KR20140006363A - Method for preparing chimeric ribonucleic acid, cdna and its derivatives - Google Patents

Abstract

The present invention relates to a hybridized ribonucleic acid, a cDNA derived therefrom, and a method for preparing the derivative thereof, and more particularly, to a hybridized ribonucleic acid and cDNA derived therefrom and a derivative thereof having an additional sequence bound to the 5 'end thereof. It relates to a manufacturing method and various application techniques using the same. According to the present invention, it is possible to provide hybridized ribonucleic acid and cDNA derived therefrom, and derivatives thereof, in which an additional sequence is bound to the 5 'end, and used to identify specific sequence information of the 5' end of the RNA transcript. Can be. For example, the present invention can be applied to the mapping of transcriptional initiation sites in genomes, identification of new transcriptional initiation sites, identification of new transcripts and rare transcripts, and characterizing global gene and protein expression in specific cells.

Description

Hybridized ribonucleic acid and its complementary DNA and its derivatives {Method for preparing chimeric ribonucleic acid, cDNA and its derivatives}

The present invention relates to a hybridized ribonucleic acid, a cDNA derived therefrom, and a method for preparing the derivative thereof, and more particularly, to a hybridized ribonucleic acid and cDNA derived therefrom and a derivative thereof having an additional sequence bound to the 5 'end thereof. It relates to a manufacturing method and various application techniques using the same.

As the whole genome sequence of humans and a large number of experimental organisms is revealed, active research is being conducted to search for new genes and analyze their function. These studies provide a lot of information to understand not only cell survival but also complex biochemical and physiological metabolism associated with disease.

In particular, many efforts have been made to identify ribonucleic acid (RNA) encoded in the genome and to characterize its function. In general, genomic DNA sequences are transcribed into RNA transcripts. While these RNA transcripts have important biochemical functions on their own, they usually direct the production of proteins that perform most of the biochemical functions of the organism. RNA transcripts encoding polypeptides are called mRNAs, and other types of RNAs include ribosomal RNA, tRNA, miRNA, and the like.

The process of identifying the entire transcript in understanding the nature of the genomic makeup of an organism is very difficult and slowing down research. The complexity and diversity of the genomes prevent computer-based accurate prediction of genes and RNA transcripts. In addition, simple predictions of gene sequences within the genome pose a problem of underestimating the number of various transcripts produced by cells. In other words, it should be noted that different transcription initiation, different RNA splicing methods, and different transcription termination in a single gene are involved in the production of more diverse transcripts. Therefore, despite the completion of the genome project, a direct study on the mRNA transcribed in living things is required, and based on the study of life phenomena and disease studies.

In this regard The most common experimental method used to identify genes and RNA transcripts is the Expression Sequence Tag (EST) analysis technique. The EST assay involves randomly synthesizing complementary DNA (cDNA) from an RNA transcript and sequencing the synthesized cDNA to determine a portion of the transcript, ie, the sequence for the EST. However, the EST assay is focused on the identification of the 3 'terminal portion of the transcript and has a limitation in analyzing sequence fragments derived from the intermediate or terminal portions of RNA. That is, EST assays usually fail to detect rare transcripts and have limitations in identifying the true 5 ′ end of the RNA transcript rather than fragments in the middle or terminal portions of the RNA transcript.

In the case of mRNA, the 5 'terminal portion of the entire transcript is important as a translation initiation part in the process of being translated into a protein, and plays an important role in the secondary structure and tertiary structure of the protein by determining the initial amino acid sequence. Therefore, it is important to provide a technique for identifying and analyzing the true 5 'terminal portion of such mRNA.

Szybalski, Gene 40: 169, 1985 Tucholski et al, Gene Vol. 157, pp. 87-92, 1995

The present invention aims to solve the problems of the conventional RNA transcript identification and analysis technique using the EST as described above, wherein the hybridized ribonucleic acid (hereinafter, “5” having an additional sequence attached to the 5 ′ end) is provided. It is an object of the present invention to provide a method for preparing cDNA derived therefrom and its derivatives.

It is also an object of the present invention to provide specific sequence information of the 5 'terminal region of an RNA transcript using the 5' terminal hybridized RNA and cDNA derived therefrom, from which, for example, initiation of transcription in the genome It provides techniques for mapping regions, identifying new transcription sites, identifying new transcripts and rare transcripts, and characterizing global gene and protein expression in specific cells.

