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WO2016070853A1 - Arn non codant synthétique comprenant une séquence répétée sineb2 inverse et son utilisation dans l'amélioration de la traduction de protéines cibles - Google Patents

Arn non codant synthétique comprenant une séquence répétée sineb2 inverse et son utilisation dans l'amélioration de la traduction de protéines cibles Download PDF

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WO2016070853A1
WO2016070853A1 PCT/CN2015/098478 CN2015098478W WO2016070853A1 WO 2016070853 A1 WO2016070853 A1 WO 2016070853A1 CN 2015098478 W CN2015098478 W CN 2015098478W WO 2016070853 A1 WO2016070853 A1 WO 2016070853A1
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rnae
protein
sequence
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吴琼
姚绎
金守红
龙海舟
余瑛婷
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Tsinghua University
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    • 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
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the present invention relates to the field of biotechnology, and more particularly to artificial non-coding RNAs containing inverted SINEB2 repeats and their use in enhancing translation of target proteins.
  • Non-coding RNA transcripts are widely present in cells, but most of the functions are unknown. Some have been found to regulate gene expression at the transcriptional level.
  • Carrieri et al. (Carrieri et al., Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat; Nature. 2012; 491 (7424): 454-7) reported a class of long-chain non-coding with new functions.
  • RNA (IncRNA) which targets the 5' end of mRNA (messenger RNA) via an overlapping antisense sequence, thereby increasing translation of the corresponding protein UCHL1 at the post-transcriptional level, possibly because this IncRNA can complement the mRNA and promote ribose Recruitment.
  • this antisense non-coding RNA can be engineered for the selected mRNA to increase the amount of translation of the mRNA of interest.
  • Carrieri Carrieri et al., Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat; Nature. 2012; 491 (7424): 454-7)
  • the prototype uchl1 non-coding RNA reported by et al. contains two essential parts, One is the 5' pairing region of mRNA and the other is the inverted SINEB2 element.
  • the pairing region is 72 nucleotides in length, and its corresponding mRNA sequence is completely complementary pairing near the translation initiation codon AUG, and the head-to-head pairing of the sensed antisense RNA transcript significantly improves the efficiency of recruiting ribosomes and promotes translation.
  • the reverse SINEB2 component is required for translation promotion, but its mechanism has not yet been elucidated.
  • RNAe ie RNA Enhancement
  • the invention provides a nucleic acid molecule which is: (1) an RNA molecule consisting of a complementary region complementary to an mRNA of interest, an inverted SINEB2 sequence, an optional poly(A) signal region, And optionally a regulatory element; or (2) a DNA molecule capable of transcribing the RNA molecule.
  • the RNA molecule consists of a complementary region complementary to the mRNA of interest, an inverted SINEB2 sequence, and a poly(A) signal region.
  • the RNA molecule consists of a complementary region complementary to the mRNA of interest, and an inverted SINEB2 sequence.
  • the total length of the pairing zone does not exceed 600 nt.
  • the pairing region may be paired with a region of -100 nt to +Xnt of the mRNA of interest, wherein X is a positive integer, such as 1, 5, 10, 20, 30, 50, 100, 200, 300, 400, and the like.
  • the regulatory element may be an ALU or the like, for example, as mentioned in Carrieri et al., Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat; Nature. 2012; 491 (7424): 454-7. ALU sequence.
  • the invention provides a method of promoting expression of a protein of interest comprising: transfecting a nucleic acid molecule provided by the invention against the protein of interest into a cell expressing the protein of interest.
  • the protein of interest may be a secreted protein or antibody, or an endogenous protein.
  • the cell can be a human cell line, a Chinese hamster cell line or a rat-derived cell line.
  • the nucleic acid molecule can exist as a plasmid, a lentiviral vector or an in vitro RNA transcript.
  • the protein of interest protein and the RNA molecule can be located on the same expression vector.
  • the protein of interest is selected from the group consisting of a plurality of proteins having the same 5' end.
  • the present invention provides an expression vector comprising: a vector backbone, a fixed tag and a multiple cloning site sequence following the immobilization tag, paired with the fixed tag and the fixed 5' untranslated region on the vector backbone A nucleic acid molecule of the invention.
  • the immobilization tag may be selected from the group consisting of HA, EGFP, His, Myc, and Flag tags.
