WO2012161144A1 - Rna配列上の修飾を識別するリボザイムおよびそれを用いたrna開裂方法 - Google Patents
Rna配列上の修飾を識別するリボザイムおよびそれを用いたrna開裂方法 Download PDFInfo
- Publication number
- WO2012161144A1 WO2012161144A1 PCT/JP2012/062878 JP2012062878W WO2012161144A1 WO 2012161144 A1 WO2012161144 A1 WO 2012161144A1 JP 2012062878 W JP2012062878 W JP 2012062878W WO 2012161144 A1 WO2012161144 A1 WO 2012161144A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- base
- rna
- ribozyme
- target rna
- hammerhead ribozyme
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
- C12N2310/121—Hammerhead
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
Definitions
- the present invention relates to a ribozyme for identifying a modification on an RNA sequence and an RNA cleavage method using the ribozyme. More specifically, the present invention relates to a hammerhead ribozyme that identifies modifications by RNA editing or the like on an RNA sequence and an RNA cleavage method using the same.
- RNA In vivo RNA is known to be chemically modified, and noncoding RNAs such as rRNA and tRNA function by being modified.
- mRNA is also modified, for example, A-to-I editing in which the nucleotide of mRNA is replaced from adenosine (A) to inosine (I), or C-- from cytosine (C) to uracil (U). Modifications such as RNA editing such as to-U editing are known.
- RNA editing is a mechanism for converting post-transcriptional genetic information.
- the base sequence of the mRNA precursor generated by transcription from the gene the base at a specific site is altered to another base by the action of an enzyme. It appears in various forms such as base insertion, deletion, and substitution (Non-patent Document 1). This phenomenon is generally a mechanism programmed in the body to change the gene product in response to changes in physiological conditions and environment.
- Non-patent Document 2 A-to-I editing, in which base substitution from adenosine (A) base to inosine (I) base at a specific site of the mRNA precursor by the action of adenosine deaminase (ADAR) is physiologically important.
- ADAR adenosine deaminase
- RNA precursors for A-to-I editing in coding RNA include serotonin 2C type receptor (HTR2CR) and glutamate receptor B subunit ⁇ ⁇ (GRIA2) ((Non-patent Document 3).
- Serotonin 2C type receptor is a 7-transmembrane G protein-coupled receptor that mediates neuronal signal transduction in the brain by serotonin, and is thought to be deeply involved in emotional control.
- the serotonin type 2C receptor (HTR2CR), when serotonin binds to the extracellular loop of HTR2CR, transmits its stimulation to the coupled G protein, and then causes changes in neuronal properties through the intracellular signaling pathway, Ultimately controls brain functions such as memory, learning, and emotion.
- HTR2CR serotonin 2C type receptor
- RNA editing the amino acid sequence of the G protein binding region of the receptor protein changes, and the signal transduction ability of the receptor changes.
- FIG. 2 combining the nucleotide sequence of HTR2CR and the encoded amino acid with a site that has undergone RNA editing (shown in white in the figure) results in amino acids from a single gene.
- a maximum of 24 types (mainly 8 types) of receptor proteins with different sequences and transfer functions are generated (Non-patent Documents 4 and 5, FIG. 2).
- Non-patent Document 6 RNA editing
- Non-Patent Literature 7 cytosine (C) is base-substituted to uracil (U), and cytosine is converted to uridine by deamination. 7). Nuclear transcripts encoding intestinal apolipoprotein B (ApoB) ⁇ ⁇ ⁇ have been reported to undergo C-to-U RNA editing, converting CAA codons to UAA stop codons and producing shorter proteins than before editing ( Non-patent document 8).
- RNA editing such as A-to-I editing and C-to-U editing is used to control genetic adaptability by generating a protein that is different from the protein generated when not editing. It is related to an important mechanism. Since RNA substitution modification in the coding region plays a decisive role in the control of biological processes, the occurrence of abnormal modification causes severe diseases (Non-Patent Document 9), especially RNA substitution. The involvement of modified schizophrenia, bipolar disorder, major depression and other mental disorders is drawing attention.
- RNA RNA precursors in addition to the serotonin type 2C receptor (HTR2C) and glutamate receptor B subunit (GRIA2) RNA RNA precursors, in particular the central nervous system mentality such as ⁇ -aminobutyric acid (GABA) receptors and potassium channels
- GABA ⁇ -aminobutyric acid
- Other RNA editing targets have been identified in sequences encoding receptors, ion channels and proteins of other proteins that are deeply involved in neural function (Non-patent Document 10). Although high-throughput sequence data suggests that RNA substitution editing controls amino acid sequence to control protein function, the detailed biological function of this RNA editing is still unclear.
- a ribozyme is known as a functional molecule that specifically recognizes and reacts with a specific site modified on an RNA sequence.
- HHR hammerhead ribozyme
- HHR hammerhead ribozyme
- HHR hammerhead ribozyme H
- HHR consists of an active region (Helix II) ⁇ ⁇ consisting of a core sequence with a conserved catalytic activity in the center, and two hybridization arms that recognize the target sequences 3 'and 5' of the active region. It consists of a recognition region ⁇ ⁇ (Helix III and Helix I, respectively) ⁇ ⁇ consisting of sequences.
- a target-specific hammerhead ribozyme kit can be prepared by converting the above-mentioned hybridization arm sequence for a target RNA according to a simple Watson-Crick base pairing rule.
- HHRs with different core sequences for target RNA can be designed, and by using a sequence complementary to the hybridization sequence sandwiching a specific triplet of target RNA, it can bind to target RNA and the triplet The phosphodiester bond existing on the 3 ′ side of can be cleaved (Non-patent Document 13).
- HHR hammerhead ribozyme
- Non-patent Document 14 Non-patent Document 14
- HHR hammerhead ribozyme
- RNA editing in brain controls a determinant of ion flow inglutamate-gated channels.
- RNA 14 (10): 2074-2085; Li, JB, Levanon, EY, Yoon, JK, Aach, J., Xie, B ., Leproust, E., Zhang, K., Gao, Y., and Church, GM 2009. Genome-wide identification ofhuman RNA editing sites by parallel DNA capturing and sequencing. Science 324 (5931): 1210-1213; Pullirsch, D. and Jantsch, MF 2010. Proteome diversification byadenosine to inosine RNA editing. RNA Biol 7 (2): 205-212. Uhlenbeck, O.C.
- HHR hammerhead ribozyme
- the hammerhead ribozyme (HHR) ⁇ according to the present invention has a target specificity of HHR for the three bases (triplets) of the target RNA that is preferentially cleaved so that the modification site such as RNA editing can be cleaved specifically. Based on the design.
- HHR hammerhead ribozyme kit
- the hammerhead ribozyme kit (HHR) designed based on the above theory can be represented by the structural formula in FIG.
- the upper molecule represented by a thick black solid line represents a hammerhead ribozyme.
- This hammerhead ribozyme (HHR) has three regions, namely, an active region (Helix II) consisting of a core sequence having catalytic activity in the center, and a target RNA located 5 'to the active region (Helix II).
- Helix I 5 'recognition region
- HelixHIII 3' recognition region
- the 5 ′ side recognition region (Helix I) and 3 ′ side recognition region (Helix III) consisting of the arm sequences are designed to hybridize with the target RNA (substrate RNA).
- the base (D) is located between the 3 'recognition region (Helix III) and the active region (Helix II), and the active region (Helix II) and 5 'Design to interpose the base (X) at the position between the side recognition region (Helix I).
- This base (D) forms a base pair with the target RNA base and pairs with the base H of the triplet represented by 3′-NHH′-5 ′ (the structural formula in FIG. 3) in the target RNA.
- the base (X) is designed to form a base pair only with the modification site (editing site) of the target RNA.
- cytosine is used as the recognition base.
- C-to-U editing specific cleavage guanosine is converted to adenosine, so adenosine is used as the recognition base.
- the base of the target RNA is converted to another base by other modifications other than A-to-I editing or C-to-U editing, it should correspond to the converted base. It goes without saying that the recognition base of HHR can be designed.
- the lower molecule represented by the black solid line represents the target RNA (substrate RNA).
- This target RNA hybridizes to the 5 'recognition region (HelixHI) and the 3' recognition region (Helix III) of HHR to form base pairs, respectively.
- the target RNA (substrate RNA) has a modification site (indicated here as “edit”), this modification site hybridizes with the recognition base of HHR to form a base pair. The bond with the base adjacent to the 3 ′ side of the modification site is cleaved.
- the triplet represented by 3′-NHH′-5 ′ indicates a site with particularly high cleavage activity, and is a site that is preferentially cleaved by the catalytic action of HHR ( Cleavage Site).
