domain of EL222, AsLOV, AuLOV and PpLOV proteins were synthesized from the whole gene, respectively, with P15 and P16, P17 and P18, P19 and P20, P21 and P22 to amplify the above DNA fragments to obtain EL222, AsLOV, AuLOV and PpLOV gene fragments.
  • the plasmids obtained are named as pCMV-LicEB, pCMV-LicAsB, pCMV-LicAuB and pCMV-LicPB, respectively encoding recombinant light-controlled nuclease factors LicEB, LicAsB, LicAuB and LicPB (Fig. 2), and its amino acid sequences are sequence SEQ ID NO: 10, 11, 12 and 13.
  • the primers for amplifying the EL222 LOV domain gene fragment are:
  • the primers for amplifying the AsLOV gene fragment are:
  • the primers for amplifying the AuLOV gene fragment are:
  • the primers for amplifying the PpLOV gene fragment are:
  • the primers for amplifying the pCMV-LicVB vector to linearize it are:
  • RNA effector containing hnRNP A1 RNA splicing regulatory domain, RS RNA splicing regulatory domain, eIF4E RNA translation initiation factor domain or PIN RNA nuclease domain as the third polypeptide
  • the coding DNA fragments of the structural domains were respectively amplified with P25 and P26, P27 and P28, P29 and P30, P31 and P32 to obtain A1-NLS and RS-NLS and eIF4E and PIN gene fragments containing the fourth polypeptide.
  • the plasmids were named pCMV-LicVA1, pCMV-LicVRS, pCMV-LicV4E, and pCMV-LicVPIN, encoding recombinant light-controlled RNA splicing regulators LicVA1-NLS and LicVRS-NLS, recombinant light-controlled RNA translation initiation factor LicV4E and recombinant light-controlled nucleic acid, respectively
  • the amino acid sequences of the enzyme factor LicVPIN ( FIG. 3 ) are SEQ ID NO: 14, 15, 16 and 17, respectively.
  • the primers for amplifying the A1-NLS domain gene fragment are:
  • the primers for amplifying the RS-NLS gene fragment are:
  • the primers for amplifying the eIF4E gene fragment are:
  • the primers for amplifying the PIN gene fragment are:
  • the primers for amplifying the pCMV-LicVB vector to linearize it are:
  • the primers for amplifying the J23117 promoter are:
  • the primers for amplifying the rrnB transcription terminator are:
  • the primers for amplifying the LicVB gene fragment are:
  • the primers to amplify the pCDFDuet1 vector to linearize it are:
  • the primers for amplifying the LicVB gene fragment are:
  • the primers for amplifying the pGADT7 vector to linearize it are:
  • 2xRAT LicT encoding DNA fragments were synthesized and amplified using P47 and P48 as templates to obtain 2xRAT LicT fragments, respectively using P49 and P50, P51 and P52 and pU5 -Gluc and pU5-mCherry (Wang et al., Nature Methods, 2012:266-269) were used as templates to amplify Gluc and mCherry gene fragments, and the 2xRAT LicT fragment was connected to Gluc and mCherry by overlapping PCR to obtain Gluc-2xRAT LicT and mCherry-2xRAT LicT fragments.
  • the pCDNA3.1 hygro(+) vector (Invitrohen) was amplified using P53 and P54 to make it linearized, and the Gluc-2xRAT LicT and mCherry-2xRAT LicT fragments were inserted into the linearized pCDNA3.1 vector using a one-step cloning kit,
  • the obtained expression plasmids were named as pCDNA3.1-Gluc-2xRAT LicT and pCDNA3.1-mCherry-2xRAT LicT respectively, which encoded Gluc-2xRAT LicT and mCherry-2xRAT LicT target regulatory units respectively, and their nucleotide sequences were the sequence SEQ ID NO: 18 and 19.
  • the primers for amplifying the 2xRAT LicT fragment are:
  • the primers for amplifying the Gluc gene fragment are:
  • the primers for amplifying the mCherry gene fragment are:
  • the primers for amplifying the pCDNA3.1 hygro(+) vector to linearize it are:
  • the Pepper fluorescent RNA system (Chen et al., Nature Biotechonogy, 2019, 37:1287-1293) was used as the reporter RNA.
  • the Pepper-RAT LicT coding DNA fragment was synthesized in a commercial company, and the Pepper-RAT LicT fragment was obtained by using P55 and P56 as a template for amplification.
  • the U6 promoter DNA fragment was synthesized in a commercial company, and the U6 promoter fragment was obtained by using P57 and P58 as a template for amplification.
  • the U6-Pepper-RAT LicT fragment was obtained by linking Pepper-RAT with the U6 promoter by overlapping PCR.
  • the primers for amplifying the Pepper-RAT LicT fragment are:
  • the primers for amplifying the U6 promoter fragment are:
  • the primers for amplifying the pEGFP-N1-FLAG vector to linearize it are:
  • the mCherry-2xRAT LicT fragment was amplified using primers P61 and P62 using pCDNA3.1-mCherry-2xRAT LicT as a template, and synthesized in a commercial company
  • the J23106 promoter DNA fragment was amplified by using primers P63 and P64 as a template to amplify the J23106 promoter fragment, and the mCherry-2xRAT LicT was connected to the J23106 promoter by overlapping PCR to obtain the J23106-mCherry-2xRAT LicT fragment.
  • the primers for amplifying the mCherry-2xRAT LicT fragment are:
  • the primers for amplifying the J23106 promoter fragment are:
  • the primers for amplifying the pJ23117-LicVB vector to linearize it are:
  • the primers used to amplify the mCherry-2xRAT LicT fragment to construct pGADT7-mCherry-2xRAT LicT are:
  • the primers for amplifying the pGADT7 vector to linearize it are:
  • 2xRAT BglG , 2xRAT SacY and 2xRAT GlcT fragments were synthesized in a commercial company, using primers P71 and P72, primers P73 and P74, primers P75 and P76 were used to amplify them respectively, using P77 and P78 to amplify the pCDNA3.1-Gluc-2xRAT LicT vector in this example to remove the 2xRAT LicT fragment to linearize it, and use the one-step cloning kit to respectively clone 2xRAT BglG .
  • the 2xRAT SacY and 2xRAT GlcT fragments were inserted into the linearized pCDNA3.1-Gluc-2xRAT LicT vector, and the resulting expression plasmids were named pCDNA3.1-Gluc-2xRAT BglG , pCDNA3.1-Gluc
  • the primers for amplifying the 2xRAT BglG fragment are:
  • the primers for amplifying the 2xRAT SacY fragment are:
  • the primers for amplifying the 2xRAT GlcT fragment are:
  • the primers for amplifying pCDNA3.1-Gluc-2xRAT LicT to linearize it are:
  • 4xRAT LicT and EGFP DNA fragments were synthesized in a commercial company, respectively, using primers P79 and P80, P81 and P82 to amplify them as templates, and then using overlapping They were joined by PCR to obtain the 4xRAT LicT -EGFP fragment.
  • the RAT LicT fragment was inserted into the pGZ3-GUM vector (a gift from the research group of Teacher Wang Zefeng, Chinese Academy of Sciences) by using primers P85 and P86 by reverse PCR. After ligation, a plasmid named pGZ3-GUM-RAT LicT was obtained, and the nucleotide sequence of the target regulatory unit encoded by it was the sequence SEQ ID NO:25.
  • the primers for amplifying the 4xRAT LicT fragment are:
  • the primers for amplifying the EGFP fragment are:
  • the primers for amplifying the pCDNA3.1-mCherry-2xRAT LicT vector to linearize it are:
  • the primers for amplifying the pGZ3-GUM vector to linearize it are:
  • the gene encoding mKalama1 blue fluorescent protein, the gene encoding CAAX intracellular membrane localization signal and the gene encoding 3xNLS nuclear localization signal were synthesized using P87, P88, P89 and P90, P91 and P92 were respectively amplified using pCMV-LicVB as a template, using P93 and P94 to amplify the LicV coding gene using pCMV-LicVB as a template, and using overlapping PCR to connect LicV with mKalama1 and CAAX or 3xNLS, respectively, to obtain LicV-mKalama1- CAAX and LicV-mKalama1-3xNLS recombinant gene fragments, use primers P95 and P96 to amplify the pEGFP-N1-FLAG vector (Addgene: 60360) to make it linear, and use
  • the primers for amplifying the mKalama1 gene fragment are:
  • the primers for amplifying the CAAX gene fragment are:
  • the primers for amplifying the 3xNLS gene fragment are:
  • the primers for amplifying the LicV gene fragment are:
  • the primers for amplifying the pEGFP-N1-FLAG vector to linearize it are:
  • Example 4 Recombinant light-controlled RNA nuclease factor regulates the degradation of target RNA
  • pCDNA3.1-Gluc-2xRAT LicT and pCMV-LicVB In order to detect the regulatory effect of recombinant light-controlled RNA nuclease factors containing different first polypeptides on target RNA degradation, pCDNA3.1-Gluc-2xRAT LicT and pCMV-LicVB, pCDNA3.1-Gluc-2xRAT BglG and pCMV- HEK293T cells were co-transfected with BglVB, pCDNA3.1-Gluc-2xRAT SacY , pCMV-SacVB, pCDNA3.1-Gluc-2xRAT GlcT and pCMV-GlcVB, and pCDNA3.1-Gluc without the RAT LicT response element was used as a control.
  • the cells were cultured for 24 hours in the dark and blue light (1.8 W/m 2 ) respectively, and the Gluc activity in the cell culture supernatant was detected.
  • the test results are shown in Figure 4, the Gluc activity in the supernatant under light conditions was significantly lower than that in the dark conditions, and the Gluc activity in the control cells did not change significantly, indicating that light can induce the recombination of LicVB, BglVB, SacVB and GlcVB
  • the light-controlled RNA nuclease factor binds to the corresponding response element and hydrolyzes the target Gluc mRNA, resulting in a decrease in the level of the target Gluc mRNA in the cell, and finally a decrease in the level and activity of the synthesized Gluc protein. This result indicates that the recombinant light-controlled RNA nuclease factors containing different first polypeptides can regulate the degradation of target RNA.
  • pCDNA3.1-Gluc-2xRAT LicT was mixed with pCMV-LicEB, pCMV-LicAsB, pCMV-LicAuB and pCMV-LicPB, respectively.
  • HEK293T cells were co-transfected, and pCDNA3.1-Gluc without RAT LicT response element was used as a control. After 6 hours of transfection, the cells were cultured for 24 hours in the dark and blue light (1.8 W/m 2 ) respectively, and the Gluc activity in the cell culture supernatant was detected.
  • the test results are shown in Figure 5, the Gluc activity in the supernatant under light conditions was significantly lower than that in the dark conditions, and the Gluc activity in the control cells did not change significantly, indicating that light can induce the recombination of LicEB, LicAsB, LicAuB and LicPB
  • the light-controlled RNA nuclease factor binds to the 2xRAT LicT response element and hydrolyzes the target Gluc mRNA, resulting in a decrease in the level of the target Gluc mRNA in the cell, which ultimately reduces the level and activity of the synthesized Gluc protein. This result indicates that the recombinant light-controlled RNA nuclease factors containing different second polypeptides can regulate the degradation of target RNA.
  • pCMV-LicVB was co-transfected with pCDNA3.1-mCherry-2xRAT LicT and pU6-Pepper-RAT LicT respectively in HEK293T cells.
  • HEK293T cells were co-transfected with pU6-Pepper-RAT LicT , and pCDNA3.1-mCherry and pU6-Pepper without RAT LicT response elements were used as controls.
  • the cells were cultured for 24 hours in the dark and blue light (1.8W/m 2 ), respectively, and the fluorescence signals of mCherry and Pepper485 in the cells were detected respectively (cells expressing the Pepper-RAT LicT target regulatory unit require Add 1 ⁇ M HBC485 dye (Chen et al., Nature Biotechonology, 2019, 37:1287-1293) to specifically label the target RNA).
  • the test results are shown in Figures 6-8, respectively.
  • the Pepper485 signal in cells under light conditions was significantly lower than that in dark conditions, and there was no significant change in the Pepper485 signal in control cells, indicating that light can induce LicVB and LicVPIN to bind to Pepper-RAT LicT target transcription
  • the RAT LicT response element in the cell hydrolyzes the Pepper target RNA, resulting in a decrease in the level of Pepper RNA in the cell and a decrease in the Pepper485 fluorescence signal.
  • pJ23106-mCherry-2xRAT LicT and pJ23117-LicVB were co-transformed into E. coli cells, and pGADT7-mCherry-2xRAT LicT and pGADT7-LicVB were co-transformed.
  • BY4741 Saccharomyces cerevisiae cells were transformed, and pJ23106-mCherry and pGADT7-mCherry without RAT LicT response element were used as controls.
  • coli and yeast cells under light conditions was significantly lower than that in dark conditions, and the mCherry signal in control cells did not change significantly, indicating that light can induce LicVB to bind mCherry-2xRAT
  • the 2xRAT LicT response element in the LicT target transcription unit hydrolyzes the target mCherry mRNA, resulting in a decrease in the level of the mCherry target mRNA in the cell, which eventually leads to a decrease in the level of the synthesized mCherry protein and a decrease in fluorescence. This result indicates that recombinant light-controlled RNA nuclease factors can regulate the degradation of target RNA in different host cells.