In addition, it is an object of the present invention to use the above-mentioned 5 'terminal hybridized RNA and cDNA derived therefrom for various uses such as the preparation of a probe that hybridizes with the sequence of the 5' terminal region of an RNA transcript.

The present invention provides a 5 'terminal hybridized RNA labeled in vitro and a method for producing a cDNA derived therefrom for achieving the object as described above.

The method for preparing 5 'terminal hybridized RNA and / or cDNA derived therefrom in one embodiment of the present invention is intended for hybridization at the 5' end of acceptor RNA, preferably full length mRNA or capped mRNA. Binding additional oligonucleotides.

The additional oligonucleotide may be single stranded RNA, single stranded DNA, single stranded hybridized RNA-DNA or a combination of these two strands. The additional oligonucleotide comprises a restriction enzyme recognition sequence selected from the group consisting of 5'-CTGGAG-3 ', 5'-GTGCAG-3', 5'-TCCGAC-3 'and 5'-CAGCAG-3'.

In one embodiment of the present invention, before binding the additional oligonucleotide to the 5 'end of the capped RNA, an enzyme capable of removing the 5' end of the phosphate group from the sample containing the RNA in advance. Is exposed to remove the phosphate group from the 5 'end of the RNA molecules without the 5' end cap, the RNA with the 5 'end cap is converted to RNA with a free 5' phosphate group and then additional oligonucleotides are added to the free 5 'phosphate group. It may comprise the step of binding to the RNA having.

In addition, an enzyme that removes phosphate groups from the 5 'end of the RNA molecules that do not have a 5' end cap includes CIP (Calf intestinal phosphatase), and RNA having a 5 'end cap is replaced with RNA having a free 5' phosphate group. To convert enzymes include tobacco acid pyrophosphatase (TAP). Meanwhile, the binding of the additional oligonucleotide to the 5 'end of the RNA may be performed by T4 RNA ligation.

In one embodiment of the present invention, the additional oligonucleotide may include a label, and the label may include an attachment label, an introduction label, and / or a label sequence.

The attachment label or introduction label can be used to purify and / or detect the additional oligonucleotides and RNA bound thereto. The label sequence may also be used as a recognition site for an enzyme or reagent that recognizes, cleaves or modifies a nucleic acid, such as a restriction enzyme. Meanwhile, cDNA is synthesized from the RNA to which the additional oligonucleotide is bound to provide a 5 ′ terminal hybridized cDNA.

In the present specification, the binding or conjugation between the acceptor RNA and the additional oligonucleotide is referred to as 'hybridization binding or conjugation'.

The present invention provides a method for identifying a sequence at or near the 5 'end of cDNA. Confirmation method of this embodiment of the present invention,

(a) a 5 'terminal hybridized cDNA is cleaved with a tag cleavage reagent, but is cleaved at an outer position in a 3' direction with respect to the recognition site of the tag cleavage reagent, and thus a 5 'portion of the hybridized cDNA and a 3' portion of the hybridized cDNA Emitting the;

(b) selectively obtaining a 5 'portion of the hybridized cDNA;

(c) sequencing a 5 'portion of the plurality of said hybridized cDNAs obtained. The 5 'portion of the hybridized cDNA includes an affinity purified label at or near the 5' end, and step (b) comprises contacting the cleavage mixture with a capture medium that binds the affinity purified label to the 5 'of the hybridized cDNA. Portions can be obtained selectively.

The tag cleavage reagent is cleaved at a position separated by at least 7, 10 or 14 base pairs in the 3 'direction from the 3' end of the recognition site.

Sequencing the 5 ′ portion of the hybridized cDNA is characterized by sequencing a sequence located immediately downstream to the hybridization binding site.

In addition, sequencing the 5 ′ portion of the hybridized cDNA comprises forming at least one nucleic acid concatemer comprising a plurality of 5 ′ portions of the hybridized cDNA, such at least one nucleic acid concatemer. Sequencing.