- N may be any nucleotide
- H represents adenosine (A), cytosine (C), or uracil (U) (Kore et al. 1998).
- the present invention provides a hammerhead ribozyme (HHR) that identifies modifications of RNA on the target mRNA, for example, modifications such as RNA editing (A-to-I editing, C-to-U editing, etc.).
- HHR hammerhead ribozyme
- Another object of the present invention is to provide a hammerhead ribozyme-target RNA construct that is a base-paired product of a hammerhead ribozyme (HHR) and a target RNA.
- HHR hammerhead ribozyme
- another object of the present invention is to provide a method for cleaving a target RNA comprising cleaving a specific site modified with a target RNA by base pairing a hammerhead ribozyme (HHR) with a target RNA. Is to provide.
- HHR hammerhead ribozyme
- the present invention is a hammerhead ribozyme capable of recognizing the target RNA modified as described above, which has the general formula [I]:
- X is a modified recognition base that recognizes the modification site of the target RNA, and means adenine (A), cytosine (C), guanine (G) or uracil (U);
- N may be the same or different, and means any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U);
- N ′ may be the same or different and is any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U), and the corresponding base N
- a hammerhead ribozyme in which the base X is adenine (A) or cytosine (C) in general formula ⁇ ⁇ ⁇ [Ia].
- HHR hammerhead ribozyme
- N ′ is any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U)), and the corresponding base in the hammerhead ribozyme (HHR) Means a base that forms a base pair with N; H means any base selected from adenine (A), cytosine (C), guanine (G), or uracil (U), and forms a base pair with the corresponding base D in the HHR.
- H ′ means any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U);
- E is any base selected from adenine (A), cytosine (C), guanine (G), uracil (U) or inosine (I), and the corresponding base X and base in HHR [I] above Means a base that forms a pair, and that, when forming a base pair with base X, cleaves the bond with the base H ′ adjacent to the 5 ′ side; m and n have the same meaning as described above) It can be expressed as
- the present invention relates to the general formula [III] constructed by forming a base pair between the hammerhead ribozyme ⁇ ⁇ and the target RNA:
- a hammerhead ribozyme-target RNA construct represented by:
- a general formula [IIIa] constructed by base pairing of a hammerhead ribozyme [Ia] and a target RNA [II]:
- a hammerhead ribozyme-target RNA construct represented by:
- the base E when the base E is the base I, the base X is the base C, and when the base E is the base U, the base X Provides a hammerhead ribozyme-target RNA construct that is base A.
- the present invention provides an RNA-modified cleavage method comprising cleaving the cleavage site of the target RNA portion of the construct using the HHR. More specifically, the present invention provides an RNA cleavage method comprising cleaving a modification site existing on a target RNA using the HHR, and cleaving a binding site adjacent to the 5 ′ upstream of the RNA modification site. To do.
- the hammerhead ribozyme (HHR) has a triplet sequence 5′-N′HH′-3 ′ (where the base N ′ is adenine (A), uracil ( U), guanine (G) or cytosine (C), and the base H and the base H ′ may be A, C or U).
- base H nor base H ′ represents guanine (G), but when base H and base H ′ are guanine (G), it is considered that there is almost no cleavage activity, If it occurs, it may be guanine (G).
- this triplet sequence is naturally included in the scope of the present invention.
- Electrophoresis diagram after synthesis of each ribozyme and substrate RNA S.
- FIG. 5 is another electrophoretic diagram showing the results of a round 1 cycle check.
- disconnection of the ribozyme with respect to a HTR2C RNA fragment is a figure which shows the base sequence of a ribozyme and an HTR2C RNA fragment.
- B The figure which shows the analysis result of A-to-I edit specific cutting
- C The figure which summarized the cutting rate obtained by the experiment with respect to HR-HTR2C and CHR-HTR2C-edit with respect to HTR2C-ade and HTR2C-ino.
- TM APOB
- A is a figure which shows the base sequence of ribozyme and APOB mRNA fragment.
- FIG. 20 The figure which shows the time-dependent cleavage reaction and dynamic analysis result about HR-APOB-edit with respect to a non-edited and edited APOB * RNA fragment.
- FIG. 20 the figure which shows the time-dependent cutting
- TM unedited APOB
- (A) is a figure which shows the base sequence of a ribozyme and a synthetic
- (B) is a figure which shows the analysis result of A-to-I edit specific cleavage by ribozyme using denaturing PAGE (15%).
- (C) The figure which summarized the cutting rate obtained by the experiment (B) with respect to HR-HRFLNA and HR- FLNA -edit against FLNA -ade and FLNA -ino.
- Diagram showing in vitro A-to-I editing-specific cleavage of ribozymes for cell-extracted FLNA mRNA (edit-specific cleavage of HR-FLNA-edit for cell-extracted FLNA mRNA by measuring the editing rate at the Q / R site Figure showing experimental method for analysis).
- the hammerhead ribozyme according to the present invention has a general formula [I]:
- X is a modification recognition base that recognizes the modification site of the target RNA, and means any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U);
- N may be the same or different, and means any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U);
- N ′ may be the same or different and is any base selected from adenine (A), cytosine (C), guanine (G), or uracil (U), and the corresponding base
- the active region (Helix II) having the core sequence of the hammerhead ribozyme of the present invention is a region that acts to catalyze the cleavage activity of the modified target RNA of the modified target RNA, and has the general formula [IV] : 3'- AAG NaNbN'a AG Nc AGUC -5 '[IV] It can also be expressed as In the above general formula [IV], the underlined bases ( A , C , G and U ) are base sequences (consensus sequence) that exert a cleavage catalytic activity necessary for cleavage of the target RNA modification site. Represents.
- the hammerhead ribozyme as one preferred embodiment of the present invention has a general formula [Ia]:
- target RNA recognized by the hammerhead ribozyme of the present invention include, for example, serotonin 2C type receptor (HTR2C), glutamate receptor, ⁇ -aminobutyric acid (GABA) receptor, FLNA (filamin A, alpha [ actin binding protein 280]: actin-binding protein 280), apolipoprotein B (ApoB), and receptors that are deeply involved in the central nervous system such as potassium channels and RNA precursors such as ion channels .
- HTR2C serotonin 2C type receptor
- GABA ⁇ -aminobutyric acid
- FLNA lactamin A, alpha [ actin binding protein 280]: actin-binding protein 280
- ApoB apolipoprotein B
- receptors that are deeply involved in the central nervous system such as potassium channels and RNA precursors such as ion channels .
- each N ′ may be the same or different and is any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U)).
- H is any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U), and forms a base pair with the corresponding base D in the above HHR [I] Means
- H ′ means any base selected from adenine (A), cytosine (C), guanine (G) or uracil (U);
- E is any base selected from adenine (A), cytosine (C), guanine (G), uracil (U) or inosine (I), and forms a base pair with the corresponding base X in the HHR Means a base that, when forming a base pair with base X,
- base E means a base that has undergone modification such as RNA editing or mutation.
- base E means inosine (I) ⁇
- C-to For -U editing it means uracil (U) ⁇ ⁇ .
- the modified recognition base X (X) ⁇ of the hammerhead ribozyme that forms base pairs with the target RNA [II] ⁇ is cytosine (C)
- the base E of RNA [II] is uracil (U) ⁇
- it is preferably adenine (A).
- the three base sequence (triplet sequence) represented by the above general formula 5′-N′HH′-3 ′ is a peripheral sequence of the cleavage site of the target RNA, and a 3′-side region of the target RNA. It is a base sequence sandwiched between 5'-side regions and shows high cleavage activity for the target RNA cleavage site. That is, the hammerhead ribozyme (HHR) of the present invention has the triplet sequence 5′-N′HH′-3 ′ (wherein the base N ′ is adenine (A), uracil (U), guanine (G) or Cytosine (C), and the base H and the base H ′ may be A, C or U).
- HHR hammerhead ribozyme
- base H nor base H ′ represents guanine (G).
- base H and base H ′ are guanine (G)
- both base H and base H ′ may be guanine (G).
- this triplet sequence is naturally included in the scope of the present invention.
- the target RNA [II] when the target RNA [II] is subjected to modification such as RNA editing or mutation, the base at that site is converted to a base different from the original base. As a result, the modified base forms a base pair with the modified recognition base of the ribozyme. As a result, the binding site between the modified base and the base adjacent to the 5'-side upstream is cleaved.