  • Example 5 Recombinant light-controlled RNA splicing factor regulates the splicing of target RNA
  • HEK293T cells were co-transfected with pGZ3-GUM-RAT LicT and pCMV-LicVA1 and pCMV-LicVRS, respectively, to express the empty vector and expression of recombinant light-controlled RNA splicing factors.
  • a plasmid without the third polypeptide recombinant light-controlled RNA splicing factor was used as a control. After 6 hours of transfection, the cells were cultured for 24 hours in the dark and blue light (1.8 W/m 2 ) respectively, and the splicing results of the target RNA in the cells were detected.
  • the test results are shown in Figure 10.
  • the ratio of the two splicing products of the target RNA in the cells is significantly different under light and dark conditions, but there is no significant difference in the control cells, indicating that the recombinant light-controlled RNA splicing factors LicVA1 and LicVRS can be used to target Alternative splicing of RNA.
  • Example 6 Recombinant light-controlled RNA translation initiation factor regulates translation of target RNA
  • HEK293T cells were co-transfected with pCMV-LicV4E and pCDNA3.1-mCherry-4xRAT LicT- EGFP to express recombinant light-controlled RNA translation without the third polypeptide Plasmids of initiation factors served as controls. After 6 hours of transfection, the cells were cultured for 24 hours in the dark and blue light (1.8 W/m 2 ), respectively, and the fluorescent signals of mCherry and EGFP in the cells were detected under different conditions. The detection results are shown in Figure 11.
  • the signal of EGFP under light conditions is much stronger than that in cultured cells under dark conditions, while the EGFP signals in control cells are very low, indicating that light can induce LicV4E recombinant light-controlled RNA
  • the translation initiation factor binds to the 4xRAT LicT response element, recruits other translation factors through the third polypeptide eIF4E to initiate the translation of the target EGFP mRNA, and synthesizes the EGFP protein. This result indicates that the recombinant light-controlled RNA translation initiation factor can be used to regulate the translation of target RNA.
  • HEK293T cells transfected with pCMV-LicV4E and pCDNA3.1-mCherry-4xRAT LicT -EGFP were cultured under blue light with different light intensity
  • the mCherry and EGFP fluorescence signals of the cells under different conditions were imaged.
  • the detection results are shown in Fig. 12 , as the intensity of blue light increases, the fluorescence signal of EGFP also increases. This result indicates that the recombinant light-controlled RNA translation initiation factor can be used to quantitatively regulate the translation of target RNA.
  • HEK293T cells transfected with pCMV-LicV4E and pCDNA3.1-mCherry-4xRAT LicT -EGFP were placed in a specific region only
  • the optical membrane was cultured under blue light, and the mCherry and EGFP fluorescence signals of the cells were imaged after 24 hours.
  • the detection results are shown in Figure 13, only cells that received blue light irradiated expressed EGFP protein at a high level, while the EGFP signal of adjacent cells was very weak. This result demonstrates that recombinant light-controlled RNA translation initiation factors can be used to spatially precisely regulate the translation of target RNAs.
  • Example 7 Recombinant light-controlled RNA localization factor regulates localization of target RNA
  • pU6-Pepper-RAT LicT was co-transfected with pCMV-LicV-mKalama1-CAAX and LicV-mKalama1-3xNLS respectively in HEK293T cells to express recombinant light-controlled RNA without LicV Plasmids of RNA-binding proteins served as controls. After 6 hours of transfection, the cells were cultured for 24 hours in the dark and blue light (1.8W/m 2 ), respectively, and labeled with 1 ⁇ M HBC620 dye (Chen et al., Nature Biotechonogy, 2019, 37:1287-1293).

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Abstract

一种光控RNA代谢调控系统，包括：a)重组光控RNA效应因子，所述重组光控RNA效应因子包括作为RNA结合结构域的第一多肽，作为光敏结构域的第二多肽和作为RNA效应结构域的第三多肽；b)靶调控单元，包括被所述第一多肽识别/结合的至少一个反应元件和被第三多肽调节的靶标RNA序列。还提供包含这种光控RNA代谢调控系统的表达载体，及用这种光控RNA代谢调控系统在宿主细胞中调控RNA代谢的方法。还提供装有所述光控RNA代谢调控系统各组分的试剂盒。所述光控RNA代谢调控系统具有高效率、高时空分辨率等优点，可用于时空精确调控活细胞中RNA的多种代谢活动。

Description

一种光控RNA代谢调控系统 技术领域
本发明涉及遗传工程、光遗传学、合成生物学等多学科交叉领域，具体地涉及RNA代谢调控领域，更具体地涉及一种光控RNA代谢调控系统和采用该系统调控宿主细胞中RNA代谢的方法。
背景技术
随着基因组测序合成的技术创新、系统生物学和各类“组学”的涌现及发展，诞生了一个全新的的交叉学科“合成生物学”。合成生物学的核心在于将遗传信息按照工程原理进行设计，关键在于对各基因元器件功能的调节和控制。合成生物学可以允许人们按照生命系统运行规则的认识，以最优化的方式对其进行重新编程，或者创造自然界原本不存在的人造法则。合成生物学可用于构建具有全新功能的生物元件、模块，甚至是“人工合成生物体系”。这不仅可以推动人们对生命本质的理解，还可以革新生物技术发展模式，培育新型生物产业。
近年来，人们通过使用光和遗传编码的光敏蛋白技术来控制细胞行为这一领域取得了激动人心的突破，大量的研究成果在国际顶级杂志发表，由此也产生了一门新兴交叉学科“光遗传学(Optogenetics)”。光遗传学是基因工程和光学技术的一种结合，它可以允许人们在时间和空间上精确调控细胞的生命活动，其时间精度可以达毫秒级而空间精度可以精确到特定单一细胞，因此相对于传统的生物研究方法具有非常大的优势。到目前为止，科学家们从细菌、植物甚至动物细胞内陆续发现各种各样的光敏蛋白，这些光敏蛋白倍用于光合作用，视觉，生物节律等各种生命过程。人们还发现在细菌与植物中，光敏蛋白还被用于控制基因的转录与表达(He,Q.et al.,Science,2002,297:840-843；Malzahn,E.et al.,Cell,2010,142:762-772.)。随着越来越多的光敏蛋白的结构被解析，科学家们对光在生命活动调控中作用的认识更加深刻。目前，科学家已经找到或者人工改造、合成了一些可用于控制真核细胞运动、蛋白质相互作用于信号转导，神经肌肉动作，肌肉蛋白聚集与细胞运动，蛋白质剪切，蛋白质降解，蛋白质表达等光敏蛋白(Arrenberg,A.B.et al.,Science,2010,330:971-974；Deng,W.et al.,American journal  of physiology Regulatory,2014,307:R1292-1302；Duebel,J.et al.,Current opinion in ophthalmology,2015,26:226-232)，并发展了各种光传导与操纵技术与设备。这使得人类对生命现象的控制程度达到了前所未有的水平。
近年来，人们对RNA分子的认识，从最初的DNA与蛋白质间遗传信息传递的中间体，到RNA剪接和编辑。基因转录与翻译调控、分子感应和应答催化等，RNA所行使的多种生物功能及作用机制不断获得深入解析。在真核细胞中，新合成的RNA会经历多个步骤才能成为信使RNA(mRNA)，在mRNA的生命周期中，RNA结合蛋白在调控不同时期RNA的代谢中发挥着至关重要的作用，如加帽、剪接、多腺苷化、编辑、转运、翻译、降解等(Arrenberg,A.B.et al.,Science,2010,330:971-974.)。除了mRNA外，还有很多非编码RNA，如转运RNA(tRNA)、核小RNA(snRNA)、核仁小RNA(snoRNA)、microRNA以及长链非编码RNA(lncRNA)等，这些非编码RNA的功能常常依赖于特定的RNA结合蛋白。因此，RNA结合蛋白广泛参了RNA的各类细胞代谢，如RNA的剪接、运输、定位、降解等。开发可以调控这些代谢活动的RNA结合蛋白将会为细胞功能研究提供独特的生物工具。
