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CN-121991944-A - Recombinant nucleic acid molecule for preparing scar-free annular RNA based on stem-loop structure and application thereof

CN121991944ACN 121991944 ACN121991944 ACN 121991944ACN-121991944-A

Abstract

The invention provides a preparation method for preparing scar-free annular RNA, a recombinant nucleic acid molecule for preparing scar-free annular RNA and application thereof. The recombinant nucleic acid molecule based on IRES stem loop structure provided by the invention can be used for preparing a circular nucleic acid molecule completely eliminating exogenous sequences, does not change any sequence of IRES, and avoids the unpredictable influence of IRES mutation on recruitment of ribosome functions.

Inventors

  • TAN WEIHONG
  • WANG WENXI
  • XIE SITAO
  • ZHANG YU
  • LV KEQIN
  • YI WEICHENG
  • FU TING

Assignees

  • 中国科学院杭州医学研究所

Dates

Publication Date
20260508
Application Date
20241107

Claims (20)

  1. 1. A recombinant nucleic acid molecule for the preparation of a scar-free circular RNA comprising elements arranged in the following order from 5 'to 3': Group 3' I intronic mutant fragments (intron fragment II), B. a first unit fragment II comprising at its 5' end a recognition fragment of the II-th ribozyme, C. A functional unit is provided for the purpose of, E. a first unit fragment I, the 3' end of which comprises a recognition fragment of the I-th ribozyme, Group f.5' I intron mutant fragment (intron fragment I); the functional units include IRES, RNA aptamer, protein binding sequence, protein coding region, non-coding region, etc., or combinations thereof; The intron fragment II is positioned at the 3' end of the intron fragment I, namely the complete group I intron mutant sequence comprises an intron fragment I-intron fragment II, wherein "-" represents a phosphodiester bond; the first unit fragment II is positioned at the 3' end of the first unit fragment I, namely the complete first unit sequence comprises a first unit fragment I-the first unit fragment II, wherein "-" represents a phosphodiester bond; wherein the 3 'end of the first unit fragment I comprises an I-th ribozyme recognition fragment, and the I-th ribozyme recognition fragment consists of a first preset number of nucleotides positioned at the 3' end of the first unit fragment I; the 5 'end of the first unit fragment II comprises a II-th ribozyme recognition fragment, and the II-th ribozyme recognition fragment consists of a second preset number of nucleotides positioned at the 5' end of the first unit fragment II; The group I intron mutant recognizes and covalently links the I-th ribozyme recognition fragment and the II-th ribozyme recognition fragment to obtain the circular nucleic acid molecule, namely the complete first unit sequence in the scar-free circular RNA comprises the I-th ribozyme recognition fragment-II-th ribozyme recognition fragment ('loop-forming fragment'), wherein '-' represents phosphodiester bond; The first unit has a partial stem structure, a partial double bond structure, or a partial hairpin structure adjacent to or comprising the loop-forming fragment; Preferably, the first unit is a nucleic acid aptamer or a translation initiation element; Preferably, the loop-forming fragment is located in the loop of the stem-loop structure of the translation initiation element, there is a double-stranded structure formed by complementary pairing sequences with each other in the 100 bases upstream and downstream of the loop-forming fragment, preferably the double-stranded structure comprises at least 5 consecutive complementary pairing bases, and further preferably the number of complementary pairing bases and non-complementary pairing bases in the stem of the stem-loop structure is more than 20bp.
  2. 2. The recombinant nucleic acid molecule of claim 1, wherein the group I intronic mutant comprises a mutation in the guide region of P1/P10, the mutated guide region recognizing the region of the original fragment in the first unit as a loop-forming fragment.
  3. 3. The recombinant nucleic acid molecule of claim 1 or 2, wherein the 3' terminal base of the I-th ribozyme recognition fragment is T, the first predetermined number of nucleotides is selected from 3-6 nucleotides, and the second predetermined number of nucleotides is selected from 0-3 nucleotides; Preferably, the group I intron is a mutant of an Ana ribozyme, the I-th ribozyme recognition fragment is 5' -N 1 N 2 N 3 T-3' or 5' -N 2 N 3 T-3', the II-th ribozyme recognition fragment is 5' -N 4 N 5 N 6 -3', the Ana ribozyme mutant comprises a mutation region N 6' N 7' ATAAN 5' N 4' GN 3' N 2' in the 5' intron fragment, wherein N is A, U, C, G or T, wherein N 3' N 2' is reverse complementarily paired with N 2 N 3 , N 5' N 4' is reverse complementarily