KR20170020091A

KR20170020091A - Method to prepare shRNA expression cassette, shRNA expression cassette and library produced thereby

Info

Publication number: KR20170020091A
Application number: KR1020150114815A
Authority: KR
Inventors: 황병준; 김성훈; 박성균; 권남훈; 유해용; 기윤
Original assignee: 재단법인 의약바이오컨버젼스연구단
Priority date: 2015-08-13
Filing date: 2015-08-13
Publication date: 2017-02-22

Abstract

The present invention relates to a method for producing a shRNA expression cassette, a shRNA expression cassette produced through the method, and a library including the same. More specifically, an asymmetric hairpin structure is induced from oligonucleotide simultaneously including DNA base sequences for coding sense and antisense RNA having different lengths (the number of bases), and a complementary sequence strand is elongated with respect to an overhung sequence in the asymmetric hairpin structure, so the shRNA expression cassette is produced. According to the method of the present invention, a conventional problem in which accuracy in RNAi library production using a synthesis of 150-200 base oligonucleotide using an Agilent SurePrint technology is just approximately 50% is resolved, so high level RNAi library production accuracy can be ensured and time and costs consumed in producing an RNAi library are reduced. Thus, high industrial usability is ensured.

Description

Methods for producing shRNA expression cassettes, shRNA expression cassettes prepared therefrom, and libraries containing the shRNA expression cassettes,

The present invention relates to a method for producing an shRNA expression cassette, an shRNA expression cassette prepared by the method, and a library containing the same, and more particularly, to a method for producing a shRNA expression cassette comprising the steps of: i) A DNA sequence comprising a sequence; ii) a DNA sequence encoding an antisense RNA for a target gene consisting of 3 to 9 bases; iii) a DNA sequence encoding a hairpin loop consisting of 10 to 33 bases; iv) a DNA sequence consisting of 19 to 24 bases and encoding a sense RNA for the same target gene as in ii); And v) an oligonucleotide consisting of a DNA sequence comprising a 3 'primer binding site comprising a recognition sequence of a second restriction enzyme type II, a method for producing an shRNA expression cassette using the oligonucleotide, an shRNA Expression cassettes and libraries comprising same.

RNA interference (RNAi) is a biological phenomenon that refers to a phenomenon that inhibits translation by small RNAs with complementary sequences of specific mRNAs. In 1998, Andrew Fire and Craig C. Mello first discovered and reported this phenomenon in C. elegans , and in 2006 he was awarded the Nobel Prize in Physics.

Small RNAs of 21 to 24 nt (nucleotides) with a nucleotide sequence complementary to the mRNA produced through transcription of the gene are bound to block mRNA or prevent translation from proceeding, resulting in protein (Gene silencing, gene silencing induction). It is also known that RNAi functions to defend foreign RNAs or transposons introduced into cells by exogenous viruses.

RNAi can be used to investigate gene function and its relationship to disease by artificially lowering the level of gene expression desired by a researcher and is therefore a valuable tool in studying life phenomena. On the other hand, an RNAi library is used for genomes of various higher organisms including humans and mice, but it is not widely used in domestic because it requires a lot of cost for producing, maintaining and using the library, and understanding of the mechanism And research on the derivation of new drug targets have also been hampered. Therefore, in the technical field to which the present invention pertains, it is possible to provide an RNAi library for genes of various plants and animals, which are not well known to genomes of humans, mice, etc., as well as genes in genomes, There is a need for new technologies that can be saved.

Currently, oligonucleotides of about 150 ~ 200 base size used in the construction of RNAi library are mostly produced by microarray chip, which is represented by Agilent Sure Print technology. The synthesis of 150 ~ 200 base oligonucleotide using this microarray chip and the accuracy Is not more than 50%. Therefore, there is a need to develop a technology for producing a genomic RNAi library having low cost, simplicity, high accuracy and high efficiency.

Therefore, the inventors of the present invention have been studying to develop a technology for producing a genomic RNAi library having low cost, simplicity, high accuracy and high efficiency, and have found that a DNA sequence encoding a sense and antisense RNA of different length (base number) After producing hairpin structures from oligonucleotides, they use enzymes widely used in several molecular biology studies Through the elongation of complementary sequence strands, we have developed a technique for producing shRNA expression cassettes with high accuracy, Using the techniques of the present invention shRNA library It can be manufactured easily and at low cost, and thus the present invention has been completed.

It is therefore an object of the present invention

I) a DNA sequence comprising a 5 'primer sequence comprising the recognition sequence of a first restriction enzyme type II;

ii) a DNA sequence encoding an antisense RNA for a target gene consisting of 3 to 9 bases;

iii) a DNA sequence encoding a hairpin loop consisting of 10 to 33 bases;

iv) a DNA sequence consisting of 19 to 24 bases and encoding a sense RNA for the same target gene as in ii); And

v) an oligonucleotide consisting of a DNA sequence comprising a 3 'primer binding site comprising a recognition sequence of a second 3'-restriction enzyme type II.

Another object of the present invention is

(a) treating a first double-stranded DNA chain consisting of the oligonucleotide and its complementary sequence with a first 5'-restriction enzyme type II and producing a single-stranded DNA chain And obtaining;

(b) inducing self-hybridization between a DNA sequence encoding a sense RNA contained in the single-stranded DNA chain obtained in the step (a) and a DNA sequence encoding an antisense RNA to form a stem- Obtaining a short asymmetrical hairpin structured single stranded DNA chain with one arm forming the stem;

(c) terminating the short arm of the stem in the asymmetric hairpin structure single stranded DNA chain obtained in the step (b) Elongating complementarily to a portion of the DNA sequence that is not hybridized and overhanging to obtain a symmetrical hairpin structured single stranded DNA chain;

(d) treating the second restriction enzyme type II on the symmetrical hairpin structured single stranded DNA strand obtained in step (c); And

(e) attaching first and second adapters to both ends of the DNA chain obtained in step (d), and amplifying by PCR;

And a method for producing a small hairpin RNA expression cassette comprising the same.

It is another object of the present invention to provide an shRNA expression cassette produced by the above method.

It is yet another object of the present invention to provide a recombinant expression vector comprising said shRNA expression cassette.

It is another object of the present invention to provide a shRNA encoding library consisting of a plurality of vectors comprising said shRNA expression cassette.

Another object of the present invention is to provide

Preparing at least one shRNA expression cassette into which a DNA sequence encoding a different sense RNA is inserted;

Cloning the prepared shRNA expression cassettes into an expression vector (RNA vector system); And

And pooling the cloned vector. The present invention also provides a method for producing an shRNA encoding library.

It is another object of the present invention to provide a pseudoviral particle in which the shRNA encoding library is packaged.

In order to achieve the above object,

iii) a DNA sequence encoding a hairpin loop consisting of 10 to 33 bases;

v) an oligonucleotide consisting of a DNA sequence comprising a 3 'primer binding site comprising a recognition sequence of a second restriction enzyme type II.

In order to achieve another object of the present invention,

(a) treating a first double-stranded DNA chain consisting of the oligonucleotide and its complementary sequence with a first restriction enzyme type II to produce and obtain a single-stranded DNA chain step;

(c) terminating the short arm of the stem in the asymmetric hairpin structure single stranded DNA chain obtained in the step (b) Without hybridization Elongating complementarily to a portion of the DNA sequence that is overhanged to obtain a symmetrical hairpin structured single stranded DNA chain;

A method for producing a small hairpin RNA expression cassette.