RNA结合蛋白通常由RNA结合结构域和功能结构域构成，前者可以特异性识别RNA序列并结合，后者发挥相应的功能进行RNA代谢调控。人们基于这样的原理，将不同的功能结构域和识别不同特异性RNA序列的RNA结合结构域结合起来，合成了一些具有特定功能的RNA结合蛋白。其中研究比较广泛的是将Pumilio/FBF重复序列(PUF)与不同的功能结构域融合，合成得到可以调控RNA稳定性、定位、剪接、翻译等的代谢活动的工程化RNA结合蛋白。2007年，Ozawa等人将绿色荧光蛋白GFP的N端结构域和C端结构域分别与两个PUF RNA结合结构域融合，用于观测哺乳动物细胞中线粒体RNA(Ozawa,T.et al.,Nature methods,2007 4:413-419.)。人们后来基于类似的技术追踪不容动物细胞b-激动蛋白的RNA和植物细胞中烟草花叶病毒的RNA(Tilsner,J.et al.,The Plant journal,2009,57:758-770；Yamada,T.et al.,Analytical chemistry,2011,83:5708-5714)。PUF结构域本身也可以作为翻译调控因子，结合到mRNA的5’端，通过空间位阻效应来影响翻译的起始(Cao,J.et al.,Nucleic acids research,2015,43:4353-4362；Cao,J.et al.,Angewandte Chemie,2014,53:4900-4904.)。Zefeng Wang课题组将PUF结构域与SMG6蛋白的PIN RNA核酸酶结构域融合，得到位点特异性的RNA核酸酶，通过控制mRNA的稳定性来调控细菌和哺乳动物细胞中的基因表达(Choudhury,R.et al.,Nature communications,2012,3:1147)。Amy Cooke等人则是将GLD2和CAF1结构域与PUF结构域融合，得到的RNA结合 蛋白可以特异性控制mRNA 3’端的多聚腺苷化，通过调控mRNA的稳定性来影响基因表达(Cooke,A.et at.,Proceedings of the National Academy of Sciences of the United States of America,2011,108:15870-15875.)。最近，人们甚至将RNA结合蛋白与火热的CRISPR/Cas9技术结合起来，开发了可以高水平激活内源基因表达的系统(Zalatan,J.G.,et al.,Cell,2015,160:339-350)。
然而，无论是自然存在或者是人工合成的RNA结合蛋白，它们的活性很难被调控。只要细胞表达这些RNA结合蛋白，它们就会发挥作用。然而，大多数RNA是在特定的时间和特定的空间里发挥响应的功能(Wang,Y.et al.,The FEBS journal,2013280:3755-3767.)，开发可以在时间和空间上精确调控RNA代谢的技术对于研究RNA功能、调控基因表达等生命科学基础研究至关重要。本申请人认为可以通过合成生物学的方法，合成光可控的RNA结合蛋白，利用光来调控其与RNA的结合，并将其与不同功能结构域结合，获得具有不同功能的RNA结合蛋白。这些RNA结合蛋白将克服现有RNA结合蛋白不可控的缺点，可以让人们在时间和空间上调控RNA的转录、翻译、稳定性等代谢活动，可被广泛应用于生物科学的基础研究以及生物技术特别是合成生物技术中。经过潜心研究，本申请人发明了一种光控RNA代谢调控系统，它由重组光控RNA效应因子及其对应的靶调控单元两部分组成，具有良好的RNA代谢行为调控能力，可以在时间和空间上精确调控RNA的各类代谢活动。
因此，本发明的第一个目的是提供一种新型光控RNA代谢调控系统。
本发明的第二个目的是提供含有所述光控RNA代谢调控系统的原核或真核表达载体。
本发明的第三个目的是提供用所述光控RNA代谢调控系统调节宿主细胞中靶标RNA代谢的方法。
本发明的第四个目的是提供装有所光控RNA代谢调控系统各组分的试剂盒。
发明概述
本发明涉及一种光控RNA代谢调控系统，包括两个部分：a)重组光控RNA效应因子，所述重组光控RNA效应因子包括作为RNA结合结构域的第一多肽，作为光敏结构域的第二多肽和作为RNA效应结构域的第三多肽；b)靶调控单元：包括被所述第一多肽识别/结合的至少一个反应元件、被第三多肽调节的靶标RNA序列。
按照本发明的光控RNA代谢调控系统，第一部分中重组光控RNA效应因子的第一多肽为RNA结合结构域，它能够特异性地识别反应元件。第一多肽可选自抗转录终 止因子蛋白的RNA识别结合结构域、RNA衰减子RNA识别结合结构域、RNA干扰酶RNA识别结合结构域、小调节RNA结合蛋白RNA识别结合结构域、RNA解旋酶RNA识别结合结构域、核酶RNA识别结合结构域、tRNA结合蛋白RNA识别结合结构域、rRNA结合蛋白RNA识别结合结构域。第二多肽为光敏结构域，通常来自以黄素类为生色团的光敏蛋白；第三多肽为RNA效应结构域，包括RNA剪接调控结构域、RNA翻译调控因子结构域、RNA核酸酶结构域、RNA表观遗传学修饰酶结构域。
第一多肽、第二多肽和第三多肽之间可以直接连接，也可操作性地通过接头肽连接。接头肽的氨基酸个数是可变的(如0,1,2,3,4,5,6,7,8,9,10个或更多)。
第一多肽、第二多肽可构成一种光控RNA结合蛋白融合蛋白(简称为光控RNA结合蛋白)，可以用于体外研究重组光控RNA效应因子的RNA结合特点。本发明的光控RNA代谢调控系统中，第二部分靶调控单元中的反应元件和靶标RNA序列之间也可直接连接，或操作性连接。
按照本发明的光控RNA代谢调控系统，第一部分中的重组光控RNA效应因子还可进一步包含附加的多肽，如促进重组光控RNA效应因子融合蛋白向不同细胞器运输的第四多肽(如细胞核定位信号肽)。第四多肽与第一、第二、第三多肽可直接连接或通过接头肽连接。
本发明还涉及含有本发明的光控RNA代谢调控系统的原核或真核表达载体。所述表达载体可以是单独含有重组光控RNA效应因子编码基因的载体，也可以是单独含有靶调控单元编码序列的原核或真核表达载体，所述靶调控单元中含有反应元件但待调控的靶核酸序列空缺。或者，也可以是同时含有重组光控RNA效应因子编码基因和靶调控单元中的反应元件但待调控的RNA编码序列空缺的原核或真核表达载体。
本发明也涉及用本发明的光控RNA代谢调控系统在宿主细胞中调控RNA代谢的方法，包括以下步骤：
a)将所光控RNA代谢调控系统构建在原核或真核质粒表达载体中；
b)将重组质粒引入宿主细胞；
c)光照诱导所述宿主细胞，调控宿主细胞中靶标RNA的代谢。
本发明的在宿主细胞中调控靶标RNA代谢的方法，所涉及的光照方法，包括光源的选择和光源的控制。光源非限制地包括LED灯、白炽灯、荧光灯、激光；光照方法包括光照量、光照时间、光照强度及光照频率的选定。用扫描、投影、光模具等方法在空间上控制靶标RNA的代谢也包含在本发明范围中。
本发明进一步涉及一种试剂盒，该试剂盒装有含本发明光控RNA代谢调控系统的原核或真核表达载体或/和引入了所述光控RNA效应因子原核或真核表达载体的宿主细胞，及相应的说明书。本发明的试剂盒还可装有含反应元件但待调控的靶核酸序列空缺的靶调控单元的原核或真核表达载体。
发明详述
本发明提供一种基于光敏多肽的光控RNA代谢调控系统，用于在时间和空间上调节在核或真核生物宿主细胞中目的RNA的各类代谢活动。本发明的光控RNA代谢调控系统涉及至少两个部分：第一部分是能够在宿主细胞中表达的重组光控RNA效应因子融合蛋白的编码核苷酸序列，该融合蛋白由三个或四个多肽组成，其中第一多肽是其RNA结合结构域，第二多肽为光敏结构域，第三多肽为RNA效应结构域，第四多肽为细胞器定位信号片段；第二部分是由反应元件-待调控的靶核酸序列组成的靶调控单元核苷酸序列，其中的反应元件为上述重组光控RNA效应因子融合蛋白第一多肽所识别/结合的RNA核苷酸基序。第一部分的三个或四个多肽优选采用有关蛋白的截短的功能活性片段(即结构域)。可通过基因工程技术将本发明的光控RNA代谢调控系统的第一部分和第二部分构建在一个原核或真核表达载体中或分别构建在二个原核或真核表达载体中。针对特定的宿主细胞类型采用不同的常规方法将其导入宿主细胞，使之表达本发明的重组光控RNA效应因子融合蛋白，用适当波长的光照射可导致其第二光敏多肽二聚化能力改变，进而使重组光控RNA效应因子的二聚化能力发生变化，二聚化的重组光控RNA效应因子可结合于本发明第二部分靶调控单元核苷酸序列中的反应元件，并通过重组光控RNA效应因子的第三多肽的RNA效应结构域调控目的RNA的剪接、修饰、运输、翻译、降解等代谢活动。
本发明提供的这种光控RNA代谢调控系统，可利用几乎不会损伤细胞或机体的光照射，在时间上和空间上调节原核或真核宿主细胞中目的RNA的代谢。
本文所用术语的定义和解释
“光控”“光可控”，“光敏”和“光诱导”的蛋白在本文中含义相同，可互换使用，指对光照敏感的、可用相应波长的光以不同强度或不同频率照射，调节该蛋白的构象或构型从而影响其活性，包括激活、增强或阻遏其活性。
“宿主”指原核生物和真核生物，原核生物包括各类细菌，真核生物包括单细胞真核生物如酵母菌，和多细胞真核生物，如植物和动物，尤其是哺乳动物，包括人。
“宿主细胞”在本专利中指所有的原核与真核细胞，原核细胞包括但不限于大肠杆 菌、枯草芽孢杆菌、乳酸菌、放线菌等，真核细胞包括但不限于酵母细胞、真菌细胞、植物细胞、线虫细胞、果蝇细胞、昆虫细胞、斑马鱼细胞、动物细胞和哺乳动物细胞，其中哺乳动物细胞可以是原始的未经改造的哺乳动物细胞，比如HEK293，Hela，H1299细胞等，也可以是在细胞株上再进行基因组改造的得到的哺乳动物细胞株，也可以是其他和本发明的光控RNA代谢调控系统相容的宿主细胞均可以。
“目的RNA”、“靶标RNA”也可称为“感兴趣RNA”指任何有功能的RNA，包括编码RNA和非编码RNA，其中非编码RNA包括rRNA，tRNA，snRNA，snoRNA和microRNA等多种已知功能的RNA，还包括未知功能的RNA。这些RNA的共同特点是都能从基因组上转录而来，但是不翻译成蛋白，在RNA水平上就能行使各自的生物学功能。
“报告RNA”为目的RNA的一种，指其表达容易被检测的有用RNA。为便于检测本发明基于光敏多肽的光控RNA代谢调控系统的效果，可以选择以下已知的的报告RNA：Pepper荧光RNA，它是基于Pepper RNA适配体特异性识别结合HBC系列染料并显著激活其荧光的原理、高斯荧光素酶(Gluc)mRNA、绿色荧光蛋白(GFP)mRNA，红色荧光蛋白(mCherry)mRNA。但本发明的光控RNA代谢调控系统不限于调控报告RNA的代谢，而可用于调控任何有功能RNA的代谢。
“报告蛋白”为报告RNA翻译产生的蛋白质，一般指活性容易被检测的蛋白质。为便于检测本发明基于光敏多肽的光控RNA代谢调控系统的效果，可以选择报告RNA编码的以下广泛应用的报告蛋白：高斯荧光素酶(Gluc)、绿色荧光蛋白(GFP)，红色荧光蛋白(mCherry)等。
“转录”在本文中专指原核或真核生物宿主细胞中通过RNA聚合酶将DNA序列转录产生相对应RNA序列的过程。真核生物基因的转录比原核生物复杂得多，真核生物的三类RNA聚合酶I、II和III分别转录三类真核基因DNA，产生三类RNA(rRNA、mRNA、tRNA)及反义RNA。文中的转录因子调节的转录过程为RNA聚合酶II启动的转录，即DNA转录为mRNA。“转录调节”本文指真核基因转录的调节，包括启动或阻遏转录，增强或抑制转录，上调或下调转录。
“RNA代谢”、“RNA代谢活动””、“RNA代谢行为”在本文中含义相同可互换使用，指的是RNA在转录生成之后经历的一系列代谢过程，包括但不限于剪接、表观遗传学修饰、运输、定位、翻译、降解等，其中表观遗传学修饰包括但不限于甲基化修饰、假尿嘧啶修饰。
“调控效果”、“RNA代谢调控效果”在本文中指重组光控RNA效应因子在蓝光光照和黑暗条件下调控靶标RNA代谢的差异，可以是直接的，也可以是间接的，例如mRNA的水平既可以通过检测mRNA的含量来直接反应，也可以通过其翻译产生的蛋白质的水平来间接反应。一般情况下，光照与黑暗条件下靶标RNA的代谢差异越大，说明重组光控RNA效应因子的调控效果越好。在实际应用中，只要光照和黑暗条件下靶标RNA的代谢存在统计学意义上的差异，就可以认为该重组光控RNA效应因子具有调控靶标RNA代谢的能力。在本发明的一具体实施列中，重组光控RNA核酸酶因子在光照条件下对靶标RNA的降解是黑暗条件下的7.4倍；在本发明的另一具体实施方式中，重组光控RNA核酸酶因子在光照条件下对靶标RNA的降解体现在其编码的蛋白质水平为黑暗条件下的13.1倍。在本发明的另一具体实施列中，重组光控RNA核酸酶因子在光照条件下对靶标RNA的降解体现在其编码的蛋白质水平为黑暗条件下的65％。在本发明的另一具体实施列中，重组光控RNA翻译起始因子在光照条件下激活靶标RNA翻译的水平是黑暗条件下的8.5倍；在本发明的另一具体实施列中，重组光控RNA剪接因子在光照条件下促进靶标RNA外显子包含的剪接是黑暗条件下的2.6倍。
“表达”、“目的蛋白基因表达”、“基因表达”在本文中含义相同可互换使用，指目的基因的DNA序列转录产生携带该基因信息的RNA(mRNA或反义RNA)和该RNA携带的信息在核糖体中被翻译产生目的蛋白二者，即转录产生信息RNA和翻译产生目的蛋白都叫做表达。本文包括这两种含义，主要指产生目的蛋白。
“RNA效应因子”、“RNA效应因子融合蛋白”、“RNA效应蛋白”“RNA效应结构域”、“RNA代谢调控因子”、“RNA代谢调控因子结构域”在本文中含义相同可互换使用，指原核或真核生物中可以调控RNA代谢的蛋白质，它可以是一个蛋白质，也可以是多个相互作用的蛋白或多肽的统称，可以是天然的或人工改造的或人为融合的，包括但不限于RNA剪接调控结构域、RNA翻译起始因子结构域、RNA翻译抑制因子结构域、RNA核酸酶结构域、RNA外切酶结构域、RNA表观遗传学修饰结构域，其中RNA表观遗传学修饰结构域包括但不限于RNA甲基化酶结构域、RNA去甲基化酶结构域、RNA假尿嘧啶合成酶结构域。RNA的效应蛋白质可以单独或募集其它RNA效应多肽一起，通过与靶调控单元中反应元件的结合和相互作用，从而调控靶标RNA的各类代谢活动。
“光控RNA结合蛋白”在本发明中指由第一多肽和第二多肽构成的一种融合蛋 白，在适当波长的光照射下，融合蛋白的二聚化能力发生改变，使得其与反应元件的结合能力发生改变。在本发明的一具体实施方式中，第一多肽LicT CAT与第二多肽VIVID(N56K+C71V+I85V)操作性连接获得重组光控RNA结合蛋白LicV，其氨基酸序列为序列SEQ ID NO：1。