On the other hand, the method for forming the nucleic acid concatemer,

(a) binding an adapter sequence to the 3 'end of the hybridized cDNA 5' moiety obtained from the cleaved 5 'terminal hybridized cDNA to generate a cDNA-adapter construct,

(b) amplifying the cDNA-adapter structure using a 5 'oligonucleotide primer comprising a first anchor cleavage reagent recognition site and a 3' oligonucleotide primer comprising a second anchor cleavage reagent recognition site, thereby amplifying the cDNA-adapter constructs; Obtaining an amplification product in which the second anchor cleavage reagent recognition sites are located at right and left;

(c) cutting the amplification product with a first anchor cleavage reagent and a second anchor cleavage reagent to obtain a double cleaved amplification product;

(d) binding the double cleaved amplification product to form a nucleic acid concatemer.

The method for identifying the 5 'terminal region or the surrounding sequence of the cDNA of one embodiment of the present invention is a 5' terminal hybridized RNA obtained from trans-splicing between the 5'-trans-splicing nucleic acid and the splice acceptor RNA. A 5 'terminal hybridized cDNA derived from is used. The cDNA also comprises 7 to 50 nucleotides, preferably 14 to 30 nucleotides, corresponding to the 5 'terminal sequence of the acceptor RNA, referred to herein as "TAG" or "5'-TAG". . In the present specification, sequence information obtained by sequencing "TAG" is referred to as "TAG sequence".

The present invention also provides a method for obtaining the 5'- and 3'-end sequences of RNA and constructing hybridized cDNAs labeled with the 5'- and 3'-ends therefrom. Method for producing a hybridized cDNA labeled with the 5 'end and 3' end,

(a) removing at least a portion of the 3 'poly-A region of the 5'-terminated hybridized cDNA comprising a cleavage site for the first tag cleavage reagent,

(b) a 3'-adapter comprising a cleavage site for the second tag cleavage reagent is coupled to the 3 'end of the 5' end labeled hybridized cDNA to obtain a hybridized cDNA labeled 5 'end and 3' end It includes a step.

 Hybridized cDNAs labeled with the 5 ′ and 3 ′ ends can be circularized, for example, by intramolecular ligation.

The present invention provides a method for preparing hybridized cDNAs labeled and circularized at the 5 'and 3' ends, wherein the labeled and hybridized cDNAs are in the form of: i) sequences corresponding to acceptor RNA, ii) first tag cleavage. 5 'terminal hybridization sequence attached to the 5' end containing the recognition site for the reagent, i) a 3'-adapter sequence attached to the 3 'end containing the recognition site for the second tag cleavage reagent. Since the cDNA is circular, the 3 'end of the 3'-adapter is coupled to the 5' end of the 5 'end hybridization sequence.

These 5 'and 3' ends are labeled and circularized hybridized cDNA is cleaved in duplicate by the first cleavage reagent and the second cleavage reagent to release the linearized product. The linearized product then contains a 5 'terminal hybridization sequence, a 5'-TAG sequence corresponding to the 5' terminal portion of the acceptor RNA, a 3'-TAG sequence corresponding to the 3 'terminal portion of the acceptor RNA, and a 3'. It is circularized in the order of the adapter sequence.

5'-3'-TAG (meaning a sequence portion in which a 5'-TAG sequence and a 3'-TAG sequence are fused as shown in FIG. 3) is hybridized to a 5 'terminal hybridization sequence and is used for the first anchor cleavage reagent. Amplification products obtained after amplifying the circularized product using a 5'-primer comprising a recognition site and a 3'-primer hybridized to a 3'-adapter sequence and containing a recognition site for a second anchor cleavage reagent. Is made by cutting with a first anchor cleavage reagent and a second anchor cleavage reagent. The 5'-3'-TAG may be cloned and sequenced directly, or may be cloned and sequenced as concatemers.

In addition, the present invention provides a method for identifying a TAG sequence in a genomic sequence and a method using a computer therefor, thereby identifying the 5 'end of a transcript corresponding to the TAG sequence and various characteristics of the transcript corresponding to the TAG sequence. Evaluate. For example, the TAG sequence can be compared with at least one nucleotide sequence database including a genomic sequence database, a cDNA database, and an EST database.

The TAG sequence is derived from a 5'-terminal hybridized cDNA or a 5'-3'-hybridized cDNA, which is compared with the genomic sequence. In one embodiment, it may be determined whether the genomic sequence corresponding to the TAG encodes a transcript with 5'-3 'orientation as the TAG sequence. In addition, if the TAG sequence is derived from a trans-splicing based method, it may be determined whether the TAG sequence is located behind a splicing acceptor site (eg, AG sequence). On the other hand, where the TAG sequence is identified at one or more positions of the genomic sequence, the position where the TAG sequence follows the splicing acceptor sequence may be considered as the correct TAG sequence position.