In order to achieve another object of the present invention, the present invention provides an shRNA expression cassette produced by the above method.

In order to accomplish still another object of the present invention, the present invention provides a recombinant expression vector comprising the shRNA expression cassette.

In order to accomplish still another object of the present invention, the present invention provides an shRNA encoding library composed of a plurality of vectors including the shRNA expression cassette.

In order to achieve still another object of the present invention,

And pooling the cloned vector. &Lt; Desc / Clms Page number 3 >

In order to achieve still another object of the present invention, the present invention provides a pseudoviral particle in which the shRNA encoding library is packaged.

Hereinafter, the present invention will be described in detail.

Unless otherwise stated in the present invention, the nucleotide sequence is described in the 5 'to 3' direction.

In the present invention, the term 'strand' is used interchangeably with 'strand', 'strand' or 'strand' and means a plurality of nucleotide polymers (ie, polynucleotides).

As used herein, the term 'sense strand' refers to a polynucleotide having the same nucleic acid sequence as a target nucleic acid, including mRNA (messenger RNA), RNA sequence (eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA) Or a polynucleotide which is identical in whole or in part to a noncoding DNA sequence.

The term "antisense strand" as used herein refers to polynucleotides that are substantially or 100% complementary to the target nucleic acid of interest, such as mRNA (messenger RNA), RNA sequences that are not mRNA (eg, , microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a polynucleotide complementary as a whole or in part to a coding or noncoding DNA sequence.

In the present invention, the term 'primer' refers to a short nucleic acid sequence capable of forming a base pair with a complementary template strand and serving as a starting point for template strand copying .

The term " 5 " primer in the present invention is used interchangeably with the term " forward primer ", and is a short sense strand for a target nucleic acid sequence to be amplified. . The 5 'primer complementarily binds to the 3'-terminal of the complementary strand (antisense strand) to the target nucleic acid sequence, and the binding site is referred to as a' 5 'primer binding site Quot;

The term " 3 " primer in the present invention is used interchangeably with the term " reverse primer ", and is a short antisense strand to a target nucleic acid sequence to be amplified, . The 3 'primer is complementary to the 3'-terminal of the target nucleic acid sequence, and the binding site is referred to as a' 3 'primer binding site.

The present invention

I) a 5 ' -phage comprising the recognition sequence of the first restriction enzyme type II, primer Containing a sequence DNA order;

ii ) For a target gene consisting of 3 to 9 bases Antisense RNA Coding DNA order;

iii ) Coding for a hairpin loop consisting of 10 to 33 bases DNA order;

iv ) 19 to 24 bases, ii ) From Sense for the same target gene RNA Coding DNA order; And

v) 3 ' including the recognition sequence of the second restriction enzyme type II primer Comprising a binding site DNA Lt; / RTI > oligonucleotides.

The sequence of the 5 'primer sequence of i) and the 3' primer binding site of v) of the present invention can be easily determined by those skilled in the art depending on the primer sequence to be used, and the number and combination of bases are not particularly limited. Preferably, the sequence of the 5 'primer sequence of i) and the 3' primer binding site of v) of the present invention may be 10 to 30 nucleotides in sequence for the accuracy of synthesis, and most preferably 15 to 25 nucleotides .

In addition, the sequence of the 5 'primer sequence of i) and the 3' primer binding site of v) of the present invention includes a restriction enzyme recognition site (sequence).

The term "restriction endonuclease" of the present invention refers to an enzyme that selectively cleaves a phosphodiester bond between double-stranded DNAs to form a fragment. All restriction enzymes recognize a specific nucleotide sequence of DNA, and this nucleotide sequence represents the selective site of action of the restriction enzyme. As cleavage of double-stranded DNA, some restriction enzymes generate a specific so-called 'sticky end'. This end can be ligation (recombination) with the corresponding complementary end of the DNA fragment obtained by itself or in another process under specific restoration conditions. When cleaved with other restriction enzymes, a double-stranded DNA having a blunt end is generated. Double-stranded DNA with a blunt end can be recombined with double-stranded DNA with another blunt end.

The restriction enzyme recognizes a specific nucleotide sequence at a recognition site and cleaves the nucleic acid at a restriction site (cleavage site). Therefore, the term 'restriction enzyme recognition site' of the present invention may include a site (restriction site) directly cleaved by restriction enzyme treatment, or may exist separately from a cleavage site (restriction site). That is, in the latter, the term 'restriction enzyme recognition site' means an arrangement of bases capable of cleaving a site in which a restriction enzyme cleaves a specific base or more from the site (sequence) through recognition of the recognition site.

The restriction enzyme of the present invention is not limited in its kind depending on the efficiency and convenience of the person skilled in the art, but may include all restriction enzymes of restriction enzyme types I, II, III and IV, for example.

Preferably, the restriction enzyme in i) and v) may be a type II restriction enzyme. The type II restriction endonuclease, known as a homodimer or tetramer, cleaves DNA in or near a specific cognitive site. Most of the type II restriction enzymes have the same restriction site as the restriction enzyme recognition site. The catalyst requires a divalent ion such as Mg ² ⁺ . The types of the type II restriction enzymes are well known in the art and include, for example, orthodox II types such as EcoRI, BamHI, HindIII, KpnI, NotI, PstI, SmaI, XhoI, IIS types such as Fok I, Alw26 I, Bbv I, Bsr I, Ear I, Hph I, Mbo I, SfaN I, and Tth 11 I I; IIF type such as NgoM IV; ⅡT type; IIG type such as Eco57I; Type IIM such as Bcg I, Bp I, Bsp 24 I, Bae I, and Cje I, and the like.

The restriction enzyme of the present invention may be preferably a type IIS restriction enzyme and may include, for example, Fok I, Alw26I, BbvI, BsrI, EarI, HphI, MboI, SfaNI, Tth111I, BsrDI, Most preferably BsrDI or EarI.

In the present invention, the terms 'first restriction enzyme' and 'second restriction enzyme' refer to the order of restriction enzyme treatment, respectively, which means that the first and second treatments are respectively treated first and second. The 'first restriction enzyme' and the 'second restriction enzyme', which are treated first and second in the present invention, are preferably different kinds of restriction enzymes.

The term " first restriction enzyme (type II) recognition sequence " in the present invention means a recognition site of a restriction enzyme (type II) to be firstly treated in the shRNA expression cassette production method described later, Enzyme (type II) recognition sequence " means the recognition site of the second restriction enzyme (type II).

In the present invention, the 3 'primer binding to the 5' primer of i) and the 3 'primer binding site of v) may be provided as one set of primers.

As a preferred example, the DNA sequence of i) above and the DNA sequence of v) of the present invention preferably comprises the DNA sequence of SEQ ID NO: 1, wherein the DNA sequence of i) is a 5 'primer sequence comprising a BsrDI recognition site , And the DNA sequence of v) may consist of the DNA sequence of SEQ ID NO: 3, which is a 3 'primer binding site sequence containing an EarI recognition site. In this case, the 3 'primer attached to the DNA sequence of v) may be a DNA sequence of SEQ ID NO: 6.