“重组光控RNA定位因子”在本发明中指将本发明所述光控RNA结合蛋白与第四多肽直接相连或操作性(即可隔开若干个氨基酸)相连获得。所述光控RNA定位因子可调控活细胞中靶标RNA的定位。在本发明的一具体实施方式中，光控RNA结合蛋白与细胞内膜定位信号操作性连接；在本发明的另一具体实施方式中，光控RNA结合蛋白与细胞核定位信号操作性连接。
“靶调控单元”指人造的由反应元件和靶标RNA序列组成的RNA序列(不是蛋白质)，其中反应元件可以位于靶标RNA序列的5’端，可以是靶标RNA序列的3’端，也可以位于靶标RNA序列的中间，二者可直接相连或操作性(即可隔开若干个核苷酸)相连。
“靶调控单元编码核苷酸序列”在本专利中指可以转录产生本专利所述靶调控单元的DNA序列。
“待调控RNA编码核苷酸序列”、“靶标RNA编码核苷酸序列”、“目的RNA编码核苷酸序列”在本文中含义相同可互换使用，指可以转录产生本专利所述待调控RNA或目的RNA或靶标RNA的DNA序列。这种DNA序列可包含在宿主细胞的染色体DNA序列中或包含在人工构建的表达载体中。
“反应元件”指RNA效应因子特异性识别/结合的一个或多个RNA基序，不同的RNA效应因子有与其相对应的不同的反应元件，RNA效应因子包含能与这种RNA基序结合的结合结构域。当RNA效应因子与其相应的反应元件特异性结合后，RNA效应因子自身或其招募的辅因子协同作用于目的RNA序列，调控RNA的代谢活动。在本发明中，反应元件指能够与重组光控RNA效应因子的第一多肽特异性识别/结合的RNA基序，例如LicT的反应元件为长25bp的RNA基序(序列2)。
“启动子”指启动和导致其下游基因转录产生RNA的DNA序列。启动子包括原核启动子和真核启动子，可以是天然基因的启动子或人工修饰的启动子。不同的启动子可指导基因在不同发育阶段的不同类型的组织或细胞中转录，或对不同环境或生理条件反应时的基因表达。启动子通常可分为“组成型启动子”、“可诱导启动子”或“可调控启动子”；按组织和细胞划分可分为“细胞特异性启动子”、“组织特异性启动子”、 “发育特异性启动子”或“细胞分化特异性启动子”。可表达的天然细胞结构蛋白基因上游都有与其相配的启动子，不同的基因DNA片段可以有相同的启动子。可用于表达本发明重组光敏转录因子的常用组成型启动子的非限制性例子有：来源于多瘤病毒、腺病毒2、巨细胞病毒CMV和猿病毒40(SV40)的启动子。多数真核基因转录起始点上游大约-25于-30位核苷酸处有富含AT的区域称为TATA盒，本文称为“最小启动子”，它确定了目的基因的转录起始位点，但本身不足以有效地启动基因转录。在TATA盒上游还有其它转录必须的核苷酸基序，即本文所述的转录因子其特异性识别/结合的反应元件，该反应元件被其相应的转录因子结合后向最小启动子传达反应性，并在转录因子募集的辅因子协同作用下激活最小启动子引起下游基因转录产生相应的目标RNA。
“载体”、“表达载体”、“基因表达载体”、“重组基因表达载体”或“质粒”在本文中含义相同可互换使用，指能在原核或真核细胞中表达重组蛋白或靶标RNA的载体，这种表达载体可以是人工构建的质粒或重组病毒载体
“转染”指宿主细胞经物理或化学方法，如电穿孔、磷酸钙共沉淀、脂质转染胺或DEAE-葡聚糖介导的转染、DNA粒子轰击和显微注射等处理，使细胞摄入外源加入的携带基因的表达载体，或通过生物学媒介，如逆转录病毒载体、腺病毒载体、受体介导的DNA摄取等将携带基因的表达载体递送入宿主细胞中。这些载体进入宿主细胞后可作为游离体形式存在于胞质中，或整合入细胞染色体中，在适当条件下该细胞可瞬时表达或长期表达载体所携带的基因编码的蛋白质或功能性RNA。这种宿主细胞就叫做被载体转染的细胞。用表达载体转染宿主细胞的方法可参见Sambrooka等人(分子克隆实验手册，第二版，冷泉港出版社(1989))，和其它有关教材。
本发明基于光敏多肽的光控RNA代谢调控系统第一部分的重组光控RNA效应因子是由三种或四种功能性多肽片段直接通过肽键串联连接或通过接头肽串联连接形成的融合蛋白。在适当波长的光照射下，该融合蛋白能结合于本发明第二部分靶调控单元中的反应元件，通过其自身或招募宿主细胞本身的其他辅因子，一起协同作用于靶调控单元中的靶标RNA序列，从而调控靶标RNA的代谢。
本文中，“重组RNA效应因子融合蛋白”与“重组RNA效应因子”含义相同，可互换使用。
本发明的重组光控RNA效应因子含有第一多肽，该多肽能特异性识别结合所述靶调控单元中的反应元件RNA序列；第一多肽选自：抗转录终止因子蛋白的RNA识别结合结构域、RNA衰减子RNA识别结合结构域、RNA干扰酶RNA识别结合结构域、 小调节RNA结合蛋白RNA识别结合结构域、RNA解旋酶RNA识别结合结构域、核酶RNA识别结合结构域、tRNA结合蛋白RNA识别结合结构域、rRNA结合蛋白RNA识别结合结构域。分析相关文献，可用作本发明的第一多肽优选包括但不限于：粗糙芽孢杆菌LicT蛋白的RNA识别结合结构域、大肠杆菌BglG蛋白的RNA识别结合结构域、枯草芽胞杆菌SacY蛋白的RNA识别结合结构域、枯草芽胞杆菌GlcT蛋白的RNA识别结合结构域、PyrR蛋白的RNA识别结合结构域、RapZ蛋白的RNA识别结合结构域、EndoA蛋白的RNA识别结合结构域、蛋白的RNA识别结合结构域，更优选LicT蛋白的RNA识别结合结构域、BglG蛋白的RNA识别结合结构域、SacY蛋白的RNA识别结合结构域和GlcT蛋白的RNA识别结合结构域。在本发明一优选实施方式中，第一多肽为LicT蛋白RNA识别结合结构域。在本发明另一优选实施方式中，第一多肽为BglG蛋白RNA识别结合结构域。在本发明另一优选实施方式中，第一多肽为SacY蛋白的RNA识别结合结构域。在本发明另一优选实施方式中，第一多肽为GlcT蛋白的RNA识别结合结构域。
除了本发明优选实施方式中使用的第一多肽，其中所述第一多肽还可选自其他抗转录终止因子蛋白的RNA识别结合结构域，包括但不限于枯草芽胞杆菌SacT蛋白的RNA识别结合结构域、菊欧氏杆菌(Erwinia chrysanthemi)Arbg蛋白的RNA识别结合结构域、乳酸菌(Lactococcus lactis)BglR蛋白的RNA识别结合结构域、干酪乳杆菌(Lactobacillus casei)LacT蛋白的RNA识别结合结构域、肉葡萄球菌(Staphylococcus carnosus)GlcT蛋白的RNA识别结合结构域。除了抗转录终止因子蛋白的RNA识别结合结构域以外，其中所述第一多肽还可选自RNA衰减子RNA识别结合结构域、RNA干扰酶RNA识别结合结构域、小调节RNA结合蛋白RNA识别结合结构域、RNA解旋酶RNA识别结合结构域、核酶RNA识别结合结构域、tRNA结合蛋白RNA识别结合结构域、rRNA结合蛋白RNA识别结合结构域。
本发明重组光控RNA效应因子融合蛋白中的第二多肽是光敏多肽，该多肽来自以黄素类(FMN或FAD)为生色团的光敏结构域。如含有光-氧-电压(LOV)结构域的光敏蛋白；类似光裂解酶的隐花色素(photolyase-like cryptochromes)；利用FAD的蓝光蛋白(blue light using FAD，BLUF)。优选含LOV结构域的光敏蛋白，经适当波长光照射后，第二多肽的二聚化能力发生改变，使重组光控RNA效应因子的二聚化能力发生变化，二聚化的重组光控RNA效应因子结合于相应的反应元件，从而调节目的RNA的代谢活动。本发明包括但不限于以下所述的优选光敏蛋白或其功能活性截短 体：粗糙链孢霉菌的VIVID LOV结构域、细菌Erythrobacter litoralis的EL222 LOV结构域、燕麦光敏色素1基因的LOV结构域AsLOV2、无隔藻金色素蛋白1的LOV结构域AuLOV、恶臭假单胞菌的PpLOV LOV结构域。
本发明第一个优选的第二多肽是粗糙链孢霉菌的VIVID蛋白的光敏结构域及其突变体。VIVID是存在于粗糙链孢霉菌(Neurospora crassa)细胞内参与蓝光调控细胞信号传导通路的一种光敏蛋白质。在蓝光照射下它能与黄素腺嘌呤二核苷酸(FAD)发生蛋白分子间反应形成二聚体。全长VIVID蛋白含有186个氨基酸，只含一个对光敏感的LOV结构域。研究表明VIVID蛋白缺失了N端36个氨基酸的截短体蛋白(VIVID-36)稳定性比全长蛋白更好，而蓝光照射后形成的VIVID-36二聚体在黑暗条件下恢复单体形式，含点突变N56K和C71V的VIVID-36二聚化能力更强。在本发明一优选实施方式中，第二多肽是含两个点突变的删除前1-36个氨基酸的VIVID(N56K+C71V)。
本发明第二个优选的第二多肽是细菌Erythrobacter litoralis EL222蛋白的LOV结构域。EL222蛋白的LOV结构域位于其N端的1-182个氨基酸，在蓝光照射下能与黄素单核苷酸(FMN)结合生成一种加成产物，进而形成同源二聚体。本发明将EL222蛋白的LOV结构域连接于第一多肽，成功导致可用光照调节重组光控RNA效应因子的第一多肽与对相应反应元件的结合能力。本发明含有EL222蛋白的LOV结构域为第二多肽的光控RNA效应因子LicEB，其在光照条件下可以结合其对应的反应元件，促进靶标RNA的降解。
本发明第三个优选的第二多肽是燕麦(Avena sativa)光敏色素1基因的LOV2结构域(AsLOV2)。燕麦细胞光敏色素1的N端为LOV1和LOV2光氧电压(LOV)结构域，在蓝光照射下均能与黄素单核苷酸(FMN)结合生成一种加成产物。本发明将燕麦光敏色素1的LOV2结构域连接于第一多肽，成功导致可用光照调节重组光控RNA效应因子的第一多肽与对相应反应元件的结合能力。本发明含有AsLOV2为第二多肽的光控RNA效应因子LicAsB，其在光照条件下可以结合其对应的反应元件，促进靶标RNA的降解。
本发明第四个优选的第二多肽是无隔藻(Stramenopile algae Vaucheria frigida)金色素1(aureochrome1)蛋白C端的LOV结构域(简写为AuLOV)。该LOV结构域在蓝光照射下能与黄素单核苷酸(FMN)结合生成一种加成产物。本发明将无隔藻金色素1的LOV2结构域连接于第一多肽，成功导致可用光照调节重组光控RNA效应因 子的第一多肽与对相应反应元件的结合能力。本发明含有AuLOV为第二多肽的光控RNA效应因子LicAuB，其在光照条件下可以结合其对应的反应元件，促进靶标RNA的降解。
本发明第五个优选的第二多肽是恶臭假单胞菌(Pseudomonas putida)的LOV结构域(简称为PpLOV)。该LOV结构域在蓝光照射下能与黄素单核苷酸(FMN)结合生成一种加成产物。本发明将PpLOV结构域连接于第一多肽，成功导致可用光照调节重组光控RNA效应因子的第一多肽与对相应反应元件的结合能力。本发明含有PpLOV为第二多肽的光控RNA效应因子LicPB，其在光照条件下可以结合其对应的反应元件，促进靶标RNA的降解。
本发明的重组光控RNA效应因子含有第三多肽，该多肽是一种RNA效应结构域，它可以是调控RNA任一代谢活动的蛋白质，包括RNA的剪接、表观遗传学修饰、运输、定位、翻译、降解等代谢活动。所述第三多肽包括但不限于RNA剪接调控结构域、RNA翻译起始因子结构域、RNA翻译抑制因子结构域、RNA核酸酶结构域、RNA外切酶结构域、RNA表观遗传学修饰结构域，其中RNA表观遗传学修饰结构域包括但不限于RNA甲基化酶结构域、RNA去甲基化酶结构域、RNA假尿嘧啶合成酶结构域。RNA的效应结构域可以单独或募集其它RNA效应多肽一起，通过与靶调控单元中反应元件的结合和相互作用，从而调控靶标RNA的各类代谢活动。在本发明的实施方案中，第三多肽为hnRNP A1RNA剪接调控结构域，它可以调控RNA的可变剪接过程；在本发明的另一实施方案中，第三多肽为丝氨酸-精氨酸(SR)蛋白的富含精氨酸-苏氨酸(RS)RNA剪接调控结构域，它可以调控RNA的可变剪接过程；在本发明的另一实施方案中，第三多肽为eIF4E RNA翻译起始因子结构域，它可以特异性地识别mRNA的5'端的帽子结构，通过招募eIF4A和eIF4G共同组成eIF4F复合物参与翻译起始；在本发明的另一实施方案中，第三多肽为解淀粉芽孢杆菌中barnase RNA核酸酶结构域，它可以催化水解RNA，导致其降解失去功能；在本发明的另一实施方案中，第三多肽为SMG6蛋白的PIN(PilT amino terminus)RNA核酸酶结构域，它可以催化水解RNA，导致其降解失去功能。
本发明的重组光控RNA效应因子融合蛋白还可包括第四多肽，该多肽是定位信号肽用以促进融合蛋白向不同细胞器运输。所述第四多肽与第一、第二、第三多肽直接或通过接头肽相连。所述第四多肽可选自细胞核定位信号肽、线粒体定位信号肽、高尔基体定位信号肽、内质网定位信号肽、细胞质定位信号肽、线粒体外膜定位信号肽、细胞 膜内膜定位信号。在本发明的一具体实施方案中，第四多肽为细胞核定位信号肽，它可以介导本发明所述重组光控RNA效应因子定位到细胞核。其中，第四多肽可以是一个或多个，如果需要或多个效果更好的话优选多个。
如上所述，本发明的重组光控RNA效应因子所含的三种或四种多肽各自可以有多种选择，将三种或四种多肽连接成融合蛋白又可以有多种组合选择，本发明优选具有良好活性的各多肽的功能结构域片段制备重组光控RNA效应因子融合蛋白，通过在宿主细胞中表达优选的RNA代谢调控能力强的，即诱导和非诱导时导致靶标RNA代谢差异大的该重组光控RNA效应因子，用于调节靶标RNA的代谢，但不论何种选择与组合，只要能实现本发明所设想的利用光调控宿主细胞中靶标RNA代谢的重组光控RNA效应因子各种组合都属于本发明的范围。