- a hammerhead ribozyme-target RNA construct represented by:
- the modified recognition base (X) of HHR forms a base pair with the modification site (E) of the target RNA, so that it is upstream of the modification site (E). Breaks the bond with the adjacent base (H ′). That is, the base (E) of the target RNA means, for example, inosine (I) in the case of A-to-I editing, and uracil (U) in the case of C-to-U editing. To do. Therefore, in the case of A-to-I editing, the modified recognition base (X) of the hammerhead ribozyme corresponding to the modified base (E) is cytosine (C), and in the case of C-to-U editing. Is adenine (A).
- a hammerhead ribozyme-target RNA construct represented by:
- the hammerhead ribozyme of the present invention recognizes the modification site of the target RNA ⁇ that has been modified by, for example, RNA editing, and the modification recognition base (X) of the hammerhead ribozyme and the modification of the target RNA.
- a hammerhead ribozyme-target RNA construct is constructed by forming a base pair with the base (E), a phosphodiester with the base (H ′) adjacent to the 5′-side upstream of the modified base (E) of the target RNA It will break the bond.
- the target mRNA that has undergone modification such as RNA editing will produce a protein having a function or action different from that of the original protein derived from the target mRNA that has not undergone such modification.
- the hammerhead ribozyme of the present invention can suppress the function and action of the modified target mRNA that may adversely affect the biological function due to the modification of the target mRNA.
- the hammerhead ribozyme of the present invention includes, for example, serotonin 2C type receptor (HTR2C), glutamate receptor, ⁇ -aminobutyric acid (GABA) receptor, FLNA (actin binding protein 280), apolipoprotein B (ApoB) Prevention of diseases such as schizophrenia, bipolar disorder, major depression and other psychiatric disorders related to receptors and ion channels that are deeply involved in central nervous system neuropsychiatric functions such as potassium channels And can be expected to help improve treatment.
- HTR2C serotonin 2C type receptor
- GABA ⁇ -aminobutyric acid
- FLNA actin binding protein 280
- ApoB apolipoprotein B
- RNA editing occurs at the following sites in five sites A to E.
- Diseases that are considered to be involved in such RNA editing include the diseases shown in Table 1 below. Therefore, the hammerhead ribozyme according to the present invention can be expected to exert effects on diseases as shown in Table 1 below in addition to the above diseases.
- RNA editing in the protein coding region is also known, and the hammerhead ribozyme [I] according to the present invention is also as shown in Table 2 below for diseases associated with these genes. Expected to have an effect on diseases.
- HHR-HTR2C RNA construct hammerhead ribozyme and serotonin 2C receptor (HTR2C) mRNA
- HTR2C2RNA hammerhead ribozyme and serotonin 2C receptor
- the present invention is not limited to the HHR-HTR2C RNA construct. Absent.
- the structure of the HHR-HTR2C RNA construct used in the present invention can be described as follows.
- the modified site (E) of HTR2C RNA is represented as adenosine (A), that is, an unmodified site, and therefore, the cytosine of the modification recognition base (X) ⁇ ⁇ ⁇ ⁇ of the hammerhead ribozyme It cannot form base pairs with C). Therefore, in the construct represented by the above structural formula, cleavage does not occur at the cleavage site of HTR2C RNA.
- the HTR2C RNA modification site (E) is base-edited to inosine (I) by A-to-I editing, it forms a base pair with the edit recognition site (C), and the cleavage site indicated by the arrow Disconnection occurs at.
- the hammerhead ribozyme (HHR)-RNA construct includes, for example, a construct of HHR and FLNA (actin binding protein 280), and a construct of HHR and ApoB (Apolipoprotein B: apolipoprotein B). Etc.
- HHRz FLNA01 HHRz FLNA01
- the hammerhead ribozyme kit (HHR) kit can be added directly, or can be complexed with a cationic lipid, encapsulated in a liposome, or otherwise delivered to target cells.
- the hammerhead ribozyme or complex thereof of the present invention is incorporated into biopolymers with or without incorporation, ex vivo, or locally in vivo by injection, infusion pump or stent. Can be administered.
- the HHR of the present invention can be expressed in a cell from either an inducible or endogenous promoter since it can be expressed from a DNA or RNA vector delivered to the cell.
- recombinant vectors are preferably DNA plasmids, adenoviruses, retroviruses or adeno-associated virus vectors.
- Other mammalian cell vectors that direct the expression of RNA can also be used for this purpose.
- Such recombinant vectors can be delivered locally as described above, and the delivered HHR cleaves the target mRNA as soon as it is expressed.
- the composition of the liposome is usually preferably a combination of steroids, particularly phospholipids combined with cholesterol, particularly a combination of high phase transition temperature phospholipids.
- the physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations.
- the constructs of the invention can also be delivered as naked gene expression vectors. This means that the construct of the present invention is not associated with a delivery vehicle (eg, liposomes or colloidal particles).
- a delivery vehicle eg, liposomes or colloidal particles.
- One of the major benefits expected with naked vectors is that there is no immune response stimulated by the vector itself.
- the present invention is applicable to gene therapy for treating diseases.
- a therapeutic method can exert a therapeutic effect by introducing an appropriate ribozyme that specifically cleaves mRNA into a target cell having the above-mentioned disease.
- Ribozymes can be delivered using recombinant expression vectors such as chimeric viruses or colloidal dispersion systems.
- the gene therapy method according to the present invention can be carried out in vivo or ex vivo according to a conventional method.
- virus vectors that can be used include RNA viruses such as adenovirus, herpes virus, vaccinia, and retrovirus.
- retroviral vector examples of the retroviral vector are those derived from mouse or avian retrovirus.
- retroviral vectors that can insert a single foreign gene include, for example, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), and Lewis sarcoma virus (RSV) .
- MoMuLV Moloney murine leukemia virus
- HaMuSV Harvey murine sarcoma virus
- MuMTV mouse mammary tumor virus
- RSV Lewis sarcoma virus
- HHRH HTR2C
- HTR2C ribozyme with A-to-I editing-specific cleavage activity
- HR-HTR2C HHRH
- HTR2C RNA for cleaving C at the 5 'position. It was designed according to a common method to include 16 complementary bases (FIG. 18A).
- the core sequence having catalytic activity (Helix II) used in this example is as shown below.
- 5′-CUGANcGAGGCC GAAAGGCCGAA-3 ′ This sequence is a consensus sequence (5′-CUGANGA ---- GAA-3 ′) And 4 base pair (bp) GCGC duplex and stem loop structure including GAAA tetraloop (Scott, WG, et al. 1995. Cell 81 (7): 991-1002). Since the sequence context of the target cleavage site (GU A triplet) in this structure is selected based on the above triplet rule, HHR is expected to show cleavage activity against HTR2C RNA.
- the modified recognition base forms a UA base pair (A is a modified site) at the C site.
- An edit-specific ribozyme is constructed by converting the recognition base U to C so that cleavage activity is produced only when A at the C site is replaced with I by A-to-I RNA editing (FIG. 18A). ).
- Inosine forms base pairs with cytosine, which is identical to GC base pairs.
- the resulting ribozyme acquires the ability to identify target RNA modification sites by base pair recognition. Therefore, it is expected that the cleavage ability of this ribozyme against editing HTR2C (HR-HTR2C-edit) is higher than that of non-editing HTR2C.
- HHSeq_F_EcoRI (26mer) 5'-C GGAATTC TAATACGACTCACTATAG-3 '
- HHSeq_R_BamHI (25mer) 5'-GCG GGATCC GGTATGTAGCAATACG-3 '
- the modified RNA was obtained from Hokkaido System Science. 5 'terminal biotin S (27nt) 5'-CAUUACGUAAUCCUAUUGAGCAUAGCC-3 ' S C-ino (41nt) 5'-GGAUCGGUAUGUAGCAAUACGUAIUCCUAUUGAGCAUAGCC-3 '
- the design (control) of the ribozyme 1 (HRz1E and HRz2E) ⁇ that cleaves the E site on HTR2C mRNA is such that the sequence around the cleavage site conforms to the 5'-Nn'HH'-3 'rule (triplet rule).
- E-site-cleaving ribozymes (HRz1E and HRz2E) were designed by shifting the cleavage site of HRz2C by 1 base to the 5 ′ side.
- ribozyme HRz1C, HRz2C, HRz1E and HRz2E
- substrate RNA substrate RNA
- S1, S2, S3, S4 and S5 target sequence
- ribozymes HRz1C and HRz2C
- the target recognition region was converted to a sequence complementary to the sequence of HTR2C mRNA so that the C site on HTR2C mRNA was a cleavage site. Since it has already been reported that the number of stems in the active region affects the cleavage activity, a ribozyme with one stem (HRz1C: 35 nt) and a ribozyme with four stems (HRz2C: 41 nt) are designed. did. However, these two types of ribozymes have low activity because the sequence around the cleavage site is (5'-UAA-3 ') and does not apply to the 5'-N'HH'-3' rule (triplet rule). It was predicted.