In the present invention, the term 'target gene' refers to a target gene whose expression or function is to be inhibited. In the present invention, shRNA specific to a target gene is expressed, and siRNA generated from the gene selectively suppresses or inactivates . Such inactivation is achieved by the siRNA binding to and ultimately removing the mRNA of the target gene. In the present invention, the target gene includes all genes capable of inhibiting its function through shRNA.

In the present invention, the term 'sense RNA' refers to a sense strand for mRNA transcribed from a target gene.

Among the oligonucleotides of the present invention, the base number and the combination of the DNA sequence encoding the sense RNA in the above iv) can be appropriately set by a person skilled in the art according to the kind of the target gene desired. Preferably, in the iv) of the present invention, the base sequence of the DNA sequence encoding the sense RNA may be 19 to 24, and most preferably 19 to 22.

The term " anti-sense RNA " in the present invention means an antisense strand for an mRNA transcribed from a target gene.

In the oligonucleotides of the present invention, the number of bases of the DNA sequence encoding the antisense RNA in ii) above is smaller than that of the DNA encoding the sense RNA of iv), so that the above i) to v) Hybridization of a DNA sequence encoding an antisense RNA and a DNA sequence encoding a sense RNA in a single-stranded oligonucleotide (and a complementary sequence thereof) consisting of an asymmetric hairpin structure.

The base number of the DNA sequence encoding the antisense RNA and the combination thereof can be appropriately set by those skilled in the art according to the DNA sequence encoding the sense RNA of iv), depending on the kind of the target gene desired. Preferably, the base number of the DNA sequence encoding antisense RNA in the above ii) of the present invention may be preferably 3 to 9, and most preferably 5 to 7.

In the present invention, the DNA sequence encoding the ii) antisense RNA complementarily binds to the DNA sequence encoding the iv) sense RNA, so that the DNA of ii) and the DNA of iv) contained on the single strand of oligonucleotide Are arranged in reverse complementary order. That is, the oligonucleotide of the present invention is a single-stranded structure including a sense strand and a self-complementary antisense strand. The term " self-complementary antisense strand "Quot; refers to an antisense strand of a reverse complementary sequence.

Therefore, the base sequence (combination) of the DNA sequence encoding the ii) antisense RNA is suitably set by a person skilled in the art according to the DNA sequence encoding the iv) sense RNA, preferably ii) DNA encoding the antisense RNA The sequence consists of a base complementary to the bases sequentially connected in the 5 'direction from the base located at the 3' end of the DNA sequence encoding the iv) sense RNA.

As used herein, the term "complementary" when used in connection with a nucleic acid means that adenine (A) binds to thymine (T) or uracil (U) and guanine (G) binds to a cytosine Quot; For example, a nucleic acid having a sequence GCAU in the 5 'to 3' direction is complementary to a nucleic acid having a sequence CGTA in the 3 'to 5' direction. As used herein, the term complementary is used to encompass both the meaning of a substantially complementary nucleic acid and a 100% complementary nucleic acid (i. E., Does not allow mismatch), and most preferably a relationship of 100% complementary nucleic acid .

The term " hairpin " of the present invention means a self-hybridized structure that takes a stem-loop structure when two complementary regions in a single chain form a double structure while being separated by a non-complementary region do. When the degree of complementarity and the orientation of the complementary site are sufficient, a sufficient base pair is formed to form a double structure called a stem, and the non-complementary region forms a loop structure And connects the two complementary regions. The loop site is made by the absence of a base pair between the nucleic acid (or nucleic acid analog) at the loop site and the loop site of the hairpin structure can be referred to as a " single strand intervening " as a single strand site between the sense and antisense strands .

In the present specification, the two complementary regions are referred to as two arms, and thus, in this specification, the stem is defined as consisting of two arms (3'arm and 5'arm). Thus, the hairpin structure is composed of a 3 'arm (3' arm, i.e., 3 'end of a single chain) and a 5' arm (5 ' Quot; means that the sequence forms a double structure separated by the non-complementary loop coding sequence.

In the iii) of the present invention The DNA sequence encoding the hairpin loop is well known in the art and can be, for example, a sequence derived from a known mir-30 (in particular a sequence derived from the loop region). The number of bases (or sequence length) of the DNA sequence of iii) can be arbitrarily set according to the working efficiency and synthesis accuracy desired by those skilled in the art, and can be, for example, 10 to 33 bases. Iii) above The DNA sequence encoding the hairpin loop may preferably consist of the DNA sequence shown in SEQ ID NO: 2.

The term " hybridization " in the present invention means a complementary (double strand) formation reaction (i.e., pairing pairing) having a complementary base sequence structure.

The term " self-hybridization " in the present invention refers to intramolecular hybridization in which complementary regions (bases) contained in a single strand nucleic acid molecule form a complementary strand Reaction.

Oligonucleotides consisting of the DNA sequences of i) to v) of the present invention are characterized in that a sense strand having an asymmetric length on both sides of a DNA sequence coding for a hairpin loop and a DNA sequence encoding an antisense RNA sequence complementary thereto are located at positions The shRNA expression cassette produced through a series of processes using oligonucleotides consisting of the DNA sequences of i) to v) of the present invention has a very high degree of nucleotide sequence synthesis accuracy. In addition, even when the GC palindrome sequence is 40% or less, the synthesis is performed well without bias.

Oligonucleotides comprising the DNA sequences of i) to v) of the present invention can be combined with various forms by those skilled in the art in view of the description of the DNA sequences of i) to v) described above. For example, several nucleotide sequences may be added between the components to properly link the components described in i) to v) without interfering with the overall features and structure of the oligonucleotides according to the present invention . For example, the oligonucleotide consisting of the DNA sequence of i) to v) may be the nucleotide sequence of SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 4 is an 88mer, and a schematic concept of the structure thereof is shown in FIG. The nucleotide sequence of SEQ ID NO: 4 is the nucleotide sequence of SEQ ID NO: A DNA sequence coding for a 6-mer antisense RNA to a target gene is positioned (indicated by 6 N) at the 5 'end centered on a DNA sequence coding for a hairpin loop, and a 20-mer It is characterized by the presence of a DNA sequence coding for sense RNA (denoted by 20 N) (see Figure 1). The 5 'end is composed of the DNA sequence shown in SEQ ID NO: 1, and the 3' end is composed of the DNA sequence shown in SEQ ID NO: 3.

The present invention

(a) an oligonucleotide comprising the oligonucleotide and a sequence complementary thereto Double strand DNA chain( double strand DNA chain ) With the first restriction enzyme type II Single strand DNA chain( single - strand DNA chain;

(b) In the step (a) The obtained Single strand DNA The sense contained in the chain RNA Coding DNA Sequence and Antisense RNA Coding DNA Sequence Self-hybridization. Induced, Stem - Loop structure Stem One arm ( arm ) This short asymmetrical hairpin structure Single strand DNA Obtaining a chain;

(c) In the step (b) The obtained Asymmetrical hairpin structure Single strand DNA In the chain Stem Short arm ( arm Lt; RTI ID = 0.0 > Not hybridized Overhang ( overhang ) Being DNA The sequence is complementary to the kidney ( elongation ) And a symmetrical hairpin structure Single strand DNA Obtaining a chain;

(d) the symmetrical hairpin structured type obtained in the step (c) Single strand DNA Treating the chain with a second restriction enzyme type II; And

(e) In the step (d) The obtained DNA And the first and second adapters ( adapter ), And amplifying by PCR;

Containing shRNA Expression cassette ( small hairpin RNA expression cassette ). &Lt; / RTI >

The term 'shRNA (small-hairpin RNA)' of the present invention refers to a small interfering RNA (siRNA) having a stem-loop structure (stem-loop structure) including first and second regions complementary to each other, Quot; The term " stem-loop structure " herein can be used interchangeably with the term " hairpin structure ", and descriptions of these structures are as described above.