When the TAG matches a location in the genomic sequence, the 5 'end of the transcript comprising the TAG can be inferred using one or more distance parameters. For example, the distance parameter may be the distance from the TAG sequence in the genome to the first closest exon or may be the distance from the TAG sequence in the genome to the closest upstream gene initiation codon (eg ATG).

The one or more distance parameters are used to determine whether the 5 'end of the transcript corresponding to the TAG is a new gene, another transcript of a known gene or a known transcript of a known gene. One or more analyzes of the TAG sequence may be performed on a computer and the TAG sequence analysis instructions may be input to a computer and / or computer readable storage medium (eg, an optical storage medium or a magnetic storage medium).

The present invention also provides oligonucleotides used to generate cDNA labeled 5 ′ ends. The oligonucleotide is 15 to 100 nucleotides in length and includes an attachment or introduction label and a recognition site for the cleavage reagent. The cleavage reagent cleaves the DNA at a position outside the recognition site but preferably cleaves the DNA at an outside position in the 3 'direction with respect to the recognition site. The cleavage reagent may be a restriction enzyme, the restriction enzyme cleaves the DNA at a position at least 7, 10 or 14 base pairs away from the recognition site, but preferably at a position outside the 3 'direction to the recognition site. Cleave the DNA. The recognition site is located within 10 base pairs close to the 3 'end of the oligonucleotide and is preferably located at the 3' end of the oligonucleotide primer. The attachment label or introduction label is selected from the group consisting of affinity purification labels and fluorescent materials.

In addition, the present invention provides a 5'-3'-TAG comprising a 5'-TAG corresponding to a sequence located at the 5 'end of RNA and a 3'-TAG corresponding to a sequence located at the 3' end of the same RNA. To provide. Preferably, the 5'-3'-TAG comprises a sequence of the 3 'end portion of the RNA encoded by the genome, except for sequences added during post-transcription, e.g., sequences such as poly-A tails. Has an axon sequence. Preferably the 5'-TAG and the 3'-TAG are located in opposite directions, more preferably the 3 'ends of these 5'-TAG and 3'-TAG are close to each other and the 5' end is close to each other. Is located far away. This direction is called the "tail-tail" direction. Meanwhile, the present invention provides a concatemer of 5'-3'-TAGs including one or more repeating units, the repeating unit comprising a first cleavage reagent recognition site, a first 5'-3'-TAG, a first Two cleavage reagent recognition sites and a second 5'-3'-TAG.

According to the present invention, it is possible to provide hybridized ribonucleic acid, a cDNA derived therefrom, and derivatives thereof, in which an additional sequence is bound to the 5 'end, and the specific sequence information of the 5' end portion of the RNA transcript can be used using these. have.

For example, the present invention can be applied to the mapping of transcriptional initiation sites in genomes, identification of new transcriptional initiation sites, identification of new transcripts and rare transcripts, and characterizing global gene and protein expression in specific cells.

1 is a diagram schematically illustrating a method of RNA-RNA ligation in vitro. In FIG. 1, Cal intestinal phosphatase (CIP) cannot remove phosphate groups at the 5 ′ end protected by m7G (ie, capped), and Tobacco acid pyrophosphatase (TAP) is used at the 5 ′ end of the capped RNA. Only the phosphate group is left. Subsequently, RNA oligonucleotides are coupled to full-length mRNA by phosphatase treatment and RNA oligonucleotides having a recognition site of a TAG restriction enzyme such as MmeI or EcoP15I.
FIG. 2 is a diagram schematically illustrating a method for ligating RNA 5 'end determination (abbreviated as 5'-LM-RED) for human RNA transcripts. This method uses two different restriction enzymes, EcoP15I (Tag Restriction Enzyme) and SnaBI (Second Anchor Restriction Enzyme), considering the high cleavage efficiency of the enzyme when two recognition sites exist in the same DNA molecule. 2 EcoP15I site is introduced at the 3 'end of cDNA.
FIG. 3 shows both 5'- and 3'-terminal portions of RNA (abbreviated as 5'-3'-Co-RED) using a circularization process to obtain paired 5'-3'-TAGs A diagram schematically illustrating a process of analyzing about.
4 shows the direction of the 5′-3′-TAG in the genomic sequence as mapped to the concatemer.
5 shows a schematic method for 5'-3'-Co-RED ortholog search using reference genomes.