  • Another aspect of the present invention provides a method for screening a high-throughput gene, characterized in that a nucleic acid molecule provided by the present invention for the gene is transfected into a cell expressing the gene, and according to the gene Influence screening.
  • the effect of the gene may be an effect on cell proliferation, and the cell proliferation rate is used as a screening condition.
  • the cell can be a HeLa or HEK293T cell.
  • the gene may be a gene that inhibits cell proliferation, such as gsk3 ⁇ , p21, e2f1, bid, c-myb, bax, c-myc or p53.
  • Figure 1 shows the tailing diagram of AT-RNAe and confirms its tailing mode.
  • A Schematic diagram of the tail region of the RNAe sequence
  • B Measurement of the tailing tendency of RNAe (measured using qRT-PCR), demonstrating that RNAe tends to undergo a short tailing mode (ie, generating AT-RNAe instead of FL-RNAe)
  • C There is variable tail-tailing verification of RNAe (measured by 3' RACE). It can be seen that there are two distinct bands in RNAe, which proves that there are two tailing modes.
  • Figure 2 shows the necessary conditions for RNAe to function by truncation.
  • A Schematic diagram of different truncated diagrams of RNAe, the lowest behavior of minRNAe.
  • B Effect of RNAe truncation on its function (measured by flow cytometry), different truncated RNAe including minRNAe can significantly promote the expression of EGFP fluorescent protein.
  • Figure 3 shows the effect of different pairing lengths of RNAe and the target mRNA on its promoting efficiency.
  • A Effect of deletion of the pairing of the target mRNA in the 5' translational region and the 5' untranslated region on RNAe function (measured by flow cytometry), demonstrating that RNAe has almost no effect after 5' translational segmentation, 5' RNAe also has a certain effect after the untranslated region is removed.
  • B Extending the effect of RNAe on the RNAe function on the RNAe function (measured by flow cytometry), it was confirmed that the total length of RNAe was almost no effect when it was 600 nt or more.
  • Figure 4 shows the specificity of RNAe promotion:
  • A Effect of pAT-RNAe-egfpc1 on intracellular protein expression (measured using mass spectrometry).
  • B Effect of pminRNAe-egfpc1 on intracellular protein expression (measured using mass spectrometry). Both showed that the significant difference protein represented by the red triangle was almost non-existent, demonstrating that the specificity of RNAe is very high.
  • Figure 5 shows the promotion of pEGFP-C1 fluorescence expression by pRNAe-egfpc1 in different cell lines (measured by flow cytometry), demonstrating RNAe in human (HEK293T, HEK293A, HeLa) cells and Chinese hamster source (CHO-K1) It can function in cells.
  • Figure 6 shows the promotion of endogenous protein SOX9 by RNAe.
  • A Expression of SOX9 protein under the action of RNAe-sox9 (measured by Western Blot), demonstrating the role of SOX9 protein in RNAe
  • B The expression of the lower expression
  • B of SOX9 protein overexpression on the extra-cartilage matrix (measured by alimin blue staining), the expression of SOX9 protein overexpressed by RNAe and SOX9 gene can directly promote the secretion of extracellular matrix.
  • Figure 7 shows the promotion of secreted luciferase by AT-RNAe and minRNAe (measured using Luciferase Assay), demonstrating that both RNAe can promote expression of this secreted protein.
  • Figure 8 shows that both AT-RNAe and minRNAe can be promoted by different forms of transfer into cells.
  • A The promotion of fluorescence (measured by flow cytometry) of two RNAe stably transfected cells with lentivirus, demonstrating that RNAe can function in the form of a lentivirus.
  • B Effect of in vitro transcribed form on RNAe function (measured using the CCK8 assay), demonstrating that both in vitro transcription and plasmid-expressed RNAe are functional.
  • Figure 9 shows the screening of proliferation-related genes by RNAe.
  • A Gene screening in HEK293T cells (measured using the CCK8 assay)
  • B Gene screening under HEK293T cell starvation conditions (measured using the CCK8 assay)
  • C Gene screening in HeLa cells (measured using the CCK8 assay)
  • D Gene screening for Hela cell starvation conditions (measured using the CCK8 assay).
  • Figure 10 shows a comparison of RNAe and RNAi methods.