本发明基于重组光控RNA效应因子的光控RNA代谢调控系统的第二部分是由重组光控RNA效应因子特异性识别/结合的反应元件-待调控RNA序列组成的靶调控单元(核苷酸序列)，具体来说，其中反应元件的核苷酸基序视本发明不同实施方式所选的重组光控RNA效应因子融合蛋白的第一多肽不同而不同。换句语说，反应元件是第一多肽的特异性反应元件，必须根据所选择的第一多肽来选择与其相应的反应元件。例如，第一多肽为LicT、BglG、SacY、GlcT蛋白的RNA识别/结合结构域时，其相应的反应元件应为“序列2、3、4、5”基序。靶转录单元中的反应元件至少为一个或可以有多个，在具体的实施方式中，反应元件为1、2、3、4或5个，如果需要或多个效果更好的话优选多个。
与反应元件操作性相连的是待调控的靶标RNA序列，可以是具有功能的任意RNA核苷酸序列。为了验证本发明系统的效果和便于检测，在本发明的实施例中，采用了示范性的报告RNA：Pepper荧光RNA、高斯荧光素酶(Gluc)mRNA、绿色荧光蛋白(GFP)mRNA，红色荧光蛋白(mCherry)mRNA，但本发明的靶标RNA不限于这些报告RNA。在本发明的本领域技术人员知道，所谓“操作性相连指反应元件与靶标RNA序列之间或多个反应元件之间不是直接相连而可以隔开若干个核苷酸，只要仍能协同作用即可。
可用标准重组DNA技术将本发明光控RNA代谢调控系统的第一部分和第二部分构建在一个原核或真核表达载体中或分别构建在二个原核或真核表达载体中。可用标准技术将这种表达载体引入各种宿主细胞群中调控靶标RNA的各类代谢活动，进一步选择产生有用的转基因生物，如转基因小鼠。本发明的光控RNA代谢调控系统可用于在 宿主细胞中内源RNA或外源RNA代谢的调控。
本领域众所周知，氨基酸的密码子核苷酸有简并性(即某些氨基酸可有二个、或三个、或四个密码子，它们称为该氨基酸的简并密码子)，本发明所述的各种重组光控RNA效应因子的编码核酸，本发明包括它们各自的所有简并核苷酸序列。本发明所述的各种重组光控RNA效应因子的氨基酸序列，本发明包括它们各自的所有含保守性缺失、添加、置换修饰但仍保留了其原有功能活性的氨基酸序列类似物。
本发明提供含待调控的靶标RNA编码核苷酸序列空缺的靶调控单元的原核或真核表达载体，待调控的靶标RNA编码核苷酸序列空缺是让用户可以自行选择所需的待调控的靶标RNA编码核苷酸序列，例如目的RNA的编码核苷酸序列，用标准的重组DNA技术将其插入本发明的这种表达载体中，通过上面所述重组光控RNA效应因子来调节转录出来的靶标RNA的代谢活动。
本发明也提供分别转化了各种重组光控RNA效应因子基因的宿主细胞表达载体或者基因组上整合了各种重组光控RNA效应因子表达框的菌株或细胞株，同时提供含有反应元件-待调控靶标RNA编码核苷酸序列空缺的表达载体。用户可用标准重组DNA技术将自行选择的待调控靶标RNA编码核苷酸序列插入该表达载体中，然后将该重新构建的载体引入已转化了各种重组光控RNA效应因子的宿主细胞或者基因组上已整合了各种重组光控RNA效应因子表达框的宿主菌株或细胞株，并培养这种菌株或细胞株表达重组光控RNA效应因子和反应元件-靶标RNA，利用光调控靶标RNA的代谢活动，探究它们的生物学功能。
本发明还提供装有各种表达载体或已转化了这种载体或者基因组上整合了各种重组光控RNA效应因子表达框的宿主细胞的试剂盒。在一个实施方式中，该试剂盒中一些容器分别装有含一种或多种重组光控RNA效应因子基因的表达载体。在另一个实施方式中，该试剂盒中的一些容器分别装有含一种或多种重组光控RNA效应因子基因的表达载体，另一些容器分别装有含靶调控单元(其中反应元件-待调控靶标RNA编码核苷酸序列空缺)的表达载体。在还有一个实施方式中，该试剂盒中一些容器装有已转化了含重组光控RNA效应因子基因的表达载体或者基因组上已整合了各种重组光控RNA效应因子表达框的宿主细胞，另一些容器装有反应元件-待调控靶标RNA编码核苷酸序列空缺的宿主细胞表达载体。本发明的试剂盒还可以包含相应的光照控制设备，例如LED灯及其调控装置。所有试剂盒都装有相应的说明书，以说明盒中的各成分、使用目的和使用方法，并提供有关的参考文献目录。
本发明还包括光控RNA代谢调控系统在宿主细胞中调控靶标RNA代谢的方法，包括步骤：
a)将所光控RNA代谢调控系统构建在原核或真核质粒表达载体中；
b)将重组质粒引入宿主细胞；
c)光照诱导所述宿主细胞，调控宿主细胞中靶标RNA的代谢。
光照诱导所述宿主细胞的方法包括光源的选择和光源的使用。光源非限制地包括LED灯、白炽灯、荧光灯、激光。在本发明的一个实施方式中，光源选用蓝色LED(460-470nm)。光照方法包括光照量、光照强度、光照时间、光照频率以及用扫描、投影、光模具等方法在空间上控制靶标RNA的代谢也包含在本发明范围中。在本发明的一个实施方式中，光照强度为0-1.8W/m 2不等；在另一个实施方式中，用打印的投影片作为光模具，在空间上调节不同位置的细胞的靶标RNA的代谢；在另一个实施方式中，用中性灰度片作为光模具，在空间上调节不同位置的细胞的靶标RNA的代谢水平。
附图简要说明
图1含不同第一多肽的重组光控RNA效应因子质粒示意图。
图2含不同第二多肽的重组光控RNA效应因子质粒示意图。
图3含不同第三多肽的重组光控RNA效应因子质粒示意图。
图4含不同第一多肽的重组光控RNA核酸酶因子对活细胞中靶标Gluc mRNA的降解效果。检测结果采用学生t检验进行光照与黑暗条件下的差异分析，*p<0.05,**p<0.01，***p<0.001，N.S.,无显著差异。
图5含不同第二多肽的重组光控RNA核酸酶因子对活细胞中靶标Gluc mRNA的降解效果。检测结果采用学生t检验进行光照与黑暗条件下的差异分析，*p<0.05,**p<0.01，***p<0.001，N.S.,无显著差异。
图6重组光控RNA核酸酶因子LicVB对活细胞中靶标mCherry mRNA的降解效果。标尺，100μm。
图7重组光控RNA核酸酶因子LicVB对活细胞中Pepper靶标RNA的降解效果。标尺，50μm。
图8重组光控RNA核酸酶因子LicVPIN对活细胞中Pepper靶标RNA的降解效果。标尺，50μm。
图9重组光控RNA核酸酶因子LicVB对大肠杆菌和酵母细胞中靶标mCherry  mRNA的降解效果。检测结果采用学生t检验进行光照与黑暗条件下的差异分析，*p<0.05,**p<0.01，***p<0.001，N.S.,无显著差异。
图10重组光控RNA剪接因子LicVA1和LicVRS对活细胞中靶标RNA剪接的调控效果。(A)剪接产物逆转录并扩增后跑胶结果；(B)对(A)电泳胶条带的统计扽西结果。检测结果采用学生t检验进行光照与黑暗条件下的差异分析，*p<0.05,**p<0.01，***p<0.001，N.S.,无显著差异。
图11重组光控RNA翻译起始因子LicV4E对活细胞中靶标RNA翻译的调控效果。标尺，200μm。
图12不同光照强度下重组光控RNA翻译起始因子LicV4E对活细胞中靶标RNA翻译的调控效果。标尺，1mm。
图13重组光控RNA翻译起始因子LicV4E在空间上精确调控靶标RNA的翻译效果。标尺，3mm。
图14重组光控RNA定位因子对靶标RNA定位的调控效果。(A)LicV-mKalama1-CAAX对Pepper-RAT LicT靶标RNA定位调控的成像效果；(B)对(A)中靶标RNA成像的统计结果；(C)LicV-mKalama1-3xNLS对Pepper-RAT LicT靶标RNA定位调控的成像效果；(D)对(A)中靶标RNA成像的统计结果。标尺，25μm。
具体实施方式
以下用实施例对本发明作进一步阐述。这些实施例仅仅用于举例说明，而不对本发明的范围构成任何限制。实施例中主要采用常规的基因工程分子生物学克隆方法，这些方法是本领域普通技术人员所熟知的，例如：简·罗斯凯姆斯等的《分子生物学实验参考手册》和J.萨姆布鲁克，D.W.拉塞尔著,黄培堂等译：《分子克隆实验指南》(第三版，2002年8月，科学出版社出版，北京)中的有关章节。本领域普通技术人员按照以下实施例，不难根据具体情况略作修改和变换而成功实施本发明，这些修改和变换均落在本申请权利要求的范围内。
实施例中所有用于PCR的引物均由上海杰瑞生物工程技术有限公司合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自东盛生物，pfu DNA聚合酶购自天根生化科技(北京)有限公司，PrimeSTAR DNA聚合酶购自TaKaRa公司，三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTP。T4连接酶、T4磷酸化酶(T4 PNK)购自 Fermentas公司，购买时附带有相对应的缓冲液等。实施例中所用的一步法快速克隆试剂盒(含同源重组酶)购自翊圣生物科技有限公司。除非特别声明，无机盐类化学试剂均购自国药集团上海化学试剂公司。卡那霉素(Kanamycin)购自Ameresco公司；氨苄青霉素(Amp)购自Ameresco公司；链霉素购自Ameresco公司；384孔发光检测白板、384孔荧光检测黑板购自Grenier公司。
实施例中所用的胶回收试剂盒购自生工公司，普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。克隆菌株MachI购自北京全式金公司，BL21(DE3)菌株购自北京全式金公司。细胞系HEK293T和HEK293购自中国科学院典型培养物保藏委员会细胞库。
实施例中用到的主要仪器：Biotek Synergy 2多功能酶标仪(美国Bio-Tek公司)，X-15R高速冷冻离心机(美国Beckman公司)，Microfuge22R台式高速冷冻离心机(美国Beckman公司)，PCR扩增仪(德国Biometra公司)，共聚焦显微镜(LEICA TCS SP8MP)(德国Leica)，光度计(日本和光公司)，核酸电泳仪(申能博彩公司)、LED蓝灯(深圳虹升光电公司定制)。
缩写词意义如下：“h”指小时，“min”指分钟，“s”指秒，“d”指天，“μL”指微升，“mL”指毫升，“L“指升，“bp”指碱基对，“mM”指毫摩尔，“μM”指微摩尔。
20种氨基酸及简称
中文名称 三字母缩写 单字母符号 中文名称 三字母缩写 单字母符号
甘氨酸 Gly G 苏氨酸 Thr T
丙氨酸 Ala A 半胱氨酸 Cys C
缬氨酸 Val V 蛋氨酸 Met M
亮氨酸 Leu L 天冬酰胺 Asn N
异亮氨酸 Ile I 谷氨酰胺 Gln Q
脯氨酸 Pro P 天冬氨酸 Asp D
苯丙氨酸 Phe F 谷氨酸 Glu E
酪氨酸 Tyr Y 赖氨酸 Lys K
色氨酸 Trp W 精氨酸 Arg R
丝氨酸 Ser S 组氨酸 His H
实施例中用到的常规分子生物学方法
(一)DNA片段5’端磷酸化反应然后自身环化反应：
从微生物中抽提出的质粒或者基因组末端都含有磷酸基团，而PCR产物没有，故需对PCR产物的5’端碱基进行磷酸基团加成反应，只有末端含有磷酸基团DNA分子才能发生连接反应。自身环化连接反应指线性化载体的3’端和5’端连接反应。
Figure PCTCN2022115877-appb-000001
T4 PNK为T4多聚核苷酸激酶的简写，用于对DNA分子的5’端磷酸基团的加成反应。使5’端磷酸化的DNA片段产物自身环化的反应体系：
Figure PCTCN2022115877-appb-000002
(二)重叠PCR
重叠PCR是常用的将两段不同或相同的基因连接起来的方法。例如图1，将基因AD和基因BC连接起来，首先设计两对引物A、D和C、B，分别用来扩增基因AD和BC，在引物D和引物C的5’端含有一定长度的互补序列。第一轮PCR得到的扩增产物AD和BC经过回收后作为第二轮PCR的模板。
第二轮按常规PCR流程扩增10轮，PCR体系为：
Figure PCTCN2022115877-appb-000003
第二轮PCR后加入引物A和引物B，再继续扩增30轮即可得到AD和BC连接了的序列。
(三)反向PCR
反向PCR是下述实施例中用于定点突变、截短突变、插入突变所用到的一种技术。其基本原理参考Takara公司的MutaBEST试剂盒的实验流程。在对应的变异部位设计反向PCR引物，其中一条引物的5’端包含变异的核苷酸序列。扩增后的产物经过胶回收纯化后，进行5’端磷酸化反应然后自身环化反应，转化入感受态细胞。
(四)一步法同源重组片段
用于同源重组的PCR目的片段的5’和3’末端序列分别和线性化载体的末端序列完全一致，使用一步克隆试剂盒进行同源重组反应，反应体系为：
Figure PCTCN2022115877-appb-000004
(五)荧光素酶(Gluc)活性的检测
检测报告基因荧光素酶Gluc的相对表达量的实验根据NEB公司试剂盒提供的使用说 明进行。细胞培养到了合适的时间，从细胞培养液中吸取10μl加到Grenier公司的384孔白板中，现场配置新鲜的Gluc检测液(1μM腔肠素，0.1M Tris-HCl缓冲液，0.3M抗坏血酸钠，pH 7.4)，用Eppendolf的12通道电动分液器吸取检测液10μl/通道加入到384孔白板中的细胞培养液中，立即使用多功能酶标仪读取相对发光强度(RLU)。
(六)细胞培养、转染与荧光检测
在适当的温度和气体混合物(通常为37℃，5％CO 2)环境下用DMEM(HyClone，目录号：SH302431)培养HEK293与HEK293T等细胞。DMEM中含有4mM谷氨酰胺，4.5g/l葡萄糖，10％胎牛血清(FBS)，丙酮酸钠和终浓度为100U/ml的青霉素以及100μg/ml的链霉素。除非另有说明，用Hieff TransTM脂质体转染试剂(Yeasen)、Lipo3000或Lipo2000转染试剂(Thermo)根据生产商的说明书进行细胞转染。在细胞成像实验中，利用Leica SP8共聚焦显微镜使用561nm的激光器对mCherry荧光进行成像，使用488nm的激光器对EGFP荧光进行成像，使用458nm的激光器对Pepper485和mKalama1的荧光进行成像。
实施例1分别构建含不同重组光控RNA效应因子编码基因的表达载体。