Hereinafter, the present invention will be described in more detail based on the embodiments of the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example

Example 1 Method for Constructing 5 ′ Terminal Hybridized RNA and cDNA in Vitro

In this example, a method for producing RNA or cDNA having a hybridized sequence at the 5 ′ end in vitro is described (see FIGS. 1 and 2). Oligonucleotides are bound to the 5 'end of the acceptor RNA in vitro to produce a 5' end hybridized RNA. Oligonucleotides known in the art can be used and such oligonucleotides can be modified using known techniques known in the art, including, for example, at least one useful sequence motif or for convenience of later analysis. The 5 'end of the RNA can be modified. The RNA used may be one type of RNA or an RNA sample containing one or more types of RNA. Such RNA samples are whole RNA or poly-A RNA derived from the cell culture, tissue or organism being analyzed. And the oligonucleotides bound to RNA may be single stranded RNA, single stranded DNA, single stranded hybridized RNA-DNA or a double stranded or triple stranded combination thereof. The oligonucleotide also includes a restriction enzyme recognition sequence selected from the group consisting of 5'-CTGGAG-3 ', 5'-GTGCAG-3', 5'-TCCGAC-3 'and 5'-CAGCAG-3'.

The oligonucleotide binding to the 5 'end of the RNA is a selective process of binding the oligonucleotide only to the RNA molecule having a cap structure at the 5' end. Most eukaryotic mRNAs have a structure called "cap" at the 5 'end, which consists of m7G (methylated guanosine at position 7) linked by a phosphate chain at the 5' end of the RNA.

However, the oligonucleotide can bind only to RNA having a free phosphate group at the 5 'end by an enzyme such as T4 RNA ligase. RNA ligation cannot bind oligonucleotides to capped RNA. Therefore, the cap structure must be removed by phosphatase such as tobacco acid pyrophosphatase (TAP) to expose the free phosphate group of RNA. RNA mixtures obtained from cells contain a mixture of RNA with a cap structure and RNA without a cap structure. RNA without a cap structure is often degraded or unwanted RNA.

In this embodiment, the oligonucleotide is selectively bound to RNA having a cap structure in consideration of the properties of the constituent RNA and the cap structure in the RNA mixture. RNA without a cap structure has a 5 'free phosphate group, so oligonucleotides can easily bind. However, this should be excluded as it is oligonucleotide binding to degraded or unwanted RNA as described above.

Therefore, in this embodiment, in consideration of this point, the RNA mixture is first treated with Cal intestinal phosphatase (CIP) to remove RNA without cap structure in advance. Then, the cap structure of the capped RNA present in the RNA mixture is removed by TAP (tobacco acid pyrophosphatase) treatment to expose the 5 'phosphate group of RNA, followed by RNA ligation to select at the 5' end of the RNA. To oligonucleotides. Since the RNA without the cap structure has already been removed by CIP, the RNA to which the oligonucleotide is bound is the first capped RNA, so that selective oligonucleotide binding to the capped RNA can be performed.

Oligonucleotides added by in vitro methods are designed to have the necessary sequences for further analysis. As an example, the added oligonucleotide includes a restriction enzyme recognition site, and the restriction enzyme cleaves DNA at a position outside of the recognition site. The recognition sequence is located at the 3 'end of the added oligonucleotide, for example within 10 bases from the 3' end. On the other hand, when oligonucleotides are bound to acceptor RNA to obtain 5 'terminal hybridized RNA, the 5' terminal hybridized RNA is separated and purified from other RNA using affinity oligonucleotide column chromatography.

In the example of Figure 1 it can be seen that the recognition site of the restriction enzyme such as EcoP15I is introduced into the added oligonucleotide. EcoP15I is used as a tag restriction enzyme to generate TAGs for human RNA transcript analysis by recognizing EcoP15I recognition sites (cognitive sequences) introduced into additional oligonucleotides in RNA-RNA and cleaving 5 'terminal hybridized RNA.