The term 'expression cassette' in the present invention refers to a nucleic acid construct which is used interchangeably with an 'expression construct' and is capable of expressing a desired nucleotide fragment (oligonucleotide consisting of i) to v) in the present invention, Depending on the host cell, a suitable promoter and operably linked nucleotide fragments. The expression cassette may comprise one or more nucleotide fragments, and may further include transcriptional regulatory elements (e.g., enhancers, etc.). In addition, the expression cassette may comprise a vector, preferably a replication origin, a selection marker and an expression regulatory element such as an operator, an initiation codon, a termination codon, a polyadenylation signal and an enhancer, to form a recombinant expression vector. The term " shRNA expression cassette " in the present invention includes at least a sense strand and an anti-sense strand of a target gene in a leader sequence of a promoter and a microRNA, , Which means a unit cassette capable of expressing shRNA.

In step (a), the first restriction enzyme type II is treated with a double strand DNA chain consisting of an oligonucleotide consisting of the DNA sequence of i) to v) and a sequence complementary thereto, and a single strand DNA chain (single-stranded DNA chain).

First, the oligonucleotide consisting of DNA sequences of i) to v) of the present invention and the double-stranded DNA chain comprising a complementary sequence thereof can be produced through a synthesis process commonly known in the art, The method is not particularly limited, but may be generated by a PCR amplification process using an oligonucleotide consisting of DNA sequences of, for example, i) to v) as a template. Specifically, in the case of providing the 88mer oligonucleotide shown in SEQ ID NO: 4 as exemplified above, a primer set of SEQ ID NO: 1 (5 'primer) and SEQ ID NO: 6 (3' primer) By PCR amplification, a double-stranded DNA chain consisting of an oligonucleotide (for example, SEQ ID NO: 4) consisting of the DNA sequence of i) to v) and a sequence complementary thereto (for example, SEQ ID NO: 5) can do.

Also, in step (a), among the oligonucleotides comprising the DNA sequences of i) to v) of the present invention, i) a restriction enzyme recognizing the recognition sequence of the first restriction enzyme type II is treated. The above-mentioned restriction enzymes are as described above.

The 'single-stranded DNA chain' obtained in the step (a) may be derived from an oligonucleotide template strand consisting of the DNA sequence of i) to v) or may be derived from a sequence complementary to the oligonucleotide. Preferably, the 'single-stranded DNA chain' obtained in the step (a) may be derived from a sequence complementary to an oligonucleotide consisting of the DNA sequence of i) to v).

Methods for producing a double stranded DNA chain (dsDNA) into a single stranded DNA chain (ssDNA) in order to obtain a single stranded DNA chain in the step (a) are known in the art . For example, a method by heating, a method by an exonuclease treatment, and the like, but are not limited thereto. The method of producing the 'double strand DNA chain (dsDNA) of the present invention with a single strand DNA chain (ssDNA)' may be preferably a method by exonuclease treatment.

The term "exonuclease" in the present invention means an enzyme which sequentially decomposes phosphodiester bonds from the ends of a nucleic acid (polynucleotide) to generate a mononucleotide. The exonuclease is 5 '→ 3 'direction, or in the 3' to 5 'direction. The exonuclease is the case where it is determined that the exonuclease is known in the art that the type is not limited but, for example, exonuclease from exonuclease I, E. coli from the E. coli of claim III, T. RecJ from thermophilus and bacteriophage lambda exonuclease.

The exonuclease of the present invention is not particularly limited as long as it is an exonuclease that acts on the double-stranded nucleic acid and produces a single-stranded nucleic acid by decomposing the nucleic acid in the 5 '→ 3' direction. For example, T7 Exonuclease (T7 Exonuclease), lambda exonuclease, and the like.

Most preferably, lambda exonuclease can be used. The enzyme recognizes a double-stranded DNA having a phosphate group at the 5'-end and decomposes only the strand to finally produce a single-stranded DNA.

The end of the nucleic acid to which the exonuclease binds is typically determined by the choice of the enzyme used and / or by methods known in the art. Typically, a chemical label such as a hydroxyl group, a cap structure, or a protein label may be used at one end of the nucleic acid sequence to prevent or facilitate binding of the exonuclease to a specific end of the nucleic acid sequence.

Specifically, in the step (a), in order to obtain a single-stranded DNA chain derived from a sequence complementary to an oligonucleotide consisting of the DNA sequence of i) to v), a series of steps as described below may be performed;

(a1) amplifying the oligonucleotide of claim 1 with a 5 'primer and a 3' primer labeled with a 5'-end to obtain a double-stranded DNA chain; And

(a2) treating the double-stranded DNA strand obtained in the step (a1) with a first restriction enzyme type II and an exonuclease to selectively obtain only a single-stranded DNA chain having a 5'- .

The 'amplification' may be performed by a known nucleic acid amplification reaction, but not limited thereto, for example, a PCR (polymerase chain reaction) reaction. The PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such methods are well known in the art and commercially available kits can be used.

The 3 'primer is provided at the 5'-terminal in a labeled state, thereby preventing the newly stretched complementary sequence from being degraded by the exonuclease later, and obtaining the desired target sequence. The labeling means may be easily labeled by a person skilled in the art depending on the kind of the exonuclease used. Preferably, the labeling may be a label of a non-phosphated compound. The markers include biotin, biotin-TEG, desthiobiotin-TEG, dual biotin, digoxigenin NHS Ester, AMCA, 6-FAM, Int 6-FAM dT, TET, HEX, TAMRA NHS Ester, ROX NHS Ester, JOE NHS Ester , Cy3, Cy5, Texas Red, Alexa 488, and preferably biotin-TEG.

The restriction enzymes and exonuclease in the above (a1) and (a2) are as described above.

In step (b), self-hybridization is induced between a DNA sequence encoding a sense RNA contained in the single-stranded DNA chain obtained in the step (a) and a DNA sequence encoding an antisense RNA, One arm forming the stem in the loop structure yields a short asymmetrical hairpin structural single stranded DNA chain.

The single-stranded DNA chain obtained in the step (a) is characterized in that the DNA sequence encoding the sense RNA contained in the chain and the DNA sequence encoding the antisense RNA are not identical in length and either one is remarkably short. The difference in the number of bases between both sequences may range from 10 to 30. Accordingly, the single-stranded DNA chain obtained in the step (a) of the present invention can be obtained by self-hybridizing a DNA sequence encoding the antisense RNA contained in a single strand and a DNA sequence encoding a sense RNA (See Fig. 6).