为构建含有以LicT、BglG、SacY或GlcT蛋白的RNA识别结合结构域为第一多肽的重组光控RNA效应因子，全基因合成LicT、BglG、SacY和GlcT蛋白的RNA识别结合结构域编码DNA片段，分别以P1和P2、P3和P4、P5和P6、P7和P8扩增上述DNA片段获得LicT CAT、BglG CAT、SacY CAT和GlcT CAT基因片段。全基因合成第三多肽barnase(K27A+N58D+R59A+E73A)(简称barnase M4)编码DNA片段，利用P9和P10扩增该片段获得barnase M4基因片段。利用引物P11和P12以pGAVPO质粒(Wang et al.,Nature Methods,2012:266-269)为模板扩增第二多肽VVD(N56K+C71V)基因片段，利用重叠PCR分别将VVD(N56K+C71V)、barnase M4和LicT CAT、BglG CAT、SacY CAT或GlcT CAT基因片段进行连接，分别获得LicT CAT-VVD(N56K+C71V)-barnase M4(简称LicVB，其氨基序列为序列SEQ ID NO：6)、BglG CAT-VVD(N56K+C71V)-barnase M4(简称BglVB，其氨基序列为序列SEQ ID NO：7)、SacY CAT-VVD(N56K+C71V)-barnase M4(简称SacVB，其氨基序列为序列SEQ ID NO：8)和GlcT CAT-VVD(N56K+C71V)-barnase M4(简称GlcVB，其氨基序列为序列SEQ ID NO：9)基因片段。利用P13和P14扩增pEGFP-N1-FLAG载体(Addgene：60360)使其线性化，利用一步法克隆试剂盒将上述重叠PCR获得的基因片段插入到线性化的 pEGFP-N1-FLAG载体中，得到的质粒命名为pCMV-LicVB、pCMV-BglVB、pCMV-SacVB和pCMV-GlcVB(图1)，分别编码重组光控核酸酶因子LicVB、BglVB、SacVB和GlcVB。
扩增LicT CAT基因片段的引物为：
上游引物(P1)：5’-agatccgctagcgctatgaaaattgcgaaggtgat-3’
下游引物(P2)：5’-agcgtagagcgtatgtcctgcggctttgctaattcttgctgatacatccttgttatcga-3’
扩增BglG CAT基因片段的引物为：
上游引物(P3)：
5’-agatccgctagcgctatgaacatgcaaatcaccaaaattc-3’
下游引物(P4)：
5’-agcgtagagcgtatgtacgtcccaggtaccgttcagttcatgactgctcaag-3’
扩增SacY CAT基因片段的引物为：
上游引物(P5)：
5’-agatccgctagcgctatgaaaattaaaagaatcttaaatc-3’
下游引物(P6)：
5’-agcgtagagcgtatgagacccaccgccatagtcaggtgtatcttttctg-3’
扩增GlcT CAT基因片段的引物为：
上游引物(P7)：
5’-agatccgctagcgctatgaatgggtccttcacagtg-3’
下游引物(P8)：
5’-agcgtagagcgtatgcaggtcacaagtacctcgacattgttccttctcgtcttttaa-3’
扩增barnase M4基因片段的引物为：
上游引物(P9)：
5’-gttccagattacgctgaattcatggcacaggttatcaacacgtttg-3’
下游引物(P10)：
5’-gatctagagtgtacattatctgatttttgtaaaggtttga-3’
扩增VVD(N56K/C71V)基因片段的引物为：
上游引物(P11)：5’-catacgctctacgctcccggcggt-3’
下游引物(P12)：5’-agcgtaatctggaacatcgtatgggtactgcagttccgtttcgcactggaaac-3’
扩增pEGFP-N1-FLAG载体使其线性化的引物为：
上游引物(P13)：
5’-tgtacactctagatcataatcagc-3’
下游引物(P14)：
5’-agcgctagcggatctgacggttcac-3’
为构建含有以EL222、AsLOV、AuLOV或PpLOV蛋白的LOV结构域为第二多肽的重组光控RNA效应因子，全基因合成EL222、AsLOV、AuLOV和PpLOV蛋白LOV结构域编码DNA片段，分别以P15和P16、P17和P18、P19和P20、P21和P22扩增上述DNA片段获得EL222、AsLOV、AuLOV和PpLOV基因片段。利用P23和P24扩增本实施例构建的pCMV-LicVB载体，去除VVD(N56K+C71V)基因序列并使其线性化，利用一步法克隆试剂盒将上述基因片段插入到线性化的pCMV-LicVB载体中，得到的质粒命名为pCMV-LicEB、pCMV-LicAsB、pCMV-LicAuB和pCMV-LicPB，分别编码重组光控核酸酶因子LicEB、LicAsB、LicAuB和LicPB(图2)，其氨基酸序列分别为序列SEQ ID NO：10、11、12和13。
扩增EL222 LOV结构域基因片段的引物为：
上游引物(P15)：
5’-aacaaggatgtatcagctgatacgatactgggtagtccgagcatgctggatatgggacaaga-3’
下游引物(P16)：
5’-gtatgggtactgcagtttgagcatctcggcggctc-3’
扩增AsLOV基因片段的引物为：
上游引物(P17)：
5’-aacaaggatgtatcagtcggtagtcagcagaattttgtgataactgatgcaagc-3’
下游引物(P18)：
5’-gtatgggtactgcagcactagcaacttggcgtaatc-3’
扩增AuLOV基因片段的引物为：
上游引物(P19)：
5’-aacaaggatgtatcaatcctagtcggtacacagaattttgtgataactgatg-3’
下游引物(P20)：
5’-gtatgggtactgcagcactagcaacttggcgtaatc-3’
扩增PpLOV基因片段的引物为：
上游引物(P21)：
5’-aacaaggatgtatcatactcacgtatgattaatgcccaactcctgcagagc-3’
下游引物(P22)：
5’-gtatgggtactgcagagcccgttcgtctggttttggtcttg-3’
扩增pCMV-LicVB载体使其线性化的引物为：
上游引物(P23)：
5’-ctgcagtacccatacgatgttccag-3’
下游引物(P24)：
5’-tgatacatccttgttatcgagcg-3’
为构建含有以hnRNP A1RNA剪接调控结构域、RS RNA剪接调控结构域、eIF4E RNA翻译起始因子结构域或PIN RNA核酸酶结构域为第三多肽的重组光控RNA效应因子，全基因合成上述结构域的编码DNA片段，分别以P25和P26、P27和P28、P29和P30、P31和P32扩增上述DNA片段获得含第四多肽的A1-NLS和RS-NLS以及eIF4E和PIN基因片段。利用P33和P34扩增本实施例构建的pCMV-LicVB载体，去除barnase M4基因序列并使其线性化，利用一步法克隆试剂盒将上述基因片段插入到线性化的pCMV-LicVB载体中，得到的质粒命名为pCMV-LicVA1、pCMV-LicVRS、pCMV-LicV4E和pCMV-LicVPIN，分别编码重组光控RNA剪接调控因子LicVA1-NLS和LicVRS-NLS、重组光控RNA翻译起始因子LicV4E和重组光控核酸酶因子LicVPIN(图3)，其氨基酸序列分别为序列SEQ ID NO：14、15、16和17。
扩增A1-NLS结构域基因片段的引物为：
上游引物(P25)：
5’-cagtgcgaaacggaaggtggcggtggctcgggcggagggggttcgggaggtatgggtcgaagtggttctggaa-3’
下游引物(P26)：
5’-gatctagagtgtacattataccttcctctttttcttgggggggaggatcccaaatcttctgccact-3’
扩增RS-NLS基因片段的引物为：
上游引物(P27)：
5’-tacgctgaattcatgcgttacagccggcgaagaagaagc-3’
下游引物(P28)：
5’-gatctagagtgtacattataccttcctctttttcttgggggggaggatcccgtccattctttcaggacttg-3’
扩增eIF4E基因片段的引物为：
上游引物(P29)：
5’-tacgctgaattcatgatggcgactgtcgaaccggaaac-3’
下游引物(P30)：
5’-gatctagagtgtacactaaacaacaaacctatttttag-3’
扩增PIN基因片段的引物为：
上游引物(P31)：
5’-cagtgcgaaacggaaatggccttgcacgccagaaacatcgccatggagctcgaaatcagacc-3’
下游引物(P32)：
5’-gatctagagtgtacactagcccacctgggcccacgtgag-3’
扩增pCMV-LicVB载体使其线性化的引物为：
上游引物(P33)：
5’-tgtacactctagatcataatcagc-3’
下游引物(P34)：
5’-catgaattcagcgtaatctgga-3’
全基因合成J23117启动子和rrnB转录终止子DNA序列，利用引物P35和P36、P37和P38对它们进行扩增获得J23117启动子和rrnB DNA片段，利用引物P39和P40以本实施例中的pCMV-LicVB质粒为模板扩增LicVB基因片段，利用重叠PCR将J23117、rrnB和LicVB连接起来，获得J23117-LicVB-rrnB重组DNA片段。利用P41和P42扩增pCDFDuet1载体(Novagen)去除原有的T7启动子和多克隆位点区使其线性化，利用一步法克隆试剂盒将J23117-LicVB-rrnB片段插入到线性化的pCDFDuet1载体中，得到的细菌表达质粒命名为pJ23117-LicVB，其编码重组光控核酸酶因子LicVB。
扩增J23117启动子的引物为：
上游引物(P35)：
5’-gatggtgtccgggatggatccgcctatgcagcgac-3’
下游引物(P36)：
5’-cttcgcaattttcatagatctctgcctgaagttatagtg-3’
扩增rrnB转录终止子的引物为：
上游引物(P37)：
5’-acaaaaatcagataagagagtagggaactgccaggcatc-3’
下游引物(P38)：
5’-cggtggcagcagttagctagcgcaaacaacagataaaac-3’
扩增LicVB基因片段的引物为：
上游引物(P39)：
5’-atgaaaattgcgaaggtgatcaac-3’
下游引物(P40)：
5’-ttatctgatttttgtaaaggtttg-3’
扩增pCDFDuet1载体使其线性化的引物为：
上游引物(P41)：
5’-taactgctgccaccgctgagcaataac-3’
下游引物(P42)：
5’-atcccggacaccatcgaatggcgc-3’
利用引物P43和P44以本实施例中的pCMV-LicVB质粒为模板扩增LicVB基因片段，利用P45和P46扩增pGADT7载体(Clontech)使其线性化，利用一步法克隆试剂盒将LicVB基因片段插入到线性化的pGADT7载体中，得到的酵母表达质粒命名为pGADT7-LicVB，其编码重组光控核酸酶因子LicVB。
扩增LicVB基因片段的引物为：
上游引物(P43)：
5’-ccaagctttgcaaagatgaaaattgcgaaggtgatcaac-3’
下游引物(P44)：
5’-catctgcagctcgagttatctgatttttgtaaaggtttg-3’
扩增pGADT7载体使其线性化的引物为：
上游引物(P45)：
5’-ctcgagctgcagatgaatcgtagatac-3’
下游引物(P46)：
5’-ctttgcaaagcttggagttgattg-3’
实施例2构建含有不同靶调控单元的表达载体
为了检测光控RNA效应因子对靶标RNA代谢的调控效果，需要构建含相应靶调控单元编码核苷酸序列的表达质粒。为了检测重组光控RNA核酸酶因子调控靶标RNA降解的效果，合成2xRAT LicT编码DNA片段，利用P47和P48以其为模板进行扩增获得2xRAT LicT片段，分别利用P49和P50、P51和P52以pU5-Gluc和pU5-mCherry(Wang et al.,Nature Methods,2012:266-269)为模板扩增Gluc和mCherry基因片段，利用重叠PCR分别将2xRAT LicT片段与Gluc和mCherry连接起来，获得Gluc-2xRAT LicT和mCherry-2xRAT LicT片段。利用P53和P54扩增pCDNA3.1 hygro(+)载体(Invitrohen)使其线性化，利用一步法克隆试剂盒将Gluc-2xRAT LicT和mCherry-2xRAT LicT片段插入到线性化的pCDNA3.1载体中，得到的表达质粒分别命名为pCDNA3.1-Gluc-2xRAT LicT和pCDNA3.1-mCherry-2xRAT LicT，其分别编码Gluc- 2xRAT LicT和mCherry-2xRAT LicT靶调控单元，其核苷酸序列为序列SEQ ID NO：18和19。
扩增2xRAT LicT片段的引物为：
上游引物(P47)：
5’-aaaatggtgggattgttactgc-3’
下游引物(P48)：
5’-ccctctagactcgaggtttaaacgggccctctagac-3’
扩增Gluc基因片段的引物为：
上游引物(P49)：