  • A Comparison of RNAe and RNAi in promoting proliferative gene screening (measured using the CCK8 assay), demonstrating that both methods are effective in the selection of promoters for promoting proliferation-related genes.
  • B Comparison of RNAe and RNAi in inhibition of proliferation gene screening (measured using the CCK8 assay). It was demonstrated that the RNAe method is more effective than RNAi in the selection of genes for inhibiting proliferation-related genes.
  • Figure 11 shows that the same RNAe can also function as a promoter for the same 40 nt as the first 32 nt of the translation region after the 5' untranslated region.
  • A Promotion of EGFP-BBCK and EGFP- ⁇ B2-crystallin proteins by RNAe (measured using flow cytometry).
  • B Promotion of EGFP-BBCK protein by RNAe (measured using Western Blot).
  • C The promotion of EGFP- ⁇ B2-crystallin protein by RNAe (measured by Western Blot), both of which demonstrate that an RNAe can promote the two different proteins.
  • Figure 12 shows the construction of the pRNAe-Plus vector.
  • A Schematic representation of the pRNAe-Plus vector
  • B Validation of the pRNAe-Plus-EGFP plasmid (measured using flow cytometry), demonstrating that pRNAe-Plus-EGFP can promote EGFP expression.
  • C Validation of pRNAe-Plus-HA-EGFP plasmid (measured using flow cytometry), demonstrating that pRNAe-Plus-HA-EGFP can promote HA-EGFP expression. .
  • SINEB2 refers to members of the SINEB2 family known in the art.
  • a preferred "SINEB2 sequence" of the present invention is the SINEB2 mentioned in the literature Carrieri et al., Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat; Nature. 2012; 491 (7424): 454-7. sequence.
  • RNAe as used herein is an abbreviation for RNA Enhancement, and in one aspect refers to a technique or method for promoting protein expression at a pre-translational level based on a long non-coding RNA complementary to a target region and an inverted SINEB2 family sequence, another In aspect, it also refers to a non-coding RNA molecule having a pre-translation promoting function or a DNA molecule expressing the RNA molecule.
  • poly(A) signal region refers to an RNA sequence at the 3' end of the mRNA that controls poly(A) tailing or a DNA sequence that expresses the sequence.
  • ALU refers to members of the ALU family known in the art.
  • a preferred “ALU” of the present invention is the ALU sequence mentioned in the literature Carrieri et al., Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat; Nature. 2012; 491 (7424): 454-7. .
  • foreign gene refers to a foreign gene that is not originally found in an organism or in a cell and can be obtained by genetic manipulation.
  • protein tag refers to a polypeptide or protein which is expressed by fusion with a protein of interest using DNA in vitro recombination technology to facilitate expression, detection, tracing and purification of the protein of interest.
  • lentiviral vector as used herein is introduced into a vector by a foreign gene which has been developed based on HIV-1 (human immunodeficiency type I virus).
  • optional means the meaning of “optional” or “non-essential”.
  • optional poly(A) signal region may or may not contain the poly(A) signal region, which may be selected by those skilled in the art depending on the situation.
  • regulatory element refers to regulatory elements commonly used in the field of molecular biology, such as promoters, enhancers, signal peptides and the like.
  • Example 1 Transcription of endogenous natural RNAe has a variable tailing pattern, thereby constructing artificial AT-RNAe
  • RNAe Unless otherwise specified, the general RNAe is named as follows. Taking pRNAe-egfpc1 as an example, the first letter lowercase “p” indicates that the molecule is a plasmid DNA molecule expressing RNAe, and no p indicates RNAe is not expressed in cells by means of plasmids, or it is an expression product RNA molecule of a certain RNAe plasmid in cells; "RNAe” stands for its type, there are RNAe (ie, long RNAe), AT-RNAe and minRNAe
  • the lowercase "egfpc1" after the horizontal line represents the pairing protein of the RNAe, which is named in lower case of the protein name.
  • RNAe sequence was initiated by the CMV promoter and thereafter contained a stretch of polyA signal sequence, which are not listed in the following sequences.
  • the DNA sequence expressing RNAe is as follows (SEQ ID NO: 11):
  • the underline is the pairing part, and replacing the pair with a sequence as described later is to replace the part (in all three RNAe).
  • the bold portion is the inverted SINEB2 conserved sequence, which is conserved among all RNAe and minRNAe and is required for RNAe to function.