- the base group in the left frame indicates Helix I (5'-recognition region)
- the underlined base group indicates the stem region
- the base group Helix III (3'-recognition region) in the right frame The same applies to the following.
- Ribozymes (HRz1E and HRz2E) can also be designed and synthesized in substantially the same manner as ribozymes (HRz1C and HRz2C).
- the following composition was heated at 98 ° C. for 5 minutes and then cooled to 25 ° C. over 1 hour for annealing.
- RNA ribozyme
- RNA sample was analyzed for purity using a denaturing gel. As a result, HRz1C was 38.8 ⁇ M, HRz2C was 35.8 ⁇ M, HRz1E was 17.0 ⁇ M, and HRz2E was 8.91 ⁇ M.
- Fig. 4 is an electrophoretic diagram after the T7 promoter reaction of each ribozyme (HRz1C, HRz2C, HRz1E, HRz2E and substrate RNA (S).
- Fig. 5 (A) and (B) are each ribozyme and substrate RNA (S ) And S5 after PAGE purification.
- HRz1C (35nt), HRz2C (41nt), HRz1E (35nt), HRz2E (41nt) and the target sequence (S (35nt)) constructed above were heated at 80 ° C for 3 minutes and then removed to 25 ° C over 15 minutes. After cooling and annealing, a cleavage reaction was performed by adding 20 mM MgCl 2 (4 ⁇ l) to an annealing sample (36 ⁇ l) having the following composition and incubating at 37 ° C.
- the reaction solution was sampled (4 ⁇ l and 8 ⁇ l each) at each time (0, 1, 2, 24 hours), and the sampled reaction solution was dissolved in 90% formamide after purification by ethanol precipitation, and a denaturing gel (6M).
- the cleavage band was analyzed by electrophoresis using Urea 15% polyacrylamide gel. Electrophoresis photographs were taken with a scanner, and the cleavage rate was calculated using image analysis software Image J.
- FIG. 6A and FIG. 6B are electropherograms showing evaluation of activity over time (EtBr staining) of each ribozyme and target RNA sequence (S).
- H1 represents HRz1
- H2 represents HRz2.
- H1 represents HRz1C
- H2 represents HRz2C.
- the number represents time (h).
- FIG. 7 is a bar graph showing the analysis results of the cut band by Image J, (A) shows the ratio of the cut band of HRz1E vs HRz1C, and (B) shows the ratio of the cut band of HRz2E vs HRz2C.
- the cleavage band of the target sequence was confirmed in all samples, but it was revealed that the control sequence cleaves the target sequence in a shorter time than HRz1 and HRz2. This is the expected result that it is difficult to construct a highly active ribozyme against the 5'-UAA-3 'sequence by molecular design alone.
- the cleavage activity was evaluated using an RI label.
- Samples purified by ethanol precipitation as described above were dissolved in 80% formamide and electrophoresed using a denaturing (8M urea, 15% polyacrylamide gel) gel.
- the target band was cut out, the gel was finely crushed with the tip of a pipette tip, 300 ⁇ l of TE buffer was added, and the mixture was stirred for 1 hour using a rotator. Centrifugation was performed at 15,000 rpm for 5 minutes, RNA was extracted from the gel, and then purified by ethanol precipitation.
- the purified sample was dissolved in 20 ⁇ l of TE to prepare substrate S radiolabeled at the 5 ′ end.
- the cleavage activity of the ribozyme obtained above was evaluated by the following method. To 2 ⁇ M ribozyme (10 ⁇ l), add 10 ⁇ cleavage buffer (2 ⁇ l), isotope-labeled substrate S (1 ⁇ l), H 2 O (5 ⁇ l), and heat at 80 ° C. for 3 minutes using a block incubator. after performs cooling and annealing reaction at room temperature for 10 minutes, the reaction was initiated by addition of 20 mM MgCl 2. Then, the cleavage reaction was performed by incubating at 37 ° C.
- FIG. 8 is a diagram showing the change over time of the cleavage activity (RI label) of each ribozyme.
- FIG. 9 is a bar graph showing the results of calculating the cleavage rate for each ribozyme cleavage activity (RI label) in FIG. 8 over time.
- RNA molecules having a target function were selected by the following procedure. (1) Selection of RNA molecules from RNA libraries under specific conditions (such as binding to target molecules or showing activity). (2) The RNA molecule selected in (1) is converted to DNA by reverse transcription. Addition and amplification of T7 promoter sequence by PCR. (3) Transcription to RNA by T7 RNA polymerase. By repeating the operations (1) to (3), an RNA molecule having a function of cleaving the C site on HTR2C mRNA was obtained.
- the basic skeleton used was a hammerhead ribozyme (Martick, M .; Scott, W. G. Cell 2006, 126, 309-20) derived from Schistosoma mansoni.
- the selection method was designed with reference to literature (Persson, T., et. Al. Chembiochem 2002, 3, 1066-71).
- composition is as shown below (H1 and H2 have the same composition).
- the libraries H1 and H2 obtained above were selected.
- the libraries H1 and H2 and the target RNA biotinylated at the 5 ′ end were respectively annealed.
- the magnetic bead (Dynabeads M-280 streptavidin: DYNAL) washed with a washing buffer (10 mM HEPES, 5 mM EDTA, 50 mM NaCl) and the annealed sample are mixed, and incubated at room temperature for 20 minutes.
- a streptavidin binding reaction was performed (1 Round: 300 ⁇ l; 2-8 Round: 50 ⁇ l).
- a cleavage buffer 50 mM Tris / HCl (pH 7.5), 50 mM NaCl, 20 mM MgCl 2 ) was added and incubated at 37 ° C. for 30 minutes to activate the ribozyme.
- the active molecule cleaves the target RNA and dissociates from the magnetic beads.
- the solution was purified by ethanol precipitation, dissolved in 6 ⁇ l of TE, and converted to cDNA by reverse transcription reaction.
- a selection sample (3 ⁇ l) and 20 ⁇ selection H (RT) (9.5 ⁇ l) were heated at 80 ° C. for 15 minutes, left at 25 ° C. for 1 minute, and then cooled at 4 ° C. and annealed.
- the annealed sample (12.5 ⁇ l) ⁇ thus obtained was mixed with 5 buffer (4 l) 10 mM dNTP (2 l) AMV RTase (1.5) ⁇ , heated at 42 ° C for 15 minutes, and then heated at 99 ° C for 5 minutes. After that, it was cooled to 4 ° C. to carry out a reverse transcription reaction.
- PCR was carried out using the following composition as a cycle of heating at 95 ° C. for 15 seconds, heating at 55 ° C. for 30 seconds, and then heating at 68 ° C. for 30 seconds.
- a cycle check was performed under the same conditions as the PCR. In the first to third rounds, the cycle check was sampled every 5 cycles up to 10 to 35 cycles, and in the 4th to 8th rounds, sampling was performed every 4 cycles up to 8 to 24 cycles.
- FIG. 11A and FIG. 11B are electropherograms showing the results of the round 1 cycle check, respectively.
- the left half shows library H1
- the right half shows library H2.
- the amplified sample is purified by phenol / chloroform extraction and ethanol precipitation, dissolved in 10 ⁇ l of TE, and 5 ⁇ l of template DNA in 10 ⁇ l is used for the next round by in vitro transcription reaction (heating at 37 ° C. for 3 hours).
- the RNA library to be used was synthesized.
- RNA molecule sequence analysis was performed (Fig. 12: Library H1 on the left half and Library H1 on the right half) Library H2 is shown). The result of sequence analysis of library H1 is as shown in FIG. 13, and the result of sequence analysis of library H2 is as shown in FIG.
- library H1 had a fragment with 10/16 shorter than the basic skeleton.
- ribozymes were obtained that were shifted by one base from the C site so that the sequence of the cleavage site obeyed the 5'-N'HH'-3 'rule.
- library H2 since the convergence of molecular species was not so much observed, in-vitro selection was performed continuously, and a decrease in the number of PCR cycles after the reverse transcription reaction was confirmed in 12 rounds, so that sequence analysis was performed again. The result of rearrangement analysis is shown in FIG.
- RNA library (2H1) in which the sequence (5'-CUGA-3 '), which is essential for the cleavage activity in the active region, is fixed and the others are diversified so that only the ribozyme that cleaves the C site can be selected. As well as 2H2).
- the product was purified by phenol / chloroform extraction and ethanol precipitation, and dissolved in 20 ⁇ l of TE.