paired with N 4 N 5 , N 5' N 4' is reverse complementarily paired with N 6' N 7' , the base complementation pairing comprising A-U, G-C, G-U, A-T, G-T base pairs; Preferably, the group I intron is a mutant of T4td ribozyme, the I-th ribozyme recognition fragment is 5' -N 1 N 2 N 3 N 4 N 5 T-3' or 5' -N 2 N 3 N 4 N 5 T-3' or 5' -N 3 N 4 N 5 T-3', the II-th ribozyme recognition fragment is 5' -N 6 N 7 -3' or N 6 or is absent, the T4td ribozyme mutant comprises the mutation region N 8' AATTGN 7' N 6' GN 5' N 4' N 3' N 2' N 1' in the 5' intron fragment, wherein N is A, U, C (U, C), G or T, N 5' N 4' N 3' N 2' N 1' reverse complement to N 1 N 2 N 3 N 4 N 5 , or N 5' N 4' N 3' N 2' reverse complement to N 2 N 3 N 4 N 5 , or N 5' N 4' N 3' reverse complement to N 3 N 4 N 5 , N 7' N 6' reverse complement to N 6 N 7 , or N 6' reverse complement to N 6 , and in the absence of N 6 N 7 , the 3' -terminal base T of the I-th ribozyme recognition fragment is immediately adjacent to the stem structure of the first unit, the P10 guide sequence N 7' N 6' may be unmutated, and N 8' reverse complement to N 6' , the base complement pair comprising A-U, G-C, G-U, A-T, G-T base pairs, preferably N 6 is U or C, N 6' is not mutated.
  4. 4. A recombinant nucleic acid molecule according to any one of claims 1-3, wherein the first unit fragment is an active sequence with the function of initiating translation of the functional unit, and the first unit fragment I and the first unit fragment II are a translation initiation element fragment I and a translation initiation element fragment II, respectively; Alternatively, the translation initiation element sequence comprises one or a combination of more than two of IRES sequences, 5' UTR sequences, kozak sequences, sequences comprising m6A modifications, the complement of ribosomal 18S rRNA, nucleic acid aptamer sequences.
  5. 5. The recombinant nucleic acid molecule of claim 4, wherein the IRES sequence comprises a ribosome entry site sequence such as HRV-B3, HRV-B92, iHRV-B37, iHRV-B97, iHRV-B4, iHRV-C11, iPV2, human XIAP, CVB3, EMCV, and mutants thereof, and artificially recombinant shuffledIRES #01, shuffledIRES #38, shuffledIRES #03, shuffledIRES #42, and mutants thereof; preferably, the nucleotide sequence of the translation initiation element is as set forth in any one of SEQ ID NOs 53, 54, 55, 56, 57, 58, 60, 62, 64 and 80; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 66, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 67; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 92, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 93; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 84, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 85; preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 96, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 97; preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 88, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 89; Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 66, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 81. Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 100, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 101. Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 102, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 103. Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 104, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 105. Preferably, the nucleotide sequence of the translation initiation element fragment I is shown as SEQ ID NO. 106, and the nucleotide sequence of the translation initiation element fragment II is shown as SEQ ID NO. 107.
  6. 6. The recombinant nucleic acid molecule of any one of claims 1-5, wherein the nucleotide sequence of intron fragment I is shown in any one of SEQ ID NOs 70, 71, 72, 86, 90, 94 and 98, and the nucleotide sequence of intron fragment II is shown in SEQ ID NOs 68 or 69; Preferably, the nucleotide sequence of the intron fragment I is shown as 70 or 71, and the nucleotide sequence of the intron fragment II is shown as SEQ ID NO. 68; Preferably, the nucleotide sequence of the intron fragment I is shown as any one of 72, 86, 90, 94 and 98, and the nucleotide sequence of the intron fragment II is shown as SEQ ID NO. 69.
  7. 7. The recombinant nucleic acid molecule of any one of claims 1-6, wherein the functional unit comprises at least one coding region, optionally the functional unit comprises at least two coding regions, each coding region encoding any type of polypeptide of interest independently of the other.
  8. 8. The recombinant nucleic acid molecule of claim 7, wherein the functional unit comprises at least two coding regions, wherein a linker is attached between any adjacent two coding regions; preferably, the linker is a polynucleotide encoding a 2A peptide.