5’-cccaagctggctagcatgggagtcaaagttctgtttg-3’
下游引物(P50)：
5’-caatcccaccattttttagtcaccaccggcccccttg-3’
扩增mCherry基因片段的引物为：
上游引物(P51)：
5’-cccaagctggctagcatggtgagcaagggcgaggag-3’
下游引物(P52)：
5’-caatcccaccattttctacttgtacagctcgtccatgccg-3’
扩增pCDNA3.1 hygro(+)载体使其线性化的引物为：
上游引物(P53)：
5’-ctcgagtctagagggcccgtttaaac-3’
下游引物(P54)：
5’-gctagccagcttgggtctccctatag-3’
为了直接观察重组光控RNA核酸酶因子对靶标RNA降解的调控效果，利用Pepper荧光RNA系统(Chen et al.,Nature Biotechonogy,2019,37:1287-1293)作为报告RNA。在商业化公司合成Pepper-RAT LicT编码DNA片段，利用P55和P56以其为模板进行扩增获得Pepper-RAT LicT片段。在商业化公司合成U6启动子DNA片段，利用P57和P58以其为模板进行扩增获得U6启动子片段。利用重叠PCR将Pepper-RAT与U6启动子连接起来，获得U6-Pepper-RAT LicT片段。利用P59和P60扩增pEGFP-N1-FLAG载体去除CMV启动子和多克隆位点区使其线性化，利用一步法克隆试剂盒将U6-Pepper-RAT LicT片段插入到线性化的pEGFP-N1-FLAG载体中，得到的表达质粒分别命名为pU6-Pepper-RAT LicT，其编码Pepper-RAT LicT靶调控单元，其核苷酸序列为 序列SEQ ID NO：20。
扩增Pepper-RAT LicT片段的引物为：
上游引物(P55)：
5’-tggaaaggacgaaacgggcccccaatcgtggcgtgtcggc-3’
下游引物(P56)：
5’-cgaggtcgagaattcaaaaaagggccccggcgccagtgcctgcctttc-3’
扩增U6启动子片段的引物为：
上游引物(P57)：
5’-gccgcccccttcaccgagggcctatttcccatgattc-3’
下游引物(P58)：
5’-gtttcgtcctttccacaagatatataaag-3’
扩增pEGFP-N1-FLAG载体使其线性化的引物为：
上游引物(P59)：
5’-gaattctcgacctcgagacaaatggcagtattc-3’
下游引物(P60)：
5’-ggtgaagggggcggccgctcgaggcta-3’
为了观察细菌和酵母细胞中重组光控RNA核酸酶因子调控靶标RNA降解的效果，利用引物P61和P62以pCDNA3.1-mCherry-2xRAT LicT为模板扩增mCherry-2xRAT LicT片段，在商业化公司合成J23106启动子DNA片段，利用引物P63和P64以其为模板扩增J23106启动子片段，利用重叠PCR将mCherry-2xRAT LicT与J23106启动子连接起来，获得J23106-mCherry-2xRAT LicT片段。利用P65和P66扩增实施例1中的pJ23117-LicVB载体去除J23117-LicVB使其线性化，利用一步法克隆试剂盒将J23106-mCherry-2xRAT LicT片段插入到线性化的pJ23117-LicVB载体中，得到的细菌表达质粒命名为pJ23106-mCherry-2xRAT LicT。利用P67和P68扩增pGADT7载体使其线性化，利用引物P69和P70以pCDNA3.1-mCherry-2xRAT LicT为模板扩增mCherry-2xRAT LicT片段，利用一步法克隆试剂盒将mCherry-2xRAT LicT片段插入到线性化的pGADT7载体中，得到的酵母表达质粒命名为pGADT7-mCherry-2xRAT LicT。
扩增mCherry-2xRAT LicT片段的引物为：
上游引物(P61)：
5’-atggtgagcaagggcgaggaggat-3’
下游引物(P62)：
5’-cagttccctactctcgtttaaacgggccctctagac-3’
扩增J23106启动子片段的引物为：
上游引物(P63)：
5’-cgatggtgtccgggaggatccgcctatgcagcgacaaa-3’
下游引物(P64)：
5’-gcccttgctcaccatagatctctgcctgaagttatagtg-3’
扩增pJ23117-LicVB载体使其线性化的引物为：
上游引物(P65)：
5’-gagagtagggaactgccaggcatc-3’
下游引物(P66)：
5’-tcccggacaccatcgaatggcgc-3’
扩增mCherry-2xRAT LicT片段用于构建pGADT7-mCherry-2xRAT LicT的引物为：
上游引物(P67)：
5’-ccaagctttgcaaagatggtgagcaagggcgaggaggat-3’
下游引物(P68)：
5’-ccctctagactcgagcggatcccattttagggttttgcctg-3’
扩增pGADT7载体使其线性化的引物为：
上游引物(P69)：
5’-ctcgagctgcagatgaatcgtagatac-3’
下游引物(P70)：
5’-ctttgcaaagcttggagttgattg-3’
为了检测含不同第一多肽的重组光控RNA核酸酶因子调控靶标RNA降解的效果，在商业化公司合成2xRAT BglG、2xRAT SacY和2xRAT GlcT片段，利用引物P71和P72、引物P73和P74、引物P75和P76分别对它们进行扩增，利用P77和P78扩增本实施例中的pCDNA3.1-Gluc-2xRAT LicT载体去除2xRAT LicT片段使其线性化，利用一步法克隆试剂盒分别将2xRAT BglG、2xRAT SacY和2xRAT GlcT片段插入到线性化的pCDNA3.1-Gluc-2xRAT LicT载体中，得到的表达质粒分别命名为pCDNA3.1-Gluc-2xRAT BglG、pCDNA3.1-Gluc-2xRAT SacY和pCDNA3.1-Gluc-2xRAT GlcT，其分别编码Gluc-2xRAT BglG、Gluc-2xRAT SacY和Gluc-2xRAT GlcT靶调控单元，其核苷酸序列为序列SEQ ID NO：21、22和23。
扩增2xRAT BglG片段的引物为：
上游引物(P71)：
5’-ctaaaaaatggtgggggattgtt-3’
下游引物(P72)：
5’-ccctctagactcgaggggccctctagactcgagcgga-3’
扩增2xRAT SacY片段的引物为：
上游引物(P73)：
5’-ctaaaaaatggtgggggtttgtt-3’
下游引物(P74)：
5’-ccctctagactcgaggggccctctagactcgagcgga-3’
扩增2xRAT GlcT片段的引物为：
上游引物(P75)：
5’-ctaaaaaatggtgggttactgatt-3’
下游引物(P76)：
5’-ccctctagactcgaggggccctctagactcgagcgga-3’
扩增pCDNA3.1-Gluc-2xRAT LicT使其线性化的引物为：
上游引物(P77)：
5’-gagtctagagggcccctcgagtctagagggcccgt-3’
下游引物(P78)：
5’-cccaccattttttagtcaccaccggcccccttg-3’
为了检测重组光控RNA翻译起始因子调控靶标RNA翻译的效果，在商业化公司合成4xRAT LicT和EGFP DNA片段，分别采用引物P79和P80、P81和P82以它们为模板进行扩增，再利用重叠PCR将它们连接起来，获得4xRAT LicT-EGFP片段。利用P83和P84扩增本实施例构建的pCDNA3.1-mCherry-2xRAT LicT载体，去除2xRAT LicT序列使其线性化，利用一步法克隆试剂盒将4xRAT LicT-EGFP片段插入到线性化的pCDNA3.1-mCherry-2xRAT LicT载体中，得到的表达质粒命名为pCDNA3.1-mCherry-4xRAT LicT-EGFP，其编码mCherry-4xRAT LicT-EGFP靶调控单元，核苷酸序列为序列SEQ ID NO：24。为了检测重组光控RNA剪接因子调控靶标RNA剪接的效果，利用引物P85和P86通过反向PCR将RAT LicT片段插入pGZ3-GUM载体(中科院王泽峰老师课题组馈赠))中，线性化的片段通过磷酸化后连接，得到了质粒命名为pGZ3-GUM-RAT LicT，其编码的靶调控单元核苷酸序列为序列SEQ ID NO：25。
扩增4xRAT LicT片段的引物为：
上游引物(P79)：
5’-gagctgtacaagtagaatacgactcactatagggag-3’
下游引物(P80)：
5’-gcccttgctcaccatggtggccaagctttgtacag-3’
扩增EGFP片段的引物为：
上游引物(P81)：
5’-atggtgagcaagggcgaggagctg-3’
下游引物(P82)：
5’-ccctctagactcgagctacttgtacagctcgtccatg-3’
扩增pCDNA3.1-mCherry-2xRAT LicT载体使其线性化的引物为：
上游引物(P83)：
5’-ctcgagtctagagggcccgtttaaac-3’
下游引物(P84)：
5’-ctacttgtacagctcgtccatgccgccggtggag-3’
扩增pGZ3-GUM载体使其线性化的引物为：
上游引物(P85)：
5’-gcaggcaaaacccttcgggcccagcatcgctgga-3’
下游引物(P86)：
5’-cgtagcagtaacaatcccattctcgagaaccatacgaactttg-3’
实施例3构建不同光控RNA定位因子表达质粒
为了检测重组光控RNA定位因子对靶标RNA定位的调控效果，全基因合成mKalama1蓝色荧光蛋白编码基因、CAAX细胞内膜定位信号编码基因和3xNLS核定位信号编码基因，利用P87和P88、P89和P90、P91和P92以其为模板分别进行扩增，利用P93和P94以pCMV-LicVB为模板扩增LicV编码基因，利用重叠PCR分别将LicV与mKalama1和CAAX或3xNLS连接，分别获得LicV-mKalama1-CAAX和LicV-mKalama1-3xNLS重组基因片段，利用引物P95和P96扩增pEGFP-N1-FLAG载体(Addgene：60360)使其线性化，利用一步法克隆试剂盒将上述重叠PCR获得的基因片段插入到线性化的pEGFP-N1-FLAG载体中，得到的质粒命名为pCMV-LicV-mKalama1-CAAX和LicV-mKalama1-3xNLS，分别编码重组光控定位因子的氨基酸序列分别为序列SEQ ID NO：26和27。
扩增mKalama1基因片段的引物为：
上游引物(P87)：
5’-tggcggtggctcgggcggtggtgaattcatgatggtgagcaagggagaggagctg-3’
下游引物(P88)：
5’-cttgtacagctcgtccatgccggg-3’
扩增CAAX基因片段的引物为：
上游引物(P89)：
5’-gacgagctgtacaagggcagcggaagatctaaga-3’
下游引物(P90)：
5’-agagtcgcggccgctttacatgatcacgcacttagtc-3’
扩增3xNLS基因片段的引物为：
上游引物(P91)：
5’-gacgagctgtacaagctgtacaaggatccaaaaa-3’
下游引物(P92)：
5’-agagtcgcggccgctttatacctttctcttcttttttggat-3’
扩增LicV基因片段的引物为：
上游引物(P93)：
5’-agatccgctagcgctatgaaaattgcgaaggtgatcaac-3’
下游引物(P94)：
5’-cccgagccaccgccaccagcgtaatctggaacatcgtatgggtactgcagttccgtttcgcactgg-3’
扩增pEGFP-N1-FLAG载体使其线性化的引物为：
上游引物(P95)：
5’-agcggccgcgactctagatcataatcagcca-3’
下游引物(P96)：
5’-agcgctagcggatctgacggttcac-3’
实施例4重组光控RNA核酸酶因子调控靶标RNA的降解
为了检测含不同第一多肽的重组光控RNA核酸酶因子对靶标RNA降解的调控效果，分别将pCDNA3.1-Gluc-2xRAT LicT与pCMV-LicVB、pCDNA3.1-Gluc-2xRAT BglG与pCMV-BglVB、pCDNA3.1-Gluc-2xRAT SacY与pCMV-SacVB、pCDNA3.1-Gluc-2xRAT GlcT与pCMV-GlcVB共转染HEK293T细胞，分别以不含RAT LicT反应元件的pCDNA3.1-Gluc作为对照。转染6h后将细胞分别置于黑暗和蓝光(1.8W/m 2)照射下 培养24小时，检测细胞培养液上清中的Gluc活性。检测结果如附图4所示，光照条件下上清中的Gluc活性显著低于黑暗条件下的Gluc活性，对照细胞中的Gluc活性无明显变化，表明光照可以诱导LicVB、BglVB、SacVB和GlcVB重组光控RNA核酸酶因子结合相对应的反应元件，水解靶标Gluc mRNA，导致细胞中靶标Gluc mRNA水平降低，最终使得合成的Gluc蛋白水平下降，活性降低。该结果表明含不同第一多肽的重组光控RNA核酸酶因子可以调控靶标RNA的降解。