In addition, as in the example of FIG. 2, the 5 'terminal hybridized RNA can be used for further amplification as well as the first strand and the second strand synthesis of the 5' terminal hybridized cDNA. Meanwhile, the sequence of the 5 'terminal hybridized RNA may be changed by oligonucleotide primers used during cDNA synthesis and / or amplification. And 5 ′ terminal hybridized RNA may be labeled using oligonucleotide primers during cDNA synthesis and / or amplification. That is, as shown in FIG. 2, during the synthesis of the 5 ′ terminal hybridized cDNA, the second EcoP15I recognition site may be introduced at the 3 ′ end of the cDNA, and the SnaBI recognition site may be introduced by binding additional adapter DNA. The first EcoP15I recognition site may be changed to a PstI recognition site.

Meanwhile, TAGs obtained by cleaving 5 'terminal hybridized cDNA with various restriction enzymes and purified can be generated in various sizes (eg, 18 bp, 20 bp, 27 bp, etc.) for matching with genomic sequences. This can be determined according to the size of the genome to be analyzed. A summary of restriction enzymes usable in this example is shown in Table 1 below.

Table 1

Finally, the 5 ′ terminal hybridized cDNA synthesized as shown in FIG. 2 may be cleaved and concatemerized and then cloned and sequenced into a vector according to methods known in the art.

Example 2: 5'-3'-Co-RNA Termination

This example describes a method for determining sequences at or near the 5 ′ and 3 ′ ends of the same RNA. In this example, experiments are performed such that the sequence at or near the 3 'end of the RNA and cDNA derived therefrom is the upstream sequence of the poly-A tail. Generally, poly-A tails are sequences that do not encode genes, but are added to RNA by enzymes in post-transcriptional processes. Therefore, it is desirable to obtain a sequence of the 3 'terminal region encoded by the genomic sequence and match it with the genomic sequence.

According to this example, TAGs derived from near the 5 'and 3' ends of a single RNA are simultaneously extracted and placed in close physical proximity within the nucleic acid. This example uses a hybridized cDNA labeled 5 ′ end generated according to the methods described herein or methods known to those skilled in the art. The poly-A tail or other 3 'end portion of the 5' end labeled hybridized cDNA can be removed.

As shown in FIG. 3, for example, the 5 ′ end of a hybridized cDNA labeled 5 ′ end may comprise a recognition site RE1 for the first tag cleavage reagent. In addition, the 5 'end of the hybridized cDNA labeled with the 5' end may be protected by binding to a capture medium (eg streptavidin), for example, via an affinity purified label (eg biotin). While the 3 'end is exposed to cleavage by nucleases for poly-A sequence removal. The nuclease is advanced and not cleaved, thereby obtaining a cDNA product that is only partially cleaved and not completely degraded. An example of such an enzyme is exonuclease III. In addition, the nuclease can leave a single strand of DNA that can be removed by treating a single strand specific nuclease such as Mung bean nuclease or S1 nuclease (Methods in Enzymology, volume 152, 94). -110).

An adapter may be attached to the cleaved 3 ′ end, which includes a recognition site for the second tag cleavage reagent. The second tag cleavage reagent recognizes and locates the recognition site to cut in the 3 'direction from the recognition site. After adapter attachment, the cDNA is called hybridized cDNA labeled 5'-3 'end. The hybridized cDNA labeled 5'-3 'end attached to the capture medium (e.g., streptavidin) is cleaved or freed by a cleavage reagent (first cleavage reagent) that cleaves the site adjacent to the 5' end. Competitive elution with an affinity purification label (e.g. biotin) results in separation from the capture medium. The 3'-adapter may be designed to include a recognition site RE1 for the first cleavage reagent, in which case the 5 'end and the 3' end that are ligated by cleavage by the first cleavage reagent are simultaneously generated. For example, the 5 ′ and 3 ′ ends may be complementary single strand ends (ie, attachment ends).