  9. 9. The recombinant nucleic acid molecule of claim 7, wherein the functional unit comprises at least two coding regions, wherein a translation initiation element is linked between any adjacent two coding regions; alternatively, the translation initiation element between any two adjacent coding regions comprises one or a combination of more of the sequences IRES sequence, 5' UTR sequence, kozak sequence, sequence comprising m6A modification, complement of ribosomal 18SrRNA, nucleic acid aptamer.
  10. 10. The recombinant nucleic acid molecule of any one of claims 1-9, wherein the functional unit comprises a coding sequence for a human or non-human protein; Preferably, the human or non-human protein is selected from the group consisting of an antigen, an antibody, an antigen binding fragment, a therapeutic polypeptide, a fluorescent protein, a Car-T molecule, a 2A peptide, a protein having disease therapeutic activity, a protein having gene editing activity; preferably, the human or non-human protein is a tandem tumor antigen peptide and a Fluc protein, more preferably, the amino acid sequence of the tandem tumor antigen peptide is shown as SEQ ID NO. 13, and the nucleotide sequence of the Fluc protein is shown as SEQ ID NO. 22.
  11. 11. The recombinant nucleic acid molecule of any one of claims 1-10, wherein the recombinant nucleic acid molecule further comprises an insertion element, the insertion element being located between the coding element and the translation initiation element; the insertion element is selected from at least one of the group consisting of (i) - (iii) below: (i) a transcriptional level regulatory element, (ii) a translational level regulatory element, (iii) a purification element; Optionally, the insert element comprises a sequence of one or a combination of two or more of the following: An untranslated region sequence, a polyA sequence, polyAC sequence, an aptamer sequence, a riboswitch sequence, a sequence that binds to a transcriptional regulator, an antisense oligonucleotide (ASO), a small interfering ribonucleic acid (siRNA), a miRNA sponge, or a LncRNA.
  12. 12. The recombinant nucleic acid molecule of any one of claims 1-11, wherein the interior of any one of the intron fragment I, the coding element, and the intron fragment II does not comprise a nucleotide sequence derived from an exon, or wherein the interior of any two of the intron fragment I, the translation initiation element fragment II, the coding element, the translation initiation element fragment I, and the intron fragment II does not comprise a nucleotide sequence derived from an exon.
  13. 13. The recombinant nucleic acid molecule of any one of claims 1-12 having a nucleotide sequence as set forth in any one of SEQ ID NOs 76, 77, 78, 82, 83, 87, 91, 95, 99, 108, 109, 110 and 111.
  14. 14. A recombinant expression vector, wherein the recombinant expression vector comprises the recombinant nucleic acid molecule of any one of claims 1-13; preferably, the vector comprises a promoter at both ends of the recombinant nucleic acid molecule; More preferably, the promoter includes one or more promoters including, but not limited to, a T7 promoter, an Sp6 promoter, a T3 promoter, a Ptac promoter, a trp promoter, a CMV promoter, a PGK promoter, a Ubc promoter, an SV40 promoter, a CAG promoter, a U6 promoter, and an H1 promoter; preferably, the promoter is a T7 promoter, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 79.
  15. 15. Use of the recombinant nucleic acid molecule according to any one of claims 1-13, or the recombinant expression vector according to claim 14, for the in vitro preparation of a circular RNA.
  16. 16. A method for preparing a circular RNA in vitro comprising the steps of: (1) The recombinant nucleic acid molecule of any one of claims 1-13 or the recombinant expression vector of claim 14 transcribed to form a circularized precursor nucleic acid molecule; (2) The cyclization precursor nucleic acid performs cyclization reaction to obtain cyclic RNA; Optionally, the method further comprises the step of purifying the circular RNA.
  17. 17. The recombinant nucleic acid molecule of any one of claims 1-13, the recombinant expression vector of claim 14, or the circular RNA prepared according to the method of claim 16.
  18. 18. A host cell expressing the recombinant nucleic acid molecule of any one of claims 1-14 or the recombinant expression vector of claim 15 or the circular RNA of claim 17.
  19. 19. A composition comprising the recombinant nucleic acid molecule of any one of claims 1-13, the recombinant expression vector of claim 14, or the circular RNA of claim 17, and one or more pharmaceutically acceptable carriers.
  20. 20. The composition of claim 19, wherein the pharmaceutically acceptable carrier is selected from a lipid, a polymer, or a lipid-polymer complex.