为了检测含不同第二多肽的重组光控RNA核酸酶因子对靶标RNA降解的调控效果，将pCDNA3.1-Gluc-2xRAT LicT分别与pCMV-LicEB、pCMV-LicAsB、pCMV-LicAuB和pCMV-LicPB共转染HEK293T细胞，以不含RAT LicT反应元件的pCDNA3.1-Gluc作为对照。转染6h后将细胞分别置于黑暗和蓝光(1.8W/m 2)照射下培养24小时，检测细胞培养液上清中的Gluc活性。检测结果如附图5所示，光照条件下上清中的Gluc活性显著低于黑暗条件下的Gluc活性，对照细胞中的Gluc活性无明显变化，表明光照可以诱导LicEB、LicAsB、LicAuB和LicPB重组光控RNA核酸酶因子结合2xRAT LicT反应元件，水解靶标Gluc mRNA，导致细胞中靶标Gluc mRNA水平降低，最终使得合成的Gluc蛋白水平下降，活性降低。该结果表明含不同第二多肽的重组光控RNA核酸酶因子可以调控靶标RNA的降解。
为了进一步检测重组光控RNA核酸酶因子对不同靶标RNA的降解调控效果，将pCMV-LicVB分别与pCDNA3.1-mCherry-2xRAT LicT和pU6-Pepper-RAT LicT共转染HEK293T细胞，将pCMV-LicVPIN和pU6-Pepper-RAT LicT共转染HEK293T细胞，分别以不含RAT LicT反应元件的pCDNA3.1-mCherry和pU6-Pepper作为对照。转染6h后将细胞分别置于黑暗和蓝光(1.8W/m 2)照射下培养24小时，分别检测细胞中mCherry的荧光信号和Pepper485的荧光信号(表达Pepper-RAT LicT靶调控单元的细胞需要加入1μM HBC485染料(Chen et al.,Nature Biotechonogy,2019,37:1287-1293)对靶标RNA进行特异性标记)。检测结果分别如附图6-8所示，在附图6中，光照条件下细胞中的mCherry信号显著低于黑暗条件下的mCherry信号，对照细胞中的mCherry信号无明显变化，表明光照可以诱导LicVB结合mCherry-2xRAT LicT靶转录单元中的2xRAT LicT反应元件，水解靶标mCherry mRNA，导致细胞中靶标mCherry mRNA水平降低，最终使得合成的mCherry蛋白水平下降，荧光降低。在附图7和8中，光照条件下细胞中的Pepper485信号显著低于黑暗条件下的Pepper485信号，对照细胞中的Pepper485信号无明显变化，表明光照可以诱导LicVB和LicVPIN结合 Pepper-RAT LicT靶转录单元中的RAT LicT反应元件，水解Pepper靶标RNA，导致细胞中Pepper RNA水平下降，Pepper485荧光信号降低。
为了检测重组光控RNA核酸酶因子对不同宿主细胞中靶标RNA降级的调控效果，将pJ23106-mCherry-2xRAT LicT和pJ23117-LicVB共转化大肠杆菌细胞，将pGADT7-mCherry-2xRAT LicT和pGADT7-LicVB共转化BY4741酿酒酵母细胞，分别以不含RAT LicT反应元件的pJ23106-mCherry和pGADT7-mCherry作为对照。分别挑取单克隆在黑暗条件下培养过夜，按照1:100稀释到新鲜培养基后分别光照和黑暗条件下培养到对数生长期后期，分别检测细胞中mCherry的荧光信号。检测结果分别如图9所示，光照条件下大肠杆菌和酵母细胞中的mCherry信号显著低于黑暗条件下的mCherry信号，对照细胞中的mCherry信号无明显变化，表明光照可以诱导LicVB结合mCherry-2xRAT LicT靶转录单元中的2xRAT LicT反应元件，水解靶标mCherry mRNA，导致细胞中mCherry靶标mRNA水平降低，最终使得合成的mCherry蛋白水平下降，荧光降低。该结果表明重组光控RNA核酸酶因子可以调控不同宿主细胞中靶标RNA的降解。
实施例5重组光控RNA剪接因子调控靶标RNA的剪接
为了检测重组光控RNA剪接因子调控靶标RNA的剪接，将pGZ3-GUM-RAT LicT分别与pCMV-LicVA1和pCMV-LicVRS共转染HEK293T细胞，以不表达重组光控RNA剪接因子的空载体和表达不含第三多肽重组光控RNA剪接因子的质粒作为对照。转染6h后将细胞分别置于黑暗和蓝光(1.8W/m 2)照射下培养24小时，检测细胞中靶标RNA的剪接结果。检测结果如附图10所示，细胞中靶标RNA两种剪接产物的比例在光照和黑暗条件下差异显著，而对照细胞中则无明显差异，表明重组光控RNA剪接因子LicVA1和LicVRS可用于靶标RNA的可变剪接。
实施例6重组光控RNA翻译起始因子调控靶标RNA的翻译
为了检测重组光控RNA翻译起始因子调控靶标RNA的翻译，将pCMV-LicV4E和pCDNA3.1-mCherry-4xRAT LicT-EGFP共转染HEK293T细胞，以表达不含第三多肽重组光控RNA翻译起始因子的质粒作为对照。转染6h后将细胞分别置于黑暗和蓝光(1.8W/m 2)照射下培养24小时，检测不同条件下细胞中mCherry和EGFP的荧光信号。检测结果如附图11所示，光照条件下EGFP的信号要远远强于黑暗条件下培养细胞中的EGFP信号，而对照细胞中的EGFP信号都很低，表明光照可以诱导LicV4E重 组光控RNA翻译起始因子结合4xRAT LicT反应元件，通过第三多肽eIF4E招募其他翻译因子一起启动靶标EGFP mRNA的翻译，合成EGFP蛋白。该结果表明重组光控RNA翻译起始因子可用于调控靶标RNA的翻译。
为了检测不同光照强度对重组光控RNA翻译起始因子调控靶标RNA翻译的影响，将转染了pCMV-LicV4E和pCDNA3.1-mCherry-4xRAT LicT-EGFP的HEK293T细胞置于不光强度的蓝光下培养24h，对不同条件下细胞的mCherry和EGFP荧光信号进行成像。检测结果如附图12所示，随着蓝光光照强度的增加，EGFP的荧光信号也随之增加。该结果表明重组光控RNA翻译起始因子可用于定量调控靶标RNA的翻译。
为了检测重组光控RNA翻译起始因子是否可以在空间上精确调控靶标RNA翻译，将转染了pCMV-LicV4E和pCDNA3.1-mCherry-4xRAT LicT-EGFP的HEK293T细胞置于只能透过特定区域光膜片的蓝光下培养，24h后对细胞的mCherry和EGFP荧光信号进行成像。检测结果如附图13所示，只有接收蓝光照射的细胞高水平表达EGFP蛋白，而旁边临近细胞的EGFP信号很弱。该结果表明重组光控RNA翻译起始因子可用于在空间上精确调控靶标RNA的翻译。
实施例7重组光控RNA定位因子调控靶标RNA的定位
为了检测重组光控RNA定位因子调控靶标RNA的定位，将pU6-Pepper-RAT LicT分别与pCMV-LicV-mKalama1-CAAX和LicV-mKalama1-3xNLS共转染HEK293T细胞，以表达不含LicV重组光控RNA结合蛋白的质粒作为对照。转染6h后将细胞分别置于黑暗和蓝光(1.8W/m 2)照射下培养24小时，加入1μM HBC620染料(Chen et al.,Nature Biotechonogy,2019,37:1287-1293)进行标记，利用荧光显微镜观察光照和黑暗条件下细胞中Pepper620的荧光分布情况。检测结果如附图14所示，在表达LicV-mKalama1-CAAX和Pepper-RAT LicT的细胞中，光照可诱导Pepper-RAT LicT靶标RNA向细胞内膜富集，而黑暗条件下Pepper-RAT LicT靶标RNA则弥散分布在整个细胞中(图14A和B)；在表达LicV-mKalama1-3xNLS和Pepper-RAT LicT的细胞中，光照可诱导Pepper-RAT LicT靶标RNA向细胞核富集，而黑暗条件下Pepper-RAT LicT靶标RNA则弥散分布在整个细胞中(图14C和D)。因此，基于光控RNA结合蛋白的光控RNA定位因子可以用于调控靶标RNA在细胞内的分布与定位。
应该理解，在实施例或实验材料方法中所显示的成分用量、反应条件等数字或是本 说明书所使用的其它参数均为近似值(除非特别注明)，可根据所需要获得的结果而加以改变。并且，这些参数并非用来限定本发明保护范围，而是应用正常的操作技术下所得到的较佳数据。除非另行定义，文中所使用的所有专业与科学用语术与本领域技术人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料及均可以应用于本发明中。文中本说明书中所述的较佳实验方法与材料仅作为示范之用。本说明书提及的所有文献都全文引入作为参考。此外应该理解，在阅读了本发明的上述内容后，本领域的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在本申请所附权利要求书所限定的范围内。

Claims (18)

  1. 一种光控RNA代谢调控系统，其特征在于，包括：
    a)重组光控RNA效应因子，所述重组光控RNA效应因子包括作为RNA结合结构域的第一多肽，作为光敏结构域的第二多肽和作为RNA效应结构域的第三多肽；
    b)靶调控单元：包括被所述第一多肽识别/结合的至少一个反应元件、被第三多肽调节的靶标RNA核苷酸序列。
  2. 根据权利要求1所述的光控RNA代谢调控系统，其中所述第一多肽、第二多肽和第三多肽之间可操作性连接，和/或其中反应元件和靶标RNA核苷酸序列之间可操作性连接。
  3. 根据权利要求1所述的光控RNA代谢调控系统，其中所述第一多肽可选自抗转录终止因子蛋白的RNA识别结合结构域、RNA衰减子RNA识别结合结构域、RNA干扰酶RNA识别结合结构域、小调节RNA结合蛋白RNA识别结合结构域、RNA解旋酶RNA识别结合结构域、核酶RNA识别结合结构域、tRNA结合蛋白RNA识别结合结构域、rRNA结合蛋白RNA识别结合结构域。
  4. 根据权利要求3所述的光控RNA代谢调控系统，其中所述第一多肽可选自BglG/SacY家族抗转录终止因子蛋白的RNA识别结合结构域。
  5. 根据权利要求4所述的光控RNA代谢调控系统，其中所述第一多肽选自粗糙芽孢杆菌LicT蛋白的RNA识别结合结构域、大肠杆菌BglG蛋白的RNA识别结合结构域、枯草芽胞杆菌SacY蛋白的RNA识别结合结构域、枯草芽胞杆菌GlcT蛋白的RNA识别结合结构域、枯草芽胞杆菌SacT蛋白的RNA识别结合结构域、菊欧氏杆菌Arbg蛋白的RNA识别结合结构域、乳酸菌BglR蛋白的RNA识别结合结构域、干酪乳杆菌LacT蛋白的RNA识别结合结构域、肉葡萄球菌GlcT蛋白的RNA识别结合结构域及其截短体或具有80％以上序列相似性且具有所限定的氨基酸序列的功能的突变体，优选为具有RNA结合活性的截短体或突变体。
  6. 根据权利要求1所述的光控RNA代谢调控系统，其中所述第二多肽选自含黄素类生色团的光敏蛋白的光敏结构域。
  7. 根据权利要求6所述的光控RNA代谢调控系统，其中所述第二多肽选自含LOV结构域的光敏蛋白的光敏结构域。
  8. 根据权利要求7所述的光控RNA代谢调控系统，其中所述第二多肽选自粗糙链孢霉菌的VIVID LOV结构域、细菌Erythrobacter litoralis的EL222 LOV结构域、燕 麦光敏色素1基因的LOV结构域AsLOV2、无隔藻金色素蛋白1的LOV结构域AuLOV、恶臭假单胞菌的PpLOV LOV结构域及其截短体或具有80％以上序列相似性且具有所限定的氨基酸序列的功能的突变体，优选为具有光诱导寡聚化能力发生改变的截短体或突变体。
  9. 根据权利要求1所述的光控RNA代谢调控系统，其中所述第一多肽和第二多肽构成一种光控RNA结合蛋白。
  10. 根据权利要求1所述的光控RNA代谢调控系统，其中所述第三多肽选自RNA剪接调控结构域、RNA翻译调控因子结构域、RNA核酸酶结构域、RNA表观遗传学修饰酶结构域。
  11. 如权利要求1所述的光控RNA代谢调控系统，其中还包含促进所述重组光控RNA效应蛋白向不同细胞器运输的定位信号肽第四多肽，所述第四多肽与第一、第二、第三多肽直接或通过接头肽相连。所述第四多肽可选自细胞核定位信号肽、线粒体定位信号肽、高尔基体定位信号肽、内质网定位信号肽、细胞质定位信号肽。
  12. 根据权利要求1所述的光控RNA代谢调控系统，其中所述反应元件为可被所述第一多肽特异性识别和结合的RNA基序。
  13. 如权利要求11所述的光控RNA代谢调控系统，其中所述反应元件选自LicT蛋白结合元件、BglG蛋白结合元件、SacY蛋白结合元件、枯草芽胞杆菌GlcT蛋白结合元件、SacT蛋白结合元件、Arbg蛋白结合元件、BglR蛋白结合元件、LacT蛋白结合元件和肉葡萄球菌GlcT蛋白结合元件。
  14. 含有权利要求1-13之一所述光控RNA代谢调控系统的表达载体。
  15. 根据权利要求14所述的表达载体，所述表达载体含有所述光控RNA效应因子编码基因，和/或所述靶调控单元编码核苷酸序列，所述靶调控单元中含有反应元件但待调控RNA编码核苷酸序列空缺。
  16. 用权利要求1-13之一所述光控RNA代谢调控系统在宿主细胞中调控靶标RNA代谢的方法，包括步骤：a)将所述光控RNA代谢调控系统构建在原核或真核质粒表达载体中；b)将重组质粒引入宿主细胞；c)光照诱导所述宿主细胞，调控宿主细胞中靶标RNA的代谢。
  17. 如权利要求16所述的调控靶标RNA代谢的方法，其中还包括光源的选择和照射方法的选择，所述光源包括LED灯、荧光灯、激光和白炽灯，所述照射方法是持续的光照或不连续的光照。其中所述光源的选择和照射方法的选择包括用光扫描、投 影、光模具在空间上控制不同位置的细胞的靶标RNA的代谢。
  18. 一种试剂盒，装有权利要求14所述表达载体或/和含有所述光控RNA效应因子表达载体的原核或真核细胞，或/和装有含反应元件但待调控靶标RNA编码核苷酸序列空缺的靶调控单元的表达载体，及相应的说明书。
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