- pBluescript (0.245 ⁇ g / ⁇ l) was treated with a restriction enzyme by treating with BamHI and EcoRI at 37 ° C. for 2 hours. After the treatment, purification was performed by phenol / chloroform extraction and ethanol precipitation. After the treatment, the result of concentration quantification by Nanoview was 0.041 ⁇ g / ⁇ l.
- PCR In PCR, after 30 cycles of heat treatment at 95 ° C for 15 seconds, 55 ° C for 30 seconds and 68 ° C for 30 seconds, 1% agarose electrophoresis was performed to determine whether the target amplification product was obtained. confirmed. 18 samples of DNA libraries H1 and H2 were selected and subjected to sequence PCR.
- HRzC-ino a ribozyme that identifies A-to-I editing at the C site on HTR2C mRNA was designed.
- HRz2E is the basic skeleton, and C-site adenosine is recognized by forming a base pair with uridine present in the target recognition region of HRzC. Therefore, by utilizing the fact that inosine forms a base pair with cytosine, and replacing the uridine that base-pairs with the C site with cytosine, the ribozyme HRzC-, which shows cleavage activity only when the C site is edited A to I, designed ino.
- the activity of ribozyme HRzC-ino obtained above was evaluated (RI labeling).
- S C-ino in which the C sites of the substrates S and S are inosine (I) was used, and the cleavage activity against each target RNA was compared (Fig. 17).
- the target RNA 5 'end labeling method and activity evaluation method were performed in the same manner as described above. *
- the in vitro trans-cleaving activity and specificity of A-to-I editing-specific ribozymes against HTR2C RNA fragments were examined.
- an in vitro assay was performed using a synthetic HTR2C RNA fragment containing a C site.
- two forms of 32 P-labeled HTR2C mRNA fragment 37 nucleotides
- one with base A at the C site as an unedited substrate HTR2C-ade
- Two formats were used, one with base I at the C site (FIG. 18A).
- Substrate RNA was transcribed in vitro and annealed in the presence of gel purified excess amounts of HR-HTR2C and HR-HTR2C-edit ribozymes.
- the cleavage reaction was initiated by adding 20 mM MgCl 2 at 37 ° C. After 1 hour, the cleavage band was analyzed using gel electrophoresis, and the cleavage rate was calculated (FIGS. 18B and 18C). In the case of HR-HTR2C, similar cleavage rates were observed for HTR2C-ade and HTR2C-ino (0.69 and 0.59).
- FIG. 19A shows the time course of cleavage products obtained from the reaction of HR-HTR2C-edit on HTR2C-ade (upper part of FIG. 19A) and HTR2C-ino (lower part of FIG. 19A).
- the kinetic analysis results of HR-HTR2C-edit are high k cat value (0.67 ⁇ 0.011 min -1 ) when using HTR2C-ino, and low k cat (0.01 ⁇ 0.001 min -1 when using HTR2C-ade. ) Values (FIG. 19B, Table 3).
- HR-HTR2C is cut k cat values for both edited and non-edited substrate (0.05 ⁇ 0.001 min -1, 0.44 ⁇ 0.022 min -1) showed catalytic activity.
- the difference in k cat values for this ribozyme was less than that for HR-HTR2C-edit.
- the product fraction at the end of the reaction (F ⁇ ) is similar when the combination of recognition base and modification site is UA (0.74), UI (0.63), CI (0.85), CA showed a significantly lower value (0.23).
- C-to-U editing-specific cleavage of APOB RNA using a ribozyme was examined.
- C-to-U editing specific ribozymes were constructed by changing the recognition base.
- C-to-U editing-specific ribozyme is based on APOB mRNA, where C-to-U editing of base C at position 6666 converts the glutamine codon (CAA) into an in-frame stop codon. Designed as a target for mRNA.
- HHR was constructed to cleave the 5 'C-to-U modification site on APOB mRNA using the same catalytically active core sequence.
- C-to-U editing specific ribozyme (HR-APOB-edit) introduces base A into the recognition nucleotide to form a base pair with base U from C-to-U editing of APOB RNA.
- HR-APOB-edit introduces base A into the recognition nucleotide to form a base pair with base U from C-to-U editing of APOB RNA.
- the cleavage activity of HR-APOB-edit against non-editing substrate (APOB-cyt) and editing substrate (APOB-uri) was analyzed by in vitro cleavage assay (FIG. 20B). As shown in FIG.
- the ribozyme for editing-specific cleavage of FLNA mRNA was designed using the method described above, and the cleavage activity and editing specificity of HR-FLNA-edit used synthetic FLNA RNA fragments. Analyzed by in vitro cleavage assay ( Figure 22). The cleavage rate constant and fraction of the product at the end point of HR-FLNA-edit for the edited FLNA RNA fragment was much larger than that of the unedited FLNA RNA fragment ( Figure 22, Table 3). These results were similar to those observed for HR-HTR2C-edit.
- edited FLNA mRNA was prepared from HEK293 cells overexpressing ADAR2.
- the inventor has already established Tet-ADAR2 cells that can be stably transfected with an ADAR2 expression vector containing a doxycycline (Dox) inducible expression system.
- Total RNA containing a mixture of edited and unedited FLNA mRNA could be obtained from Tet-ADAR2 cells cultured with any concentration of Dox. If ribozyme specifically cleaves edited FLNA mRNA in the total RNA extracted, the ratio of edited FLNA mRNA to unedited FLNA mRNA should decrease.
- Dox doxycycline
- FIG. 23A An experimental scheme for quantifying the editing rate is shown in FIG. 23A.
- 150 ng of extracted RNA was annealed with an excessive amount of HR-FLNA-edit (final concentration: 2.5 ⁇ M), and a cleavage reaction was performed in a buffer containing MgCl 2 20 mM.
- RNA samples are subjected to RT-PCR using FLNA-specific primers, and PCR fragments are directly sequenced using the fluorescent dideoxy sequencing method. (FIG. 23A).
- the ribozyme recognition base is designed to form a base pair only with the modified base of the target RNA.
- RNA editing-specific cleavage Ribozymes designed for A-to-I editing-specific cleavage were more than 10 times more catalytically active against editing HTR2C and editing FLNA RNA fragments than against non-editing RNA.
- ribozymes for C-to-U editing of APOB RNA were found to have very high catalytic activity against the edited APOB RNA fragment.
- Converting the ribozyme recognition base as in the present invention is useful for designing editing-specific and mutation-specific cleavage, even if its cleavage activity is limited by the triplet rule.
- the cleavage sites of all the target RNAs exemplified were selected so as to match the triplet rule that retains the ribozyme cleavage activity. Since HHR has N'HH 'cleavage specificity, the present invention cannot be applied when G is present in the nucleotide at the 5'-side 1 base or 2 bases upstream of the target sequence mutation site. For the same reason, not all combinations of mutations can be identified using the mutant recognition method by HHR applying the N'HH 'specificity.
- the ribozyme is designed based on the strategy adopted in the present invention and considering which base is the target mutation site or exists in the context of the surrounding sequence, substitution of almost any nucleotide can be achieved. It can be specifically recognized by an artificial ribozyme. Thus, if the target mutation specificity increases, the possibility that the ribozyme can be applied to mutation or substitution editing that selectively suppresses gene expression can be increased.
- the present invention shows that editing-specific cleavage by ribozymes can be applied not only to short RNA fragments but also to physiological mRNAs in vitro.
- the effectiveness of editing-specific cleavage of cell-derived target mRNA was evaluated as a change in editing rate using the peak height of the modified site based on the DNA sequencing chromatogram.
- This method analyzed edit-specific cleavage of HR-FLNA-edit and required quantification of the total amount of FLNA mRNA, but roughly estimated that 51% of FLNA mRNA was HR in the 24-hour reaction. -Disconnected by FLNA-edit.
- the present invention utilizes the minimal hammerhead liposome (HHR) framework to design ribozymes for target RNA cleavage based on A-to-I and C-to-U RNA substitution editing.
- HHR minimal hammerhead liposome
- the basic framework for designing the ribozyme of the present invention is to convert the recognition base of the ribozyme to produce a ribozyme exhibiting high specific cleavage activity for both synthetic editing RNA fragments and physiological mRNAs.
- the strategy of the present invention is not limited to RNA substitution editing, but by selecting specific combinations of recognition bases and target bases, substitution of bases, including other types of mutations, such as mutations such as SNPs, It can be widely applied to modifications such as deletion and addition. Therefore, the present invention is expected to be useful not only for diseases caused by RNA editing and the like, but also for research and development of new drugs that contribute to the prevention and treatment of diseases caused by other types of modifications such as mutations.