The term 'asymmetric' means that in the stem-loop structure of a hairpin, the lengths of both arms (or strands) of the stem structure due to the binding between complementary sequences in a single chain are different. That is, one of the two arms constituting the stem has a shorter sequence length than the other. Therefore, when the length of the stem is longer than one of the stem structures, some of the bases are exposed as overhang. Specifically, when the single-stranded DNA obtained in step (a) is derived from the oligonucleotide template strand, the short arm constituting the stem is a DNA sequence encoding antisense RNA having a relatively small number of bases And some of the bases of the DNA encoding the sense RNA are exposed in an overhang state. Also, when the single-stranded DNA obtained in step (a) is derived from the complementary strand of the oligonucleotide, the short arm forming the stem is a DNA sequence encoding a sense RNA having a relatively small number of bases , A complementary sequence to the antisense RNA coding DNA of the oligonucleotide template strand), and some of the bases of the DNA encoding the antisense RNA are exposed in an overhanged state.

The term 'overhang' in the present invention means nucleotides existing in the free end state without forming a base pair at the 5 'end (5' arm) or the 3 'end (3' arm). In one embodiment of the invention, the overhang is a 3 ' terminal or 5 ' terminal overhang on the DNA strand that encodes the antisense RNA or the DNA strand that encodes the sense RNA.

Specifically, when the single-stranded DNA obtained in the step (a) is derived from the oligonucleotide template strand, the overhang may be a 3'-terminal overhang on the DNA strand encoding the sense RNA, Where the single stranded DNA is derived from a complementary strand of the oligonucleotide, the overhang may be a 5 ' terminal overhang on the DNA strand encoding the antisense RNA.

The self-hybridization of step (b) is carried out on the basis of the hybridization technique of nucleic acid sequences known in the art, and hybridization of the nucleic acid sequence can be carried out under suitable hybridization conditions generally determined by an optimization procedure. The hybridization conditions may be controlled or adjusted according to various factors such as the sequence composition of the nucleic acid, the GC content and length, the hybridization temperature, the composition of the hybridization reagent or solution (ionic strength of the solution), the washing temperature and the desired degree of hybridization specificity It can be deformed. In the step (b) of the present invention, various degrees of hybridization stringency (e.g., high, medium and low) may be applied. The more stringent the conditions (eg, the higher the hybridization run temperature), the greater complementarity is required to form double strands. Preferably, the hybridization of step (b) of the present invention is carried out under suitable high stringency conditions by techniques known in the art, whereby the complementary hybridization in the absence of mismatch 100% hybridization may occur between sequences. The term " moderate to high stringency " hybridization conditions refers to conditions capable of achieving hybridization specificity equal to or substantially the same as the conditions used in the examples of the present invention.

The hybridization stringency is for specific hybridization between complementary regions present on a single stranded DNA chain, and the term " specific hybridization " refers to the use of other nucleic acids having similar sequences Refers to the ability to hybridize only to the desired nucleic acid sequence without hybridization.

In step (c), the terminal of the short arm forming the stem of the asymmetric hairpin structured single strand DNA obtained in step (b) is complementarily extended to the overhanging DNA sequence part without hybridization and elongated to obtain symmetrical hairpin structured single stranded DNA chains.

The term " elongation " in the present invention means that oligodeoxynucleotides or nucleic acids complementary to the template nucleic acid are apposited, and the above-mentioned conjugation means that the polymerase polymerizes the nucleotides to form complementary Thereby forming a nucleic acid molecule.

The 'elongation' process of step (c) may be performed by a known molecular biologic DNA synthesis method, for example, using DNA polymerase and dNTP (deoxynucleotide triphosphate) And then polymerizing a complementary sequence to the template nucleic acid sequence. A variety of DNA polymerases known in the art can be used in the present invention and include, but are not limited to, for example, a Klenow fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, and a bacteriophage T7 DNA Polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Pyrococcus furiosus Thermus spp. 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermus spp. 17, Thermus spp. 17, Thermus spp. 17, Thermus spp. 17, Thermus calbiophilus, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Includes DNA polymerase from Thermosipho africanus.

When carrying out the elongation reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. An excess of the required components means an amount such that the elongation reaction is not substantially restricted to the concentration of the component. A joinder such as Mg ² ⁺ , dATP, dCTP, dGTP and dTTP can be provided to the reaction mixture to such an extent that the intended elongation efficiency can be achieved. The elongation reaction of the DNA base sequence may be performed at a temperature suitable for the elongation reaction of the polymerase.

In the step (c), the distal end of the short arm forming the stem is complementarily stretched to the overhanging arm of the opposite arm (the long arm forming the stem) (See Fig. 7).

Through the enzymatic sequence synthesis method of this step, the synthesis accuracy of the shRNA expression cassette and the shRNA library using the shRNA expression cassette becomes very high.

Compared with the conventional synthesis of about 200-base oligonucleotides using Agilent Sure Print technology and the accuracy of producing RNAi library using the oligonucleotides of only about 200 bases, the present invention is very remarkable due to the method including the step (c) The accuracy of the RNAi library was improved.

In step (d), the second restriction enzyme type II is treated on the symmetrical hairpin structural single strand DNA chain obtained in step (c).

The restriction enzyme is as described above, and the restriction enzyme is treated to obtain a fragment of the desired nucleic acid (see FIG. 8).

In order to obtain the nucleic acid fragment with high efficiency, an optional purification process may be further performed. For example, an electrophoresis method using polyacrylamide gel may be used, but the present invention is not limited thereto.

In step (e), first and second adapters are attached to both ends of the DNA chain obtained in step (d), and amplified by PCR.

The term " adapter " as used herein means any oligonucleotide linking molecule linking a nucleic acid and a nucleic acid. In the present invention, manipulation such as insertion into the vector of the final shRNA expression cassette construct is facilitated through connection with the adapter molecule.

In the present invention, an adapter can use a synthetic oligonucleotide adapter according to a conventional method, and a method for synthesizing the adapter is well known in the art. Adapters of the present invention may have a variety of combinations of sequence configurations as desired by those skilled in the art, and may include, for example, a sequence complementary to a primer used in an amplification procedure and / or a sequence identical to a primer, Sequence. Preferably, the adapters of the present invention are provided as a pair (first and second adapters), wherein the first adapter (in other cases, the second adapter) has the same sequence and restriction enzyme as the 5 ' And the second adapter (in other cases, the first adapter) may comprise a sequence complementary to the 3 'primer used in the amplification procedure and a restriction enzyme recognition sequence.

The first and second adapters of step (e) preferably include recognition sequences for different restriction enzymes. The restriction enzyme is as described above, and the type of the restriction enzyme in the restriction enzyme recognition sequence included in the first and second adapters of step (e) is not particularly limited, but is preferably AcuI or EcoR I May be included.

For example, the first adapter of step (e) may comprise the recognition sequence of the restriction enzyme AcuI and the DNA sequence of SEQ ID NO: 7, the second adapter may contain the recognition sequence of the restriction enzyme EcoR I, And the DNA sequence represented by SEQ ID NO: 8.

Methods for linking nucleic acid sequences are well known in the art and may be, for example, by enzymatic linkage through ligase treatment, but are not limited thereto. The ligase is known in the art and includes, for example, E. coli ligase, T4 DNA ligase, mammalian ligases (DNA ligase I, DNA ligase III, DNA ligase IV) and thermostable ligase T4 DNA ligase can be used.

PCR amplification is as described above. For example, PCR amplification of the single strand generated by attaching the first adapter shown in SEQ ID NO: 7 and the second adapter shown in SEQ ID NO: (Sense strand fragment of SEQ ID NO: 7) and SEQ ID NO: 10 (complementary strand fragment of SEQ ID NO: 8).