  • the remaining italic is the tail portion, which is full length RNAe, which is different from ATRNAe and minRNAe.
  • the pAT-RNAe-egfpc1 sequence is (SEQ ID NO: 12):
  • the pminRNAe-egfpc1 sequence is (SEQ ID NO: 13):
  • Equation 2 downstream of the control group Ct -Ct upstream pRNAe-egfpc1 Ct ⁇ 1 upstream downstream -Ct (average value) measured by the plasmid (about Equation 1), the experimental group of intracellular transcription of ⁇ 1 RNAe-egfpc1 (Average) (about Equation 2), because there is no variable tailing in the upstream primer region, so the upstream of the Ct in the plasmid and the cell is equal, and about Equation 2 is subtracted from Equation 2 to obtain the downstream Ct in the cell.
  • the full-length RNAe at the end also has an equal amount of front end, so about 1/4 of the RNAe is tailed in poly(A), and about 3/4 is variable-tailed before the poly(A) after the ALU, so only the front end
  • the ratio of AT-RNAe to full-length RNAe was 3:1.
  • RNAe RNAe molecule is tailed at position 480 (the iterative and uppercase AATAAA position in the pRNAe-egfpc1 sequence above), so RNAe tends to prepend before poly(A). This result also suggests that the sequence following the tailing point may not be necessary for the function of RNAe.
  • AT-RNAe (RNAe got by alternative tailing) includes a 72 nt (nucleotides) pairing region, a 167 nt inverted SINEB2 region, some remaining regulatory sequences and a poly(A) signaling region, except for polys obtained spontaneously after tailing in vivo.
  • the sequence is 480 nt in total, which is an RNAe that is similar to the original full-length RNAe-enhanced translation efficiency but has a shorter sequence structure.
  • Example 2 The pairing region of Example 2 and the target mRNA and the inverted SINEB2 sequence are sufficient conditions for RNAe to function, thereby constructing artificial minRNAe
  • Carrieri et al. (Carrieri et al., Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat; Nature. 2012; 491 (7424): 454-7) demonstrated the necessity of the inverted SINEB2 sequence for AS Uchl1. Based on this, we demonstrated that the inverted SINEB2 sequence is a sufficient condition for the function of the RNAe molecule outside the pairing region, and constructed artificial minRNAe.
  • RNAe-egfpc1 shown as minRNAe in Figure 2A
  • this shortest RNAe is the RNAe with the strongest effect (about 450% enhancement).
  • this shortest RNAe is the RNAe, which only includes the 72nt pairing region, 167nt SINEB2 region.
  • the poly(A) signal region, except for the poly(A) tail obtained after spontaneous tailing in vivo, the other sequences are 282 nt, which is an optimized shortest and more powerful RNAe.
  • Example 3 optimizes the RNAe pairing region to improve its translational enhancement efficiency
  • RNAe molecule The portion paired with the 5'-TR (translation region) is more important for the function of the RNAe molecule than the portion paired with the 5'-UTR (untranslated region). And optimization of the pairing region can improve the efficiency of translation promotion of RNAe molecules.
  • RNAe 40:0,100:0
  • pRNAe-egfpn1-40:0 the paired partial sequence is (SEQ ID NO: 18), pRNAe-egfpn1-100: 0 (the pairing sequence is (SEQ ID NO: 19)
  • RNAe (0:32, 0:100, 0:200)
  • pRNAe-egfpn1-0:32 paired with EGFP mRNA 5'-TR in the pEGFP-N1 plasmid
  • Sequence is (SEQ ID NO: 20), pRNAe-egfpn1-0: 100 (the pairing sequence is (SEQ ID NO: 21), pRNAe-egfpn1-0: 200 (the pairing sequence is (SEQ ID NO: 22)).
  • the pairing sequence is (SEQ ID NO: 23) as a control (40:32 group in Figure 3)
  • the pairing sequence is (SEQ ID NO: 23) as a control (40:32 group in Figure 3)
  • the pairing sequence is (SEQ ID NO: 23) as a control (40:32 group in Figure 3)
  • 0.3 ⁇ g of pEGFP-N1 plasmid and 1.2 ⁇ g of each RNAe plasmid were transfected into HEK293T cells as in Example 1, and the mean fluorescence intensity of the cells was measured by flow cytometry 48 hours later. The results are shown in Figure 3A.