Abstract
Description
Cはシトシン(C)を意味し;
Gはグアニン(G)を意味し;
Uはウラシル(U)を意味し;
Dは、アデニン(A)、 シトシン(C)、グアニン(G) またはウラシル (U) を意味し;
Xは、標的RNAの修飾部位を認識する修飾認識塩基であって、アデニン(A)、 シトシン(C)、グアニン(G) またはウラシル (U) を意味し;
Nは、いずれも同一であってもまたは異なっていてもよく、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基を意味し;
N’は、同一であってもまたは異なっていてもよく、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基であって、対応する塩基Nと塩基対を形成する塩基を意味し;
aは、1~10の整数、好ましくは2~6の整数、より好ましくは2~4の整数を意味し;
bは、1~6の整数、好ましくは2~4の整数を意味し;
cは、1~4の整数、好ましくは1~2の整数を意味し;
mは、2~50の整数、好ましくは5~30の整数、さらに好ましくは5~20の整数を意味し;および
nは、2~50の整数、好ましくは5~30の整数、さらに好ましくは5~20の整数を意味する)
で表されるハンマーヘッド型リボザイムを提供する。
で表されるハンマーヘッド型リボザイムが提供される。
Hは、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基であって、上記HHRにおいて対応する塩基Dと塩基対を形成する塩基を意味し;
H’は、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基を意味し;
Eは、アデニン(A)、シトシン(C)、グアニン(G)、ウラシル(U)またはイノシン(I)から選ばれるいずれかの塩基であるとともに、上記HHR [I] において対応する塩基Xと塩基対を形成する塩基であって、塩基Xと塩基対を形成したときに、5’側に隣接する塩基H’との結合を切断する塩基を意味し;
mおよびnはいずれも前記と同じ意味を有する)
で表わすことができる。
で表されるハンマーヘッド型リボザイム-標的RNA構築物を提供する。
で表されるハンマーヘッド型リボザイム-標的RNA構築物を提供する。
Cはシトシン(C)を意味し;
Gはグアニン(G)を意味し;
Uはウラシル(U)を意味し;
Dは、アデニン(A)、 シトシン(C)、グアニン(G) またはウラシル (U) を意味し;
Xは、標的RNAの修飾部位を認識する修飾認識塩基であって、アデニン(A)、 シトシン(C)、グアニン(G) またはウラシル (U) から選ばれるいずれかの塩基を意味し;
Nは、いずれも同一であってもまたは異なっていてもよく、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基を意味し;
N’は、同一であっても、または異なっていてもよく、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基であって、対応する塩基Nと塩基対を形成する塩基を意味し;
aは、1~10の整数、好ましくは2~6の整数、より好ましくは2~4の整数を意味し;
bは、1~6の整数、好ましくは2~4の整数を意味し;
cは、1~4の整数、好ましくは1~2の整数を意味し;
mは、2~50の整数、好ましくは5~30の整数、さらに好ましくは5~20の整数を意味し;および
nは、2~50の整数、好ましくは5~30の整数、さらに好ましくは5~20の整数を意味する)
で表すことができる。
3’-AAGNaNbN’aAGNcAGUC-5’ [IV]
で表すこともできる。上記一般式 [IV] において、下線を施した塩基(A、C、GおよびU)は、標的RNA の修飾部位の切断に必要な切断触媒活性作用を及ぼす塩基配列(共通配列:consensus sequence)を表している。
で表わすことができる。
binding protein 280]:アクチン結合タンパク質280)、アポリポタンパクB (ApoB) や、カリウムチャンネル等の中枢神経系の精神神経機能に深く関与している受容体やイオンチャンネル等のRNA前駆体などが挙げられる。
Hは、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基であって、上記HHR [I] において対応する塩基Dと塩基対を形成する塩基を意味し;
H’は、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基を意味し;
Eは、アデニン(A)、シトシン(C)、グアニン(G)、ウラシル(U)またはイノシン(I)から選ばれるいずれかの塩基であって、上記HHRにおいて対応する塩基Xと塩基対を形成する塩基であって、塩基Xと塩基対を形成したときに、5’側上流に隣接する塩基H’との結合を切断する塩基を意味し;
mおよびnはいずれも前記と同じ意味を有する)
で表わすことができる。
で表されるハンマーヘッド型リボザイム-標的RNA構築物を提供する。
で表されるハンマーヘッド型リボザイム-標的RNA構築物を提供する。
5′-CUGANcGAGGCC GAAAGGCCGAA-3′
この配列は、コンセンサス配列 (5′-CUGANGA----GAA-3′)
および4塩基対 (bp) GCGCデュプレックス (duplex) とGAAAテトラループ (tetraloop) を含むステム・ループ構造から構成されている (Scott,
W.G., et al. 1995. Cell 81(7): 991-1002)。この構造における標的切断部位(GUAトリプレット)の配列の前後関係は上記トリプレットルールに基づいて選択されているので、HHRは、HTR2C RNAに対して切断活性を示すことが期待される。そこで、得られたHHR構築物において、その修飾認識塩基は、C部位でU-A 塩基対(Aは修飾部位)を形成する。編集特異的リボザイムは、C部位のAがA-to-I RNA編集によってIで置換されたときだけ切断活性が出るように、認識塩基のUをCに変換することによって構築される(図18A)。イノシンは、G-C 塩基対と同一であるシトシンと塩基対を形成する。その結果、得られるリボザイムは、塩基対認識による標的RNA修飾部位を識別する能力を取得する。したがって、このリボザイムの編集HTR2C(HR-HTR2C-edit) に対する切断能力は、非編集HTR2Cよりも高いことが期待される。
(in vitro 転写試薬)
RNase-Free water、10× AmpliScribe T7 Reaction Buffer, 25mM NTP、100 mM DTT、RiboGuard RNase Inhitor、AmpliScribe T7または Enzyme Solution : AmpliScribe
Taq DNA Polymerase、10×Thermo Pol Reaction Buffer:NEB
2.5mM dNTP, 2mM dNTP:
(逆転写試薬)
AMV Reverse Transcriptase 10U/μl、AMV RT 5×BUFFER、10mM dNTP:Promega
Antarctic Phasshatase 5000U/ml、10×Antarctic Phasphatase buffer:NEB
(リン酸化試薬)
T4 Polynucleotide Kinase、10×T4 Polynucleotide Kinase Buffer, :TAKARA
NEG 502Z 〔gamma-32P〕 222TBq/mmol( 6000Ci/mmol ) 370MBq/ml:PerkinElmer
BamHI 10U/μl:TOYOBO
EcoRI 70U/μl:Promega 10×H buffer:TAKARA
(その他)
2×Ligation mix
HRz1C (53nt)
5’-GCAATACGTATTCGAAAACTCATCAGTCCTATTGCTATAGTGAGTCGTATTAG-3’
HRz 2C (59nt)
5’-GCAATACGTATTCGGCCTTTCGGCCTCATCAGTCCTATTGCTATAGTGAGTCGTATTAG-3’
HRzC 1E (53nt)
5’-AGCAATACGTTTCGAAAACTCATCAGATCCTATTCTATAGTGAGTCGTATTAG-3’
HRzC 2E (59nt)
5’-AGCAATACGTTTCGGCCTTTCGGCCTCATCAGATCCTATTCTATAGTGAGTCGTATTAG-3’
5’-CAATAGGATTACGTATTGCTACTATAGTGAGTCGTATTAG-3’
S5 (59nt)
5’-GGCTATGCTCAATAGGATTACGTATTGCTACATACCGATCCTATAGTGAGTCGTATTAG-3’
T7proG cap
5’-CTAATACGACTCACTATAG-3’
5’-CTAATACGACTCACTATAGGCTATGCTCA-3’
(Library H1(-)) (59mer)
5’-GGTATGTAGCAATACGTA(N24)TCCTATTGAGCATAGCC-3’
(Library H2(-)) (63mer)
5’-GGTATGTAGCAATACGTA(N24)TCCTAT(N4)TGAGCATAGCC-3’
5’-AGCAATACGTTTCGGCCTTTCGGCCTCATCAGGTCCTATTCTATAGTGAGTCGTATTAG-3’
(Library H(+)) (29 mer)
5’-CTAATACGACTCACTATAGGCTATGCTCA-3’
Selection H RT 16nt
5’-GGTATGTAGCAATACG-3’
5’-CGGAATTCTAATACGACTCACTATAG-3’
HHSeq_R_BamHI (25mer)
5’-GCGGGATCCGGTATGTAGCAATACG-3’
5’末端biotin S (27nt)
5’-CAUUACGUAAUCCUAUUGAGCAUAGCC-3’
S C-ino (41nt)
5’-GGAUCGGUAUGUAGCAAUACGUAIUCCUAUUGAGCAUAGCC-3’
なお、リボザイム(HRz1CおよびHRz2C) の設計は、HTR2C mRNA上C部位が切断部位となるように、標的認識領域をHTR2C mRNAの配列と相補的な配列に変換した。活性領域に存在するステムの数が切断活性に影響を与えることが既に報告されているため、ステム数が1つのリボザイム(HRz1C:35nt)および、ステム数が4つのリボザイム(HRz2C:41nt)を設計した。しかし、この2種のリボザイムは、切断部位周辺の配列が(5’-UAA-3’)となり、5’-N’HH’-3’ルール(トリプレットルール)には当てはまらない為に活性が低いと予測された。
図7は、Image Jによる切断バンドの解析結果を示す棒グラフであり、(A)は、HRz1E vs HRz1Cの切断バンドの割合、(B)はHRz2E vs HRz2Cの切断バンドの割合を示している。
まず、上記で得られたサンプルに、H2O (79μl)、10×Antarctic Phasphatase buffer (10μl)、10μM S (10μl)、Antarctic Phasphatase
(1μl) を加え、37℃にて1時間インキュベートして5’末端の脱リン酸化を行った。その後、フェーノール/クロロホルム抽出、エタノール沈殿により精製を行ったサンプルをH2O 14μlに溶解し、ブロックインキュベーターを用いて65℃で10分間加熱することによりAntarctic Phasphataseを失活させた。