The present invention relates to a process for the preparation of shRNA Expression cassettes are provided.

The shRNA expression cassette comprises all or part of the following DNA sequence as described above in the method for producing an shRNA expression cassette comprising the steps (a) to (e);

A first adapter DNA sequence;

A DNA sequence encoding a sense RNA;

A DNA sequence encoding a hairpin loop;

A DNA sequence encoding an antisense RNA that binds complementarily to the sense RNA; And a second adapter DNA sequence;

Lt; RTI ID = 0.0 > expression cassette. &Lt; / RTI >

The DNA sequence encoding the sense RNA contained in the shRNA expression cassette and the DNA sequence encoding the antisense RNA complementarily binding to the sense RNA are characterized in that the number of bases, that is, the length of the sequence is the same.

The present invention relates to shRNA RTI ID = 0.0 > expression cassette. &Lt; / RTI >

As used herein, the term " recombinant expression vector " refers to a vector capable of expressing a target RNA in a host cell, including a necessary regulatory element operatively linked to the expression of the gene insert.

The term 'operably linked' in the present invention refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired RNA to perform a general function. For example, the promoter and the nucleic acid sequence encoding the RNA may be operatively linked to affect the expression of the coding nucleic acid sequence. Operational linkages with recombinant vectors can be made using recombinant DNA techniques well known in the art, and site-specific DNA cleavage and linkage can be achieved using enzymes generally known in the art, such as restriction An enzyme or a ligase is used.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Preferably a plasmid vector or a viral vector. The viral vector may be by a viral vector known in the art, for example, but not limited to, an adenovirus vector, an adeno-associated viral vector, a lentivirus vector, a retrovirus vector, and a herpes virus vector.

Suitable expression vectors include signal sequence or leader sequences for membrane targeting or secretion, as well as expression control sequences such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer (promoter gene) . The promoter of the vector may be constitutive or inducible. The expression vector also includes a selection marker for selecting a host cell containing the vector and, if the expression vector is a replicable vector, a replication origin.

The expression vector of the present invention may add any sequence motif for efficient processing of the shRNA into the siRNA for efficient (intracellular) expression of the shRNA expression cassette contained in the vector. The motif sequence is involved in the processing of shRNA into siRNA, and the type thereof is not limited as long as it is known in the art. For example, a CNNC motif (C means a cytosine residue and N means any nucleotide , C, G, T, and A).

The recombinant vector of the present invention can be introduced into a host cell using a method known in the art. Methods for introduction into host cells are preferably selected from the group consisting of calcium chloride method, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome mediated method liposome-mediated method) can be used.

Examples of host cells include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. But are not limited to, host cells, fungi (e.g., Aspergillus), yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Cells derived from higher eukaryotes, including, but not limited to, eukaryotic cells such as Schizosaccharomyces, Neurospora crassa and the like, insect cells, plant cells, mammals and the like can be used as host cells, May be a human cell.

The present invention relates to shRNA Consisting of a plurality of vectors comprising an expression cassette shRNA Coding library ( shRNA encoding library ).

The plurality of vectors constituting the shRNA encoding library include an shRNA expression cassette into which a DNA sequence encoding a different sense RNA is inserted. Since each vector contains a DNA sequence encoding a different sense RNA, it is obvious to those skilled in the art that each of the vectors also includes a DNA sequence encoding an antisense RNA that is different from (and is complementary to) the sense RNA I understand.

The present invention

Different senses RNA Coding DNA The sequence inserted shRNA Producing at least one expression cassette;

The fabricated shRNA Expression cassettes were used as expression vectors ( RNA vector system ) &Lt; / RTI > cloning step;

remind Cloned Vector Pooling Step shRNA Coding library ( shRNA encoding library ). &Lt; / RTI >

Meanwhile, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, NY (1984), 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, ; and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

The present invention relates to shRNA Coding library ( shRNA encoding library )end Packaged Viral particle( pseudoviral particle ).

The shRNA coding library of the present invention may be provided in the form packaged in pseudoviral particles. Methods for producing the above viral particles and the types of viruses that are based on the production are known in the art.

The foreign gene of the shRNA coding library of the present invention can be effectively transferred into eukaryotic cells through the above viral particles.

The shRNA expression cassette of the present invention solves the problem that the accuracy of the synthesis of 150-200 base oligonucleotide using Agilent Sure Print technology and the production of RNAi library using it is only about 50% As well as reducing the time and cost of constructing an RNAi library, which is highly industrially applicable.

Figure 1 shows the 88mer oligonucleotide designed in Example 1 (88mer oligo DNA).
Fig. 2 is a schematic diagram showing a process for producing the shRNA expression cassette and shRNA expression library of the present invention.
Fig. 3 shows a 1- ^st PCR amplification reaction of Example < 2-l >, showing primer pair (set) and amplification product used in the above reaction.
FIG. 4 shows the BsrDI restriction enzyme treatment in Example < 2-2 >, which shows the cleavage site and the resulting DNA double chain fragment resulting from the treatment of the restriction enzyme.
FIG. 5 shows the lambda exonuclease treatment of Example < 2-2 > and shows the DNA single chain removed or obtained by the lambda exonuclease treatment.
FIG. 6 shows an asymmetric hairpin structure single strand DNA chain produced by the hybridization reaction, showing the self-hybridization induction reaction of Example < 2-3 >.
FIG. 7 shows the DNA sequence stretching reaction of Example < 2-4 >, showing a symmetrical hairpin structured single stranded DNA chain obtained by a T4 DNA polymerase reaction.
FIG. 8 shows the EarI restriction enzyme treatment of Example < 2-5 >, which shows the cleavage site due to the restriction enzyme treatment and the generated fragment of the structured nucleic acid of the hairpin structure.
FIG. 9 shows the branched adapter binding reaction of Example < 2-6 >, and shows the hairpin structured nucleic acid generated by the above-mentioned coupling reaction.
Fig. 10 shows the 2 ^nd PCR amplification reaction of Example <2-7>, showing the primer pair (set) and the amplification product used in the reaction.
Fig. 11 is a graph showing the accuracy of shRNA confirmed by NGS analysis in Example < 2-8 >. The x-axis is the% perfect match (the percentage of perfect matches for each shRNA as a percentage of the total read) and the y-axis is the shRNA numbers. The interval on the x-axis represents tens [less than or equal to] (for example, the first interval is the interval with 0% perfect match less than 10), and the last interval contains 100.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

&Lt; Example 1 >

88mer oligo DNA design

&Lt; 1-1 > sense RNA Coding DNA Nucleotide sequence collection

Prior to synthesis of the 88mer template oligo DNA used in the present invention, DNA sequence information encoding 20,000 sense RNAs expected to inhibit the expression level of a specific gene is collected and prepared. In this example, the target genes were all human genes, and the sense RNA was selected by reference to the library of TRC (The RNAi Consortium). Three to five different sense RNA sequences per target gene were included.