  • the 40:0 and 100:0 groups could not enhance the fluorescence of EGFP, indicating that only the RNAe paired with the EGFP mRNA 5'-UTR could hardly promote EGFP expression.
  • RNAe function in the 5'-UTR pairing region and the necessity of RNAe function in the 5'-TR pairing region; 0:32, 0:100, 0:200 group compared with the empty control 0:0 group It can enhance the expression of EGFP, indicating that RNAe paired with EGFP mRNA 5'-TR can promote the expression of EGFP, and the enhancement of EGFP in 0:200 group also proves that the length of pairing can exceed the original pairing length after 100nt. Promoting protein expression (Fig. 3A), i.e., the 5'-TR pairing region is sufficient to perform pairing functions and the 5'-UTR pairing region is not necessary for RNAe function.
  • RNAe is paired with the target mRNA sequence from -40nt to +32nt (40:32, with A in the ATG as the first nucleotide) to study the effect of the length of the pairing region on RNAe function, because it is paired with the 5'-TR.
  • the importance of the region was stronger.
  • the total length of pairing When the total length of pairing is less than 400 nt, it can promote the expression of the target protein, but when the total length exceeds 600 nt, it will inhibit the protein of interest. Expression, this inhibition may result from the inhibition of translation or degradation of the corresponding mRNA by long antisense RNA.
  • HIV monoclonal antibody 10E8 (Yu, Y., Tong, P., Li, Y., Lu, Z. & Chen, Y.10E8-like neutralizing antibodies against the RNAe after changing the length of the pairing region. HIV-1 induced using a precisely designed conformational peptide as a vaccine prime. Sci China C Life Sci 57, 117-127 (2014)) The effect of expression promotion.
  • pminRNAe-Ab-40:32-uni (a common minRNAe directed against the light and heavy heavy chain of the antibody (paired with the same secretion signal sequence of the light and heavy chain), the paired sequence is (SEQ ID NO: 27)), pminRNAe-Ab-40: 100-10e8h (minRNAe for antibody heavy chain, paired sequence is (SEQ ID NO: 28)), pminRNAe-Ab-40: 100-10e8l (minRNAe directed against the antibody light chain, partial sequence is (SEQ ID NO: 29)), pminRNAe-Ab-100: 32-uni (for the common minRNAe of the antibody light heavy chain, the paired sequence is (SEQ ID NO: 30)), pminRNAe-Ab-100: 200-10e8h (minRNAe for antibody heavy chain, paired sequence is (SEQ ID NO: 31)), pminRNAe-Ab-100: 200-10e8l (minRNAe directed against the antibody light chain, the paired sequence is (SEQ ID NO: 32)), pminRNAe-Ab-
  • ELISA enzyme-linked immunosorbent assay
  • RNAe has a relatively good promotion of translation effects.
  • the RNAe of the original paired length can be directly used, but if it is necessary to perform the optimal RNAe screening for a specific protein in production, it needs to be considered.
  • the length of the paired region with the target mRNA is optimized to some extent.
  • RNAe form has a strong or weak effect on the protein, but for a specific protein.
  • the promotion effect can be inferred. If an RNAe form has a promoting effect on a certain protein, the corresponding two other RNAe forms are also promoted in the same manner.
  • RNAe method has a very strong specificity, which may be caused by the pairing region of RNAe with the target mRNA (usually with 40 nt 5'-UTR (untranslated region) and 32 nt 5'-TR (translation area) pairing) guarantee.
  • we selected 23 genes in human genes see Example 10) and designed the corresponding RNAe, using NCBI's Blast scoring function to compare these 23 RNAe sequences with the human full transcriptome. It is found that there is only one high-scoring corresponding gene in each RNAe, which is the gene that can be promoted by the RNAe itself. The rest may be derived from the mismatched scores are very low (Table 2). The scores represent the Blast scoring function. The strength of its pairings suggests that RNAe may be predicted to bind only to a specific gene corresponding to it.
  • HEK293T cells were transfected into pRNAe-Mock according to the method of Example 1 (provided by Weishangde Biotech Co., Ltd.) (an empty vector without RNAe sequence, derived from the following literature (named pN1 in the literature) Yao, Y., He, Y., Guan, Q. & Wu, QA tetracycline expression system in combination with Sox9 for cartilage tissue engineering. Biomaterials 35, 1898-1906 (2014)), pAT-RNAe-egfpc1 (without matching region) RNAe) or pminRNAe-egfpc1, and carry it all Protein mass spectrometry (Guo, Q. et al.