RNAは、逆転写でDNAに変換することができ、DNAはPCRで増幅することも可能である。この性質を利用し、以下のような手順で目的機能を有するRNA分子の選出を行った。(1) RNAライブラリーからの特定条件下(標的分子に結合するや、活性を示すなど)でのRNA分子の選別。(2) (1)で選択した RNA分子を逆転写反応によるDNAへの変換。PCRによるT7プロモーター配列の付加、増幅。(3) T7 RNAポリメラーゼによるRNAへの転写。(1)~(3)の操作を繰り返し行うことで、HTR2C mRNA上C部位を切断する機能をもつRNA分子を得た。
活性領域に存在する24塩基を全てランダム化したライブラリーH1と、標的認識領域に4塩基のループを導入したライブラリーH2を設計した。
まず、ライブラリーH1およびH2と、5’末端をbiotin化した標的RNAをそれぞれアニーリングした。続いて、洗浄用バッファー(10mM HEPES、5mM EDTA、50mM NaCl)で洗浄したマグネットビーズ(Dynabeads M-280 streptavidin: DYNAL社)とアニーリングしたサンプルを混合し、室温で20分間インキュベートすることにより、biotinとstreptavidinの結合反応を行なった(1Round:300μl;2~8Round:50μl)。
8ラウンドのPCRにより増幅したDNAライブラリーに制限酵素をふ化するために、HH-FwとHH-Rvの2種類のプライマーを用いてPCRを行った。その際、最適なPCR条件を決定するために、サイクルチェックを行なった。サイクルチェックは、下記組成を用いて、95℃で15秒間加熱した後、55℃で30秒間、続いて68℃で30秒間加熱処理し、2~10ラウンドまでは2サイクルごとにサンプリングした。その結果、6ラウンドが最適条件であったので、以下の組成で制限酵素ふ化を行った。
pBluescript(0.245μg /μl)を、BamHIとEcoRIを含む組成を用いて、37℃で2時間処理して制限酵素処理を行った。処理後、フェノール/クロロホルム抽出、エタノール沈殿により精製を行った。処理後、ナノビューによる濃度定量の結果は、0.041μg /μlであった。
pBluescriptと、DNAライブラリー(H1、H2)とを、16℃で4時間31分間ライゲーションを行った。なお、mol比がpBluescript:DNAライブラリー=1:3であった。その後、フェノールクロロホルム抽出、エタノール沈殿により精製を行った後、H2O 3μlで溶解した。
ライゲーション後のサンプル3μlから1.5μlを氷上にて溶解したJM83に加え、エレクトロキュベットに移し、エレクトロポレーションを行った。その後、直ちにSOCに注ぎ、37℃にて30分間培養を行った。培養後のサンプルをLB培地(アンピシリン、X-gal含)に撒き、37℃にて一夜培養を行った。
一夜培養を行った後、青コロニーおよび白コロニーのうち、白コロニーのみを爪楊枝を用いて、DNAライブラリーH1およびH2をそれぞれ24個ずつPCRチューブにピックアップした。チューブには、以下の組成のPCRミックスをそれぞれ10μlずつ分注した。
HRz2Eを基本骨格とし、C部位のアデノシンは、HRzCの標的認識領域に存在するウリジンと塩基対を形成することにより認識される。そこで、イノシンがシトシンと塩基対を形成することを利用し、C部位と塩基対合するウリジンをシトシンに置換することにより、C部位がA to I編集された時にのみ切断活性を示すリボザイムHRzC-inoを設計した。
HR-HTR2C-editの切断活性と編集特異性を評価するために、インビトロアッセイを、C部位を含む合成HTR2C RNAフラグメントを用いて行った。このアッセイでは、2形式の32P標識 HTR2C mRNAフラグメント(37個のヌクレオチド)、つまり、1つは、非編集基質(HTR2C-ade)としてのC部位に塩基Aを有するものと、編集基質としてのC部位に塩基Iを有するものとの2形式を使用した(図18A)。
保存A-to-I修飾部位(Q/R部位)を含むFLNA mRNAを、内在性FLNA mRNAが編集されて、細胞中でRNA 2上で作用するアデノシン・デアミナーゼ(ADAR2)を過剰発現する標的mRNAとして使用した
(Nishimoto, Y., et al. 2008. Neurosci Res 61(2): 201-206)。FLNA mRNA (HR-FLNA-edit) の編集特異的切断のためのリボザイムは、上述した方法を用いて設計し、HR-FLNA-editの切断活性ならびに編集特異性は、合成FLNA RNAフラグメントを使用したインビトロ切断アッセイにより分析した(図22)。編集FLNA RNAフラグメントに対するHR-FLNA-editの最終ポイントでの産物の切断率定数ならびに分画は、非編集FLNA RNAフラグメントのそれよりもずっと大きかった(図22、表3)。これらの結果は、HR-HTR2C-editに対して観察された結果と類似していた。
Claims (10)
- 一般式 [ I ] :
Cはシトシン(C)を意味し;
Gはグアニン(G)を意味し;
Uはウラシル(U)を意味し;
Dは、アデニン(A)、 シトシン(C)、グアニン(G) またはウラシル (U) を意味し;
Xは、標的RNAの修飾部位を認識する修飾認識塩基であって、アデニン(A)、 シトシン(C)、グアニン(G) またはウラシル (U) から選ばれるいずれかの塩基を意味し;
Nは、いずれも同一であってもまたは異なっていてもよく、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基を意味し;
N’は、同一であっても、または異なっていてもよく、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基であって、対応する塩基Nと塩基対を形成する塩基を意味し;
aは、1~10の整数、好ましくは2~6の整数、より好ましくは2~4の整数を意味し;
bは、1~6の整数、好ましくは2~4の整数を意味し;
cは、1~4の整数、好ましくは1~2の整数を意味し;
mは、2~50の整数、好ましくは5~30の整数、さらに好ましくは5~20の整数を意味し;および
nは、2~50の整数、好ましくは5~30の整数、さらに好ましくは5~20の整数を意味する)
で表されるハンマーヘッド型リボザイム。
- 請求項1に記載のハンマーヘッド型リボザイムであって、 一般式 [Ia] :
Hは、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基であって、上記HHR [I] において対応する塩基Dと塩基対を形成する塩基を意味し;
H’は、アデニン(A)、シトシン(C)、グアニン(G)またはウラシル(U)から選ばれるいずれかの塩基を意味し;
Eは、アデニン(A)、シトシン(C)、グアニン(G)、ウラシル(U)またはイノシン(I)から選ばれるいずれかの塩基であり、かつ、上記HHR [I] において対応する塩基Xと塩基対を形成する塩基であって、塩基Xと塩基対を形成したときに、5’側に隣接する塩基H’との結合を切断する塩基を意味し;
A、C、G、U、D、X、N、N’、a、b、c、mおよびnはいずれも前記と同じ意味を有する)
はいずれも前記と同じ意味を有する。)
で表されるハンマーヘッド型リボザイム-標的RNA分子構築物。
- 請求項1または2に記載のハンマーヘッド型リボザイムであって、一般式 [Ia] において、塩基Xがアデニン(A)またはシトシン(C)であるハンマーヘッド型リボザイム。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013516358A JP6145957B2 (ja) | 2011-05-20 | 2012-05-19 | Rna配列上の修飾を識別するリボザイムおよびそれを用いたrna開裂方法 |
US14/118,562 US9238814B2 (en) | 2011-05-20 | 2012-05-19 | Ribozyme for identifying modification on RNA sequence and RNA cleavage method using same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161488345P | 2011-05-20 | 2011-05-20 | |
US61/488345 | 2011-05-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012161144A1 true WO2012161144A1 (ja) | 2012-11-29 |
Family
ID=47217221
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2012/062878 WO2012161144A1 (ja) | 2011-05-20 | 2012-05-19 | Rna配列上の修飾を識別するリボザイムおよびそれを用いたrna開裂方法 |
Country Status (3)
Country | Link |
---|---|
US (1) | US9238814B2 (ja) |
JP (1) | JP6145957B2 (ja) |
WO (1) | WO2012161144A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2776450A4 (en) * | 2011-11-10 | 2015-07-22 | Shire Human Genetic Therapies | ANTISENSE OLIGONUCLEOTIDE MODULATORS OF SEROTONIN 2C RECEPTOR AND USES THEREOF |
WO2019111957A1 (ja) * | 2017-12-06 | 2019-06-13 | 学校法人福岡大学 | オリゴヌクレオチド、その製造方法及び標的rnaの部位特異的編集方法 |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2015367378A1 (en) | 2014-12-17 | 2017-06-15 | Proqr Therapeutics Ii B.V. | Targeted RNA editing |
WO2016140967A1 (en) * | 2015-03-02 | 2016-09-09 | The Regents Of The University Of California | Catalytic strands of minimal hammerhead ribozymes and methods of using the same |
KR102418185B1 (ko) | 2016-06-22 | 2022-07-06 | 프로큐알 테라퓨틱스 Ⅱ 비.브이. | 단일 가닥 rna-편집 올리고뉴클레오타이드 |
WO2018041973A1 (en) | 2016-09-01 | 2018-03-08 | Proqr Therapeutics Ii B.V. | Chemically modified single-stranded rna-editing oligonucleotides |
US11274300B2 (en) | 2017-01-19 | 2022-03-15 | Proqr Therapeutics Ii B.V. | Oligonucleotide complexes for use in RNA editing |
WO2021113270A1 (en) | 2019-12-02 | 2021-06-10 | Shape Therapeutics Inc. | Therapeutic editing |