Another method for removing the poly-A tail of the 5'-end labeled hybridized cDNA is to synthesize cDNA from a mixture comprising phosphorothioate derivatives of cytosine and guanosine, adenosine and thymidine. . In this case, the poly-A tail portion of the 5 'end labeled hybridized cDNA has little phosphorothioate linkage, whereas the remaining portion (5' portion) of the 5 'end labeled hybridized cDNA is G and C. Has phosphorothioate linkages at a frequency proportional to the amount present. Phosphorothioate linkages are resistant to restriction enzymes such as exonuclease III (Putney et al. (1981) Proc. Natl. Acad. Sci. USA 78, 7350-7354), hybridization labeled 5 ′ end Treatment of phosphorothioate analogues for cDNA with exonuclease III removes the poly-A tail but protects sites with phosphorothioate linkages without being cut by exonuclease III. Other nucleotide analogues resistant to cleavage by exonuclease can be used in place of the phosphorothioate analogs. The cleaved nucleic acids have a blunt end and bind to the 3'-adapter as described above.

The cDNA treated as described above is circularized in such a way that the 3 'end is attached to the 5' end to obtain a 5'-3'-TAG including a 3'-TAG and a 5'-TAG. For example, circularization can be performed by intramolecular ligation, which can be accomplished simply by diluting the DNA concentration to favor intramolecular ligation over intermolecular ligation. Such circularization is known to be possible for nucleic acids between 0.3 and 45 kb, and it is known that almost 100% of DNA undergoes intramolecular ligation under diluted conditions (PNAS, 1984, 81, 6812-6816).

After circularization, the cDNA is cleaved by the first tag cleavage reagent and the second tag cleavage reagent (but they may be identical) to generate nucleic acids having the 5 'and 3' ends of the cDNA but with the middle portion deleted. do. The cDNA thus obtained is a primer that is again circularized (rounded into smaller circles) and hybridized to the 5'-terminal hybridization and 3'-adapter moieties, for example, by the T4 DNA ligase conjugating the blunt ends. Is amplified using. The amplification product should include recognition sites for the first anchor cleavage reagent and the second anchor cleavage reagent, which may initially be present in the 5 'terminal hybridization portion and the 3'-adapter portion, or may be introduced via PCR amplification.

When the recognition sites are introduced through amplification, 5'-3'-TAG is released upon restriction enzyme treatment, which corresponds to sequences near the 3 'and 5' ends of the acceptor RNA. In the 5'-3'-TAG, the 5'-TAG sequence and the 3'-TAG sequence are arranged (tail-tailed) so that the 3 'end is adjacent to each other. The 5'-3'-TAGs are concatemerized to form a concatemer, the concatemer comprising one or more repeating units, the repeating unit comprising a first anchor cleavage reagent recognition site, a first 5'-3 '-TAG', second anchor cleavage reagent recognition site, and second 5'-3'-TAG.

As illustrated in FIG. 3, the recognition sites may be cleaved using, for example, a first anchor cleavage reagent such as BpmI and BsgI and a second anchor cleavage reagent, but other restriction enzymes of type II and III may also be used. . The first restriction enzyme (RE 1) preferably has a recognition site of 8 bp or more, but is not limited thereto, and any one may be used as long as it minimizes random cutting of the cDNA corresponding to the acceptor RNA. For example, restriction enzymes with unusual recognition sites include NotI, FseI, and AscI.

The 5'-3'-TAG sequences obtained through the process as described above are matched with the genomic sequence as shown in FIG. 5, in which the transcript (the reference of the organism under test) in the genome of the reference organism ("reference genome") Identify genomic regions that are likely to encode log transcripts. At this time, the organism to be tested may have incomplete or meaningless genomic sequences. In general, a large number of genomic sequences, particularly complete genome sequences, can be obtained from reference organisms for use in matching. Sequences matching the 5'-3-TAG sequences obtained from the transcripts of the organism to be tested are called orthologous sequences. Such orthologous sequences have 80%, 90%, 95% or more homology with the corresponding TAG sequence. In rare cases, 100% match.

On the other hand, the higher the degree of similarity of the sequence, the smaller the number of ortholog sequences. If a genomic region is found to contain orthologous genomic sequences near the 5 'end and orthologous genomic sequences near the 3' end, the genomic region is a transcript corresponding to the transcript of the organism under test. It can be said to encode. The size of the genomic region depends on how the genes are organized in the reference organism. For example, a reference organism with a large number of identified genes can produce a size distribution of genomic regions occupied by each gene. In this embodiment, the cutoff value of the genomic region size is selected such that at least 80% of known and / or predictive genes in the reference genome are included in a region equal to or less than the cutoff value of the genomic region size. More stringent standards may apply 85%, 90%, 95%, or more.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> Scripps Korea Antibody Institute <120> Method for preparing chimeric ribonucleic acid, cDNA and its          derivatives <130> P12-0291KR <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> RE 1 recognition seq. <400> 1 ctggag 6 <210> 2 <211> 6 <212> DNA <213> Artificial Sequence <220> RE 2 recognition seq. <400> 2 gtgcag 6 <210> 3 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> RE 3 recognition seq. <400> 3 tccgac 6 <210> 4 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> RE 4 recognition seq. <400> 4 cagcag 6