  • the protein with a large change in expression is particularly small (10/4440 in Figure 4A, 5/4440 in Figure 4B), indicating that there is almost no significant difference between the three groups of proteins outside EGFP, ie, the RNAe molecule is intracellular. It only affects the expression of the target mRNA without affecting the expression of other proteins, thus verifying the specificity of the RNAe method to promote protein expression.
  • Table 2 uses the NCBI database Blast function to evaluate the specificity of RNAe
  • Example 5 RNAe method can function in cells of various species
  • RNAe method As a protein regulation technology, the RNAe method is widely applicable and can be applied to cells of various species. It can be used in human HEK293T, HEK293A, HeLa cells, Chinese hamster cell line CHO-K1, rat source cell line C5. .18 and primary chondrocytes (see Example 6 for details) function as follows.
  • RNAe-Mock empty vector without RNAe sequence
  • RNAe in HEK293T and HEK293A cells showed that the fluorescence intensity of RNAe in HEK293T and HEK293A cells was about 200% in all cell lines tested, and the fluorescence intensity of RNAe in HeLa cells was about 150%, and RNAe in CHO-K1 cells.
  • the fluorescence intensity was promoted by about 40%, and although the specific promotion ratio was different, RNAe-egfpc1 significantly enhanced the expression of EGFP.
  • AT-RNAe and prolonged RNAe can be introduced in these types of cells.
  • Example 6 RNAe can promote the expression of endogenous proteins
  • RNAe can enhance the expression of endogenous genes and does not affect its physiological functions, it can be extended.
  • SRY se determining region Y
  • SOX9 SRY (sex determining region Y)-box 9
  • Col2a type II collagen
  • pRNAe-sox9 plasmid the pairing sequence is (SEQ ID NO: 58), and transfected into the rat pre-chondral cell line C5.18 in an amount of 1 ⁇ g of well per 12-well plate, using Lipofectamine 3000 reagent (Invitrogen, USA).
  • the internal standard protein glyceraldehyde-3-phosphate dehydrogenase GPDH
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Example 7 RNAe can promote the expression of secreted proteins
  • RNAe method can promote a variety of intracellular non-secretory protein expression, and further we verified that the RNAe method can be applied to the promotion of secreted proteins.
  • a secreted reporter gene a secreted luciferase protein (we constructed pAT-RNAe-metluciferase (AT-RNAe, the paired sequence is (SEQ ID NO: 59) and pminRNAe-metluciferase (minRNAe, the paired sequence is (SEQ ID NO: 59))
  • a secreted luciferase protein we constructed pAT-RNAe-metluciferase (AT-RNAe, the paired sequence is (SEQ ID NO: 59) and pminRNAe-metluciferase (minRNAe, the paired sequence is (SEQ ID NO: 59)
  • a plasmid expressing a secreted luciferase protein (Clonetech Inc.
  • Example 1 purchased the plasmid to clone the gene into the pEGFP-C1 vector) and the RNAe plasmid designed and constructed in the above manner as in Example 1.
  • the transfection method was used to transfect HEK293T cells, and the cell supernatant was collected for detection. Detection of secreted luciferase protein using [Promega Biotechnology Co., Ltd. The dual luciferase reporter gene detection system (E1910)], after normalizing the relative fluorescence intensity by the control group, it can be seen that the fluorescence intensity of pAT-RNAe-metluciferase in the enzymatic reaction of the secreted luciferase protein is exceeded.
  • pminRNAe-metluciferase significantly promoted the fluorescence intensity of secreted luciferase protein in the enzymatic reaction by more than 50%, demonstrating expression in secreted luciferase protein in cell supernatants The amount has been significantly improved, demonstrating that both RNAe enhance the expression of secreted luciferase protein in the cell and ultimately increase its secretion (Fig. 7).
  • Example 8 RNAe can promote antibody expression and has potential for industrial production applications
  • Antibodies have important value in scientific research and medical applications, and promote the expression and production of antibodies.
  • the antibodies have tetramers (two heavy chains and two light chains) and require glycosylation and other secretions. It works outside the cell and is a special protein.