CN112063694B (zh) * | 2020-05-09 | 2022-08-09 | 西安交通大学 | 一种rna a-i编辑的酶识别检测方法 |
-
2012
- 2012-05-19 JP JP2013516358A patent/JP6145957B2/ja not_active Expired - Fee Related
- 2012-05-19 WO PCT/JP2012/062878 patent/WO2012161144A1/ja active Application Filing
- 2012-05-19 US US14/118,562 patent/US9238814B2/en not_active Expired - Fee Related
Non-Patent Citations (13)
Title |
---|
ECKSTEIN, F. ET AL.: "In vitro selection of hammerhead ribozyme sequence variants", CHEMBIOCHEM, vol. 2, 2001, pages 629 - 635 * |
IWAMOTO, K. ET AL.: "RNA editing of serotonin 2C receptor and major mental disorders", YAKUGAKU ZASSHI, vol. 128, no. 4, 2008, pages 521 - 525 * |
MAAS, S. ET AL.: "A-to-I RNA editing and human disease", RNA BIOLOGY, vol. 3, no. 1, January 2006 (2006-01-01), pages 1 - 9 * |
MOSHIRI, H. ET AL.: "A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens", NUCLEIC ACIDS RESEARCH, vol. 38, no. 13, 2010, pages E138, 1 - 13 * |
NISHIMOTO, Y. ET AL.: "Determination of editors at the novel A-to-I editing positions", NEUROSCIENCE RESEARCH, vol. 61, 2008, pages 201 - 206 * |
PAN, W.-H. ET AL.: "Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner", MOLECULAR THERAPY, vol. 7, no. 1, January 2003 (2003-01-01), pages 129 - 139 * |
SCOTT, W. G. ET AL.: "The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage", CELL, vol. 81, June 1995 (1995-06-01), pages 991 - 1002 * |
SOWDEN, M. P. ET AL.: "Apolipoprotein B RNA sequence 3' of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing", NUCLEIC ACIDS RESEARCH, vol. 26, no. 7, 1998, pages 1644 - 1652 * |
TANG, J. ET AL.: "Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection", RNA, vol. 3, 1997, pages 914 - 925 * |
THOMSON, J. B. ET AL.: "In vitro selection of hammerhead ribozymes containing a bulged nucleotide in stem II", NUCLEIC ACIDS RESEARCH, vol. 24, no. 22, 1996, pages 4401 - 4406 * |
VAISH, N. K. ET AL.: "In vitro selection of a purine nucleotide-specific hammerhead-like ribozyme", PROC. NATL. ACAD. SCI. USA, vol. 95, March 1998 (1998-03-01), pages 2158 - 2162 * |
WERRY, T. D. ET AL.: "RNA editing of the serotonin 5HT2c receptor and its effects on cell signaling, pharmacology and brain function", PHARMACOLOGY & THERAPEUTICS, vol. 119, 2008, pages 7 - 23 * |
YAMANAKA, S. ET AL.: "RNA Editing Tenshago no Iden Joho Shushoku", PROTEIN, NUCLEIC ACID AND ENZYME, vol. 42, no. 6, 1997, pages 803 - 811 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2776450A4 (en) * | 2011-11-10 | 2015-07-22 | Shire Human Genetic Therapies | ANTISENSE OLIGONUCLEOTIDE MODULATORS OF SEROTONIN 2C RECEPTOR AND USES THEREOF |
WO2019111957A1 (ja) * | 2017-12-06 | 2019-06-13 | 学校法人福岡大学 | オリゴヌクレオチド、その製造方法及び標的rnaの部位特異的編集方法 |
Also Published As
Publication number | Publication date |
---|---|
JPWO2012161144A1 (ja) | 2014-07-31 |
US9238814B2 (en) | 2016-01-19 |
JP6145957B2 (ja) | 2017-06-14 |
US20140228556A1 (en) | 2014-08-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6145957B2 (ja) | Rna配列上の修飾を識別するリボザイムおよびそれを用いたrna開裂方法 | |
Gaudelli et al. | Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage | |
CN115651927B (zh) | 编辑rna的方法和组合物 | |
US20220073962A1 (en) | Methods for rna analysis | |
AU2018251187B2 (en) | Compositions and methods for transient gene therapy with enhanced stability | |
Chen et al. | RNA editing of apolipoprotein B mRNA. Sequence specificity determined by in vitro coupled transcription editing. | |
CN113939591A (zh) | 编辑rna的方法和组合物 | |
WO2017181107A2 (en) | Modified cpf1 mrna, modified guide rna, and uses thereof | |
JP7058839B2 (ja) | ローリングサークル増幅産物を使用した無細胞タンパク質発現 | |
EP3702360A1 (en) | Novel processes for the production of oligonucleotides | |
US20220307007A1 (en) | Modified bacterial retroelement with enhanced dna production | |
Pan et al. | Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries | |
JP2022527814A (ja) | Rna編集のための化学修飾したオリゴヌクレオチド | |
CN113994000A (zh) | 包括胞苷类似物的反义rna编辑寡核苷酸 | |
KR20180131577A (ko) | 신규의 최소 utr 서열 | |
Xu et al. | Targeted RNA editing: novel tools to study post-transcriptional regulation | |
Kawahara et al. | Extensive adenosine-to-inosine editing detected in Alu repeats of antisense RNAs reveals scarcity of sense–antisense duplex formation | |
Diaz Quiroz et al. | Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing | |
EP3673084B1 (en) | Method for introducing mutations | |
CN107873056A (zh) | 新型表达调节性rna分子及其用途 | |
Chavali et al. | Functional categories of RNA regulation | |
WO2024006978A2 (en) | Improved methods for in vitro transcription | |
Hipolito | Improving DNAzyme catalysis through synthetically modified DNAzymes and probing DNA polymerase function to improve selection methodology | |
WO2023183627A1 (en) | Production of reverse transcribed dna (rt-dna) using a retron reverse transcriptase from exogenous rna | |
CN117321202A (zh) | 具有宽松pam要求的双链dna的编辑 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12789353 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2013516358 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14118562 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 12789353 Country of ref document: EP Kind code of ref document: A1 |