Claims (15)

As a method for preparing 5 'terminal hybridized RNA or cDNA in vitro,
Selectively binding additional oligonucleotides for hybridization to the 5 ′ end of the capped mRNA,
The additional oligonucleotide comprises a recognition site of a cleavage reagent,
And said cleavage reagent cleaves said additional oligonucleotide at a location at least seven base pairs away from said recognition site. The method of claim 1,
Said additional oligonucleotide is selected from the group consisting of single stranded RNA, single stranded DNA, single stranded hybridized RNA-DNA and double stranded nucleic acid. The method of claim 1,
Selectively binding the additional oligonucleotide for hybridization to the 5 'end of the capped mRNA,
(a) exposing a sample comprising RNA to an enzyme that removes phosphate groups from the 5 'end of the RNA without the 5' end cap to obtain a first treated RNA sample,
(b) exposing the first treated RNA sample to an enzyme that converts RNA having a 5 'end cap to RNA having a free 5' phosphate group to obtain a second treated RNA sample;
(c) reacting said second treated RNA sample with a mixture comprising said additional oligonucleotide and ligase. The method of claim 3,
Wherein said ligase is a T4 RNA ligase. The method of claim 3,
And wherein said sample comprising RNA is rich in poly-A RNA. The method of claim 3,
The enzyme that removes the phosphate group from the 5 'end of the RNA having no 5' end cap is CIP (Calf intestinal phosphatase). The method of claim 3,
The enzyme for converting the RNA having a 5 'terminal cap to RNA having a free 5' phosphate group is characterized in that TAP (Tobacco acid pyrophosphatase). The method of claim 1,
The additional oligonucleotide comprises a restriction enzyme recognition sequence selected from the group consisting of 5'-CTGGAG-3 ', 5'-GTGCAG-3', 5'-TCCGAC-3 'and 5'-CAGCAG-3' How to. The method of claim 1,
The cleavage reagent is a restriction enzyme. 10. The method of claim 9,
The restriction enzyme is selected from the group consisting of type II restriction enzyme and type III restriction enzyme. The method of claim 1,
Synthesizing cDNA of said 5 ′ terminal hybridized RNA. As a method of identifying the 5 'terminal region or the surrounding sequence of the hybridized cDNA labeled with the 5' terminal having a hybridization bond,
(a) cleaving the 5'-end labeled hybridized cDNA with a tag cleavage reagent, wherein the cleaved cDNA is cleaved at a position at least 7 base pairs away in the 3 'direction from the recognition site of the tag cleavage reagent to release the 5' portion of the cDNA. To do that,
(b) selectively obtaining a 5 ′ portion of the cDNA,
(c) sequencing the 5 'portion of the cDNA. The method of claim 12,
The 5 'portion of the cDNA includes an affinity purified label at or near the 5' end, and the cDNA is contacted with a capture medium that binds the affinity purified label to selectively obtain the 5 'portion of the cDNA. How to feature. The method of claim 12,
Sequencing the 5 'portion of the cDNA,
Forming at least one concatemer comprising a 5 'portion of the plurality of cDNAs,
Sequencing the at least one concatemer. 15. The method of claim 14,
Forming the at least one concatemer (concatemer),
Binding an adapter sequence to the 3 'end of the cDNA 5' to generate a cDNA-adapter;
Amplifying the cDNA-adapter using a 5 'oligonucleotide primer comprising a first anchor cleavage reagent recognition site and a 3' oligonucleotide primer comprising a second anchor cleavage reagent recognition site, thereby amplifying the first anchor cleavage reagent recognition site. And obtaining an amplification product including the second anchor cleavage reagent recognition site.
Cutting the amplification product with a first anchor cleavage reagent and a second anchor cleavage reagent to obtain a double cleaved amplification product;
Combining the double cleaved amplification products to form concatemers.

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