  • the application of RNAe in it further proves its applicability.
  • RNAe After analysing the results of the ELISA experiment by the control group, it can be seen that both RNAe have a stimulating effect on the luminescence intensity of the ELISA test more than 100% (40:32 group in Fig. 3C), which proves that the 10E8 in the cell supernatant is increased. , to prove that RNAe promotes the expression of 10E8 in cells and eventually brings about an increase in its secretion.
  • Example 9 RNAe can be loaded into cells by various forms.
  • the RNAe method can play a role after transcription into a nucleus through a plasmid or a lentiviral vector, or it can be transcribed in vitro to obtain transcript RNA and then directly transfected into the cytoplasm to directly function, which greatly broadens its application. range.
  • RNAe method can be transfected into a cell by a plasmid vector to function.
  • RNAe can be transfected into cells to function by means of stable transfection such as lentivirus. Therefore, it is possible to obtain a cell line with enhanced expression of a certain protein by constructing a stably transfected cell line, which can facilitate the application of RNAe in actual production.
  • the fluorescence expression of the cell line stably transfected with both RNAe was increased by more than 100% compared with the normal HEK293T cells of the control group, which proved that the stably transfected RNAe also had physiological functions.
  • RNAe can be first transcribed by in vitro transcription and then transfected into cells, and the absence of 5' capped RNAe reduces the cost of capping in vitro. Since the plasmid needs to enter the nucleus to transfect and function, and the plasmid enters the nucleus is a limiting step in its functional process. In contrast, the transfected RNA does not need to enter the nucleus for transcription, and can function directly in the cytoplasm. Therefore, the final efficiency of transfection will be higher, and the types of cells used will be more.
  • T7RNA Efficient Synthesis Kit #E2040S, NEB (Beijing) Co., Ltd.
  • transcribe the pminRNAe-egfpc1 plasmid in vitro see Example 10 for the specific construction of the plasmid in the following experiment
  • capping of RNA by adding the cap analog m7G ( 5') ppp (5') G (#S1404S, NEB (Beijing) Co., Ltd.) to achieve.
  • RNAe transcribed in vitro can successfully function in cells.
  • Fig. 8B capped in vitro transcribed minRNAe-bax, minRNAe-bid (Bax, Bid inhibits cell proliferation), can reduce cell proliferation rate, demonstrating that RNAe transcribed in vitro can successfully function in cells.
  • Example 10 The RNAe method allows for large-scale gene screening with the potential for high-throughput gene screening
  • RNAe method has high The application prospect of flux gene screening.
  • RNAe for a specific gene requires approximately 550CNY and 7 days. This construction cost and time is similar to the RNAi method (high-throughput gene screening using the RNAi method is currently the more common method), and its cost and The time period is suitable for high throughput gene screening.
  • Table 3 The number of genes related to cell proliferation from the literature (see Table 3), and conducted a verification process by whether these gene functions are consistent with the literature reports, since the verification is the same for large-scale genes. Validation was performed in the same manner as the high-throughput screening mode, so if the screening was successful, the RNAe method has potential for high-throughput gene screening applications. (Figure 9).
  • RNAe-gsk3 ⁇ paired sequence is (SEQ ID NO: 49)
  • pRNAe-p21 pairing sequence is (SEQ ID NO: 53)
  • pRNAe-e2f1 pairing sequence is (SEQ ID NO: 45)
  • pRNAe-cxcr4 pairing sequence is (SEQ ID NO: 44)
  • pRNAe-eif5a2 pairing sequence is (SEQ ID NO: 47)
  • pRNAe-hsp70 pairing sequence is (SEQ ID NO: 51
  • pRNAe-smad3 pairing sequence is (SEQ ID NO: 55)
  • pRNAe-c-fos pairing sequence is (SEQ ID NO: 40)
  • pRNAe-ccne1 pairing sequence is (SEQ ID NO: 39)
  • pRNAe-bid pairing sequence is (SEQ ID NO: 38)
  • RNAe method we proposed with a widely accepted method of RNAi to verify the effect of RNAe.
  • 15 genes epidermal growth factor receptor 1 ⁇ , p21, e2f1, bid, c-myb, bax, c-myc, p53, seven proliferative genes cxcr4, smad3, ccne1, bcl-xL, bcl-2 , src, sp1 and synthesized the RNAi molecules corresponding to these genes (designed by Shanghai Jima Pharmaceutical Technology Co., Ltd.), synthesizing and ensuring the inhibition efficiency of three RNAi mixed inhibition to ensure its function).