<1-2> 88 mer oligo DNA Design and synthesis

As shown in FIG. 1, a 5 'primer DNA sequence (SEQ ID NO: 1) including the BsrDI restriction enzyme recognition sequence and a 20mer DNA sequence (SEQ ID NO: 1) collected in the above Example <1-1> the reverse complementary DNA sequence consisting of six nucleotides (i.e., 6mer DNA sequences encoding the anti-sense RNA), DNA sequence encoding a hairpin loop (SEQ ID NO: 2), collected in example <1-1> A 20-mer DNA sequence And the 3 'primer binding site (3' primer binding site) containing the EarI restriction enzyme recognition sequence A single stranded 88mer oligonucleotide (SEQ ID NO: 4) to which the DNA sequence (SEQ ID NO: 3) was linked was designed.

Single-stranded 88mer oligo DNA was synthesized using CustomArray B3 ™ Synthesizer (Custom array) based on the 88mer oligo DNA sequence information designed above.

&Lt; Example 2 >

shRNA Expression cassette ( short hairpin RNA expression cassette )

<2-1> 1 ^st PCR amplification

Stranded 88mer oligo DNA (SEQ ID NO: 4) prepared in Example <1-2> as a template and subjected to PCR amplification using the primer set described in Table 1 below (see FIG. 3). At this time, the 5 'end of the 3' primer (i.e., reverse primer) is labeled with biotin TEG, a modification of biotin.

PCR conditions: 95 ° C / 30 sec - [95 ° C / 15 sec 74 ° C / 30 sec] _x20 72 ° C / 60 sec 4 ° C / ∞

(In this specification, the symbol '∞' means 0 to infinity, meaning that the time (sec) is not particularly limited.)

The amplification product obtained by the PCR is double-stranded 88mer oligo DNA. At this time, the newly synthesized complementary strand (SEQ ID NO: 5) is synthesized with the biotin TEG labeled with the 3 'primer (see FIG. 3).

<2-2> BsrDI Restriction enzymes and lambda exonuclease process

The product obtained by PCR in Example <2-1> was treated with BsrDI (NEB) restriction enzyme (treatment at 65 ° C for 3 hours or longer, see FIG. 4), lambda exonuclease (NEB) To obtain single-stranded DNA finally labeled with biotin TEG at the 5 ' -end (see Fig. 5).

<2-3> Self-hybridization ( self - hybridization ) Judo

A 2X hybridization buffer (see Table 2) was added to single-stranded DNA labeled with biotin TEG at the 5'-end obtained in Example <2-2>, and the mixture was incubated at 85 ° C for 5 minutes Allow to cool to room temperature. After the sample is allowed to cool to room temperature, transfer the sample to 4 ° C, leave it for 20 minutes, and leave it at O / N (overnight) at 20 ° C.

Through the above procedure, a 20-mer DNA sequence is complementarily combined with a 6-mer DNA sequence (a DNA sequence encoding an anti-sense RNA for a target gene) to form a single-stranded DNA chain having a stem- Is created. During the hybridization process, some of the bases (15 bases) in the 20mer remain overhanged, so that the arm (especially the arm 3 ') constituting the stem structure is short asymmetric hairpin structure single strand DNA chain (See FIG. 6).

<2-4> DNA Sequence height

The reaction elements shown in the following Table 3 were applied to the asymmetric hairpin structure single stranded DNA chain obtained in Example <2-3>, and the reaction was carried out at 12 ° C for 30 minutes.

Through this process, the ends of the shorter arms (i.e., the 3'-ends of the 6-mer DNA sequence) constituting the stem structure in the asymmetric hairpin structure single strand DNA chain obtained in Example <2-3> In the arms (especially the 5 'arms), they are not hybridized, are overhanged, and complementarily elongate with the remaining part as a template. This process yields a symmetrical hairpin structured single stranded DNA chain (see FIG. 7).

<2-5> Purification of EarI restriction enzyme treatment and section

The symmetrical hairpin structure single strand DNA chain obtained through the procedure of Example <2-4> is treated with EarI restriction enzyme and the desired fragment sample (nucleic acid of 30 bp band) is prepared by separating into polyacrylamide gel.

Through this process, the biotin TEG tagged in the DNA chain is cleaved and a sticky end to which an adapter sequence described later can bind is generated (see FIG. 8).

<2-6> Preparation and connection of branched adapter

To prepare the branched adapter, 100 oleole each of Branch oligo A and Branch oligo B was added, and 2X hybridization buffer was added to the mixture to obtain a final concentration of 10 pmole / ul. The mixture was incubated at 85 캜 for 5 minutes After allowing to stand, it was allowed to cool slowly until it became normal temperature. After it was allowed to stand at room temperature, it was left at 4 ° C for 30 minutes and stored at 20 ° C after O / N storage. Branch oligo A and branch oligo B contain AcuI and EcoRI restriction sites, respectively.

The sample prepared in Example <2-5> was mixed with the branched adapter at a ratio of 1:10 (for example, when the sample was 1 pomole, 10 pmole of the branched adapter was used) and treated with T4 DNA ligase (NEB) O / N at < / RTI > In the ligation mixture samples prepared above, Branch oligo A and Branch oligo B bind to both ends of the single-stranded DNA chain prepared in Example <2-5> (see FIG. 9).

<2-7> 2 ^nd PCR Amplification

Using the ligation mixture obtained in Example <2-6> as a template, PCR was performed with the primer sets set forth in Table 5 below to obtain shRNA cassettes as final products (see FIG. 10).

PCR conditions: 95 ° C / 30 sec - [95 ° C / 15 sec 74 ° C / 30 sec] _x15 74 ° C / 60 sec 4 ° C / ∞

<2-8> Measurement of accuracy of shRNA cassette base sequence through NGS

The nucleotide sequence of the shRNA cassette encoding 20,000 different 20-mer sense RNAs obtained in the above Example <2-7> was analyzed, and the accuracy of the shRNA expression cassette prepared according to the method of the present invention was measured.

The NGS analysis was carried out using Illumina HiSeq 2000, commissioned by Selamix Co., Ltd. Each of the shRNA cassettes obtained as a PCR product in Example <2-7> acts as a template in NGS, and the individual nucleotide sequence amplified and analyzed by NGS is referred to as "read". As a measure of the accuracy of the nucleotide sequence, 'perfect match' is the nucleotide sequence between the AcuI of branch adapter A and the restriction site of EcoRI of branch adapter B in each shRNA cassette, that is, the base of the part to be inserted in the expression vector The sequence was determined to be exactly 100% identical to the predicted sequence.

The NGS analysis results are summarized in Table 6 below. The total number of readings was 11,811,812, and the template included in the read, i.e., shRNA, was 19,956 out of the total 20,000 kinds, 99.78%. Also, 591 readings per template were analyzed.

Meanwhile, the number of perfect matches among the total read was 8,143,805, and the template containing the perfect read was found to be 19,531 corresponding to 97.66% of the total 20,000. An average of 407 perfect reads were generated per template.

As a result of analyzing the accuracy of the nucleotide sequence of the shRNA cassette prepared by the method of the present invention for each template, the perfect match ratio (% perfect match) of the shRNA was 60% or more and less than 80% (See FIG. 11), and the overall average perfect match was 63.25% (see FIG. 11). That is, according to the method of the present invention, an accurate sequence of shRNA cassettes having no specific error in the nucleotide sequence of the specific sense of the target gene and the antisense sequence, the restriction site of the restriction enzyme and the hairpin structure sequence at a probability of about 63% . This indicates that accuracy is greatly improved considering that the accuracy of existing shRNA library production is less than 50%.