Description

Recombinant nucleic acid molecule for preparing scar-free annular RNA based on stem-loop structure and application thereof Technical Field The invention relates to the fields of molecular biology, bioengineering technology and gene recombination technology, in particular to a preparation method and application of a circular nucleic acid molecule capable of completely eliminating exogenous sequences. Background MRNA drugs, which are a very potential drug approach, can efficiently express proteins in cells and are very suitable for vaccine and protein supplementation therapies, but also present challenges including mRNA stability, specific expression in tissues and organs, and the like. Unlike linear RNAs, circular RNAs (circrnas) do not have a 5 'cap and a 3' polyadenylation polyA tail, are covalently closed circular structures that allow the circrnas to be protected from exonuclease degradation, have a relatively longer half-life and greater stability in cells. The circRNA can also be used as a transcript for translation to produce a protein, and in the circRNA, an Internal Ribosome Entry Site (IRES) is inserted into the upstream of the ORF of the protein coding sequence, and the ribosome can be recruited to complete protein translation, so that comparison evaluation on known IRES activity and recombination optimization are reported. The circRNA is more stable and is able to translate the properties of the protein, making it a potential alternative to mRNA molecules. There are various methods of chemistry and biology for the cyclization of RNA, including the use of T4 RNA ligase, group I intron ribozymes, group II intron ribozymes, and Tornado expression systems. The natural group I intron ribozyme can catalyze the cleavage from the RNA precursor by two continuous transesterification reactions, a PIE (permuted intron-exon) strategy is developed by utilizing the group I intron ribozyme, and the PIE splicing strategy is used for reversing the half-intron sequences with partial exons (E) at two sides of the group I ribozyme to obtain linear RNA containing 3 '-intron-E2-target RNA-E1-5' intron structure, and can splice the introns to obtain E2-target RNA-E1 loop RNA by only adding GTP and Mg 2+ as cofactors. For PIE method, further engineering improvement is carried out to improve cyclization efficiency, daniel G.Anderson et al researchers mainly use Anabaena tRNA ribozyme intron, homologous arms which can be complemented are added at two ends of 3 'intron-E2-target RNA-E1-5' intron to pull two ends of linear RNA, spacer sequences (comprising polyA or polyAC and complementary paired homologous arms) are added between two sides of target RNA and E2 and E1 to ensure that ribozyme structure is not interfered by other RNA sequences and independent splicing bubbles are formed, so that PIE method can realize efficient cyclization in vitro and is more suitable for cyclization of long RNA. However, the E2 and E1 exons and the spacer sequence that assist cyclization remaining in the circular RNA in the PIE method are redundant sequence structures, and may cause unnecessary effects such as immunogenicity, interference with the circular RNA structure, and influence on translation. Based on PIE method, left-hand H-H etc. researchers use T4Td ribozyme intron to develop a method system for screening target protein coding region sequence, screening the exon sequence (5 '-TTGGGTCT-3') or its similar sequence at T4Td ribozyme recognition site position in target coding region sequence, meeting the requirement of less hairpin structure and small free energy in upstream and downstream sequence, cutting it at T4Td ribozyme recognition site at said position, splicing it to form "3 'intron-coding region cut-off segment 1-IRES-coding region cut-off segment 2-5' intron" linear structure, making intron splice in coding region to obtain circular RNA without redundant sequence, but the method has its limitation that the exon sequence (5 '-TTGGGTCT-3') or its similar sequence must contain T4Td ribozyme recognition site position in coding region sequence, limiting its application range. In addition, zuo Chijian and other researchers also use similar ideas to screen positions with fewer hairpin structures and small free energy in IRES sequences of Enterovirus A, caprine kobuvirus and Echovirus E29, base substitution is carried out on sequences similar to a T4Td ribozyme recognition site in the positions, the base substitution is mutated into a ribozyme recognition site 5'-TTGGGTCT-3', and the base substitution is truncated at the position to splice into a linear structure of a 3 'intron-IRES truncated fragment II-coding region-IRES truncated I-5' intron, so that the intron is spliced in an IRES region, but the IRES is mutated by the method, and the function of IRES recruiting ribosome to initiate translation is influenced [7] unprecedentedly. Thus, eliminating redundant exon sequences in the construction of circular RNAs, there remains a nee