  • RNAe plasmid or the mixed three siRNAs into HEK293T cells according to the Lipofectamine 3000 instructions, and passed the Cell Counting Kit (CCK8) CCK-8/WST-8 kit of Shanghai Shengsheng Biotechnology Co., Ltd. (40203ES60)] The cell proliferation rate was measured.
  • the results of the experiment are shown in Figure 10.
  • the CCK8 values were down-regulated after RNAi transfection, and the CCK8 values were up-regulated after RNAe transfection.
  • the RNAe method promoted gene expression. The result is to promote the physiological effects of gene expression, while RNAi reveals the physiological effects of gene expression inhibition, which has the opposite effect on gene function.
  • RNAe is identical to RNAi and can perform functional function verification.
  • the RNAi transfer has almost no effect on the CCK8 value of the cells, and the CCK8 value is significantly down-regulated after RNAe transfection.
  • the gene expression level that inhibits cell proliferation in cells under normal conditions is low, and its silencing does not necessarily bring about significant physiological functions, but its expression increases, but it can produce obvious physiological effects, so the gene screening for inhibiting proliferation is After RNAi inhibited these genes, the cells were almost indistinguishable from normal growth, and RNAe had a significant advantage in this particular case (Fig. 10).

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Abstract

La présente invention concerne un ARN non codant synthétique comprenant une séquence répétée SINEB2 inverse et son utilisation dans l'amélioration de la traduction de protéines cibles. Spécifiquement, la présente invention concerne une molécule d'acide nucléique. La molécule d'acide nucléique est : (1) une molécule d'ARN formée des éléments suivants : une région d'appariement complémentaire de l'ARNm cible, une séquence SINEB2 inversée, une région de signal poly(A) optionnelle, et un élément de régulation optionnel; ou (2) une molécule d'ADN qui peut transcrire la molécule d'ARN.
PCT/CN2015/098478 2014-11-04 2015-12-23 Arn non codant synthétique comprenant une séquence répétée sineb2 inverse et son utilisation dans l'amélioration de la traduction de protéines cibles Ceased WO2016070853A1 (fr)

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EP3992289A1 (fr) * 2020-10-30 2022-05-04 Transine Therapeutics Limited Molécules d'acide nucléique fonctionnelles comprenant des domaines de liaison de protéines

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CN111696629B (zh) * 2020-06-29 2023-04-18 电子科技大学 一种rna测序数据的基因表达量计算方法

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YI, YAO ET AL.: "RNAe: an Effective Method for Targeted Protein Translation Enhancement by Artificial Non-coding RNA with SINEB2 Repeat", NUCLEIC ACIDS RESEARCH, vol. 1, 26 February 2015 (2015-02-26), pages 1 - 18, XP055277853, ISSN: 0305-1048 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT201800002411A1 (it) * 2018-02-05 2019-08-05 Scuola Int Superiore Di Studi Avanzati Sissa Domini strutturali di molecole di rna antisenso che aumentano la traduzione
WO2019150346A1 (fr) * 2018-02-05 2019-08-08 Scuola Internazionale Superiore Di Studi Avanzati - Sissa Domaines structurels de molécules d'arn antisens régulant positivement la traduction
EP3749772A1 (fr) * 2018-02-05 2020-12-16 Scuola Internazionale Superiore di Studi Avanzati - SISSA Domaines structurels de molécules d'arn antisens régulant positivement la traduction
CN112236524A (zh) * 2018-02-05 2021-01-15 国际高等研究院 上调翻译的反义rna分子的结构域
US12134768B2 (en) 2018-02-05 2024-11-05 Fondazione Istituto Italiano Di Tecnologia Structural domains of antisense RNA molecules up-regulating translation
EP3992289A1 (fr) * 2020-10-30 2022-05-04 Transine Therapeutics Limited Molécules d'acide nucléique fonctionnelles comprenant des domaines de liaison de protéines
WO2022090733A1 (fr) * 2020-10-30 2022-05-05 Transine Therapeutics Limited Molécules d'acide nucléique fonctionnelles incorporant des domaines de liaison à des protéines

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