As described above, the present invention relates to a method for producing an shRNA expression cassette, an shRNA expression cassette produced by the method, and a library containing the shRNA expression cassette, and more particularly, to a method for producing a shRNA expression cassette, A DNA sequence comprising a 5 ' primer sequence; ii) a DNA sequence encoding an antisense RNA for a target gene consisting of 3 to 9 bases; iii) a DNA sequence encoding a hairpin loop consisting of 10 to 33 bases; iv) a DNA sequence consisting of 19 to 24 bases and encoding a sense RNA for the same target gene as in ii); And v) an oligonucleotide consisting of a DNA sequence comprising a 3 'primer binding site comprising a recognition sequence of a second restriction enzyme type 2S, a method for producing an shRNA expression cassette using said oligonucleotide, an shRNA Expression cassettes and libraries comprising same.

The shRNA expression cassette of the present invention solves the problem that the synthesis of 150-200 base oligonucleotide using the existing Agilent Sure Print technology and the accuracy of producing the RNAi library using it is only about 50% As well as reducing the time and cost of constructing an RNAi library, which is highly industrially applicable.

<110> Medicinal Bioconvergence Research Center <120> Method to prepare shRNA expression cassette, shRNA expression cassette and library produced thereby <130> NP14-0022 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 88mer 5 ' primer with BsrD1 site <400> 1 cgatggacgt ccgtgcaatg 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 88mer hairpin loop sequence <400> 2 tacatctgtg gcttcacta 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 88mer 3 'primer binding sequence with EarI site <400> 3 agaagagaac ccgcccgcc 19 <210> 4 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> 88mer oligo DNA <400> 4 cgatggacgt ccgtgcaatg nnnnnntaca tctgtggctt cactannnnn nnnnnnnnnn 60 nnnnnggtga gaagagaacc cgcccgcc 88 <210> 5 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> complementary DNA of 88mer oligo DNA <400> 5 ggcgggcggg ttctcttctc accnnnnnnn nnnnnnnnnn nnntagtgaa gccacagatg 60 tannnnnnca ttgcacggac gtccatcg 88 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 88mer 3 'reverse primer <400> 6 ggcgggcggg ttctcttct 19 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> branch oligo A adapter <400> 7 atagagagta cgtgctcctg ctgaaggagg gtacgtagg 39 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> branch oligo B adapter <400> 8 gtgcctactg cctcggaatt ctgccctcac gagtcgaagg cacag 45 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 2nd PCR forward primer <400> 9 atagagagta cgtgctcctg ctgaaggagg 30 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 2nd PCR reverse primer <400> 10 ctgtgccttc gactcgtgag ggcag 25

Claims

I) a DNA sequence comprising a 5 'primer sequence comprising the recognition sequence of a first restriction enzyme type II;
ii) a DNA sequence encoding an antisense RNA for a target gene consisting of 3 to 9 bases;
iii) a DNA sequence encoding a hairpin loop consisting of 10 to 33 bases;
iv) a DNA sequence consisting of 19 to 24 bases and encoding a sense RNA for the same target gene as in ii); And
v) a DNA sequence comprising a 3 'primer binding site comprising a recognition sequence of a second restriction enzyme type II
&Lt; / RTI >

The oligonucleotide according to claim 1, wherein the DNA sequence of i) is the DNA sequence of SEQ ID NO: 1.

2. The oligonucleotide according to claim 1, wherein the DNA sequence encoding the hairpin loop of iii) has the nucleotide sequence shown in SEQ ID NO: 2.

2. The oligonucleotide according to claim 1, wherein the DNA sequence of v) is a DNA sequence represented by SEQ ID NO: 3.

The oligonucleotide according to claim 1, wherein the oligonucleotide is the nucleotide sequence shown in SEQ ID NO: 4.

(a) treating the double-stranded DNA chain comprising the oligonucleotide of claim 1 and a sequence complementary thereto with a first restriction enzyme type II to produce a single-stranded DNA chain and ;
(b) inducing self-hybridization between a DNA sequence encoding a sense RNA contained in the single-stranded DNA chain obtained in the step (a) and a DNA sequence encoding an antisense RNA to form a stem- Obtaining a short asymmetrical hairpin structured single stranded DNA chain with one arm forming the stem;
(c) The terminal of the short arm forming the stem in the asymmetric hairpin structured single stranded DNA chain obtained in the step (b) is complementarily extended to an overhanging DNA sequence part without hybridization elongation to obtain a symmetrical hairpin structured single stranded DNA chain;
(d) treating the second restriction enzyme type II on the symmetrical hairpin structured single stranded DNA strand obtained in step (c); And
(e) attaching first and second adapters to both ends of the DNA chain obtained in step (d), and amplifying by PCR;
Lt; RTI ID = 0.0 > expression cassette. &Lt; / RTI >

7. The method of claim 6, wherein step (a)
(a1) amplifying the oligonucleotide of claim 1 with a 5 'primer and a 3' primer labeled with a 5'-end to obtain a double-stranded DNA chain; And
(a2) treating the double-stranded DNA strand obtained in the step (a1) with a first restriction enzyme type II and an exonuclease to selectively obtain only a single-stranded DNA chain having a 5'- ;
Wherein the shRNA expression cassette is produced by a method comprising the steps of:

7. The method of claim 6, wherein the first and second adapters of step (e) comprise different restriction enzyme cleavage sequences.

7. The shRNA expression cassette according to claim 6, wherein the first and second adapters of step (e) each comprise the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8, respectively. ).

8. The method of claim 7, wherein the exonuclease is T7 exonuclease or exonuclease lambda. 9. The method of claim 7, wherein the exonuclease is T7 exonuclease or exonuclease lambda.

8. The method of claim 7, wherein the label is a label by a non-phosphorylated compound.

An shRNA expression cassette produced by the method of claim 6.

13. The method of claim 12, wherein the shRNA expression cassette comprises
A first adapter DNA sequence;
A DNA sequence encoding a sense RNA;
A DNA sequence encoding a hairpin loop;
A DNA sequence encoding an antisense RNA that binds complementarily to the sense RNA; And
A second adapter DNA sequence;
Lt; RTI ID = 0.0 > expression cassette. &Lt; / RTI >

A recombinant expression vector comprising the shRNA expression cassette of claim 12.

15. The recombinant expression vector according to claim 14, wherein the expression vector further comprises a CNNC motif.

15. The recombinant expression vector according to claim 14, wherein the expression vector is a plasmid vector or a viral vector.

17. The recombinant vector according to claim 16, wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated viral vector, a lentivirus vector, a retrovirus vector, and a herpes virus vector.

An shRNA encoding library consisting of a plurality of vectors comprising the shRNA expression cassette of claim 12.

19. The shRNA encoding library of claim 18, wherein the plurality of vectors comprises an shRNA expression cassette into which a DNA sequence encoding a different sense RNA is inserted.

Preparing at least one shRNA expression cassette of claim 12 inserted with a DNA sequence encoding a different sense RNA;
Cloning the prepared shRNA expression cassettes into an expression vector (RNA vector system);
And pooling the cloned vector. &Lt; RTI ID = 0.0 > 8. < / RTI >

A pseudoviral particle in which the shRNA encoding library of claim 18 is packaged.