TITLE OF THE INVENTION
METHOD FOR SUBTRACTING cDNAs BY SUPPRESSING THE SYNTHESIS OF SPECIFICALLY TARGETED mRNAS
FIELD OF THE INVENTION
The present invention relates to genetic engineering and moie particularly to a method to specifically modify a mRNA population The present invention can therefore be applied to the field of cDNA synthesis and cDNA cloning More specifically, the invention relates to methods of customizing an mRNA population by selectively eliminating chosen mRNAs The invention further relates to customized cDNA libraries generated from such a customized mRNA population The invention also relates to a method of specifically targeting and cleaving and/or degrading mRNAs in order to customize the mRNA population In addition, the invention relates to a method for subtracting cDNAs by suppressing the synthesis of specifically targeted mRNAs
BACKGROUND OF THE INVENTION
The isolation and rapid mapping of complementary DNAs (cDNAs) is central to characterizing the information that is of significant biological relevance in the genome of an organism A full length cDNAallows one to predict transcription initiation start sites, translation initiation start sites, deduce certain protein characteristics based on primary ammo acid sequences, predict transcription termination sites, and visually inspect the 5' and 3' untranslated regions for elements which may be nvolved in post-transcπptional regulation of gene expression The analysis of several complete cDNAs of a given gene enables one to gather information on alternative splicing, alternative promoter usage, and alternative polyadenylation signals - all events knownto be important in gene expression regulation In addition, the comparison of genomic and cDNA sequence is essential to determine exon-intron structure and document the
occurrence of RNA editing - a post-transcriptional regulatory mechanism on which only little information is available
The cloning of mRNA into cDNA for the purposes of functional studies is a complex interrelated series of enzyme-catalyzed reactions involving the in vitro synthesis of a DNA copy of mRNA, its subsequent conversion to duplex cDNA and insertion into an appropriate prokaryotic vector The procedure usually involves the following series of steps
I) Isolation of high quality mRNA from the tissue or cell line cf interest II) Annealing of a DNA o gonucleotide, either a random hexamer, an oligo d(T) primer or specific oligo, to the mRNA When full-length cDNAs are required, oligo d(T) is utilized, since this is expected to anneal to the 3' poly (A) tail of the mRNA
III) Reverse transcπptase is then utilized to prime from the DNA primer and copy the RNA template into cDNA
IV) Second strand synthesis is performed utilizing RNAse H, DNA polymerase I, and DNA ligase
V) The ends of the cDNAs are polished, prepared for cloning, and the cDNAs are introduced into an appropriate vector Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover expressed sequences that, in turn, once mapped, greatly expedite the process involved in the identification and cloning genes linked to human diseases However, an impediment to this process is due to the different abundance of mRNAs within a given cDNA library The mRNAs of a typical cell are distributed into 3 frequency classes (1 , 2) that are presumably maintained in representation in cDNA libraries The frequency distribution of mRNAswithin these classes are as follows
I) a highly abundant class consisting of 10 - 15 mRNAs that altogether represent 10-20% of the total mRNA mass,
II) a middle abundance class consisting of 1000 - 2000 mRNAs making up 30-40% of the total mRNA and
III) a low abundance class consisting of 15,000 - 20,000 mRNAs covering -50% of the total mRNA (2) The distribution of the different mRNAs in these three classes implies that within any given cDNA library about 20% of the total number of clones in that library come from only -10-15 abundant mRNA species Thus, random picking of clones in a cDNA library, often leads to the re-isolation of cDNAs corresponding to the same abundant mRNAs thereby reducing the frequency at which new genes can be efficiently isolated
As seen above, reassociation-kinetic analysis indicates that the population of mRNAs of a typical cell can be subdivided into three fequency classes (ref 2) For gene identification endeavors, a procedure which "normalizes" the relative abundance of genes in a given library can be an important issue in order to provide more "depth" to cDNA libraries
To date, a small number of normalization protocols in which removal of abundant cDNAs from libraries is performed during their constuction have been prepared For example, current approaches involve a complex series of steps utilizing reassociation kinetics between 2 copies of the libraries or between cDNA and RNA Since hybridization of RNA or DNA is concentration dependent, rarer species anneal less rapidly and abundant species anneal more rapidly By controlling the time of annealing, one can achieve a point where abundant mRNA or cDNA are annealed to their complement while less abundant cDNAs are not (refs 3,4) The major drawback of the current methods of normalization may be 1) the complexity of the procedure, and 2) artifacts which arise during the procedure For example the length of some cDNAs may be noticeably shortened in some normalized libraries (ref 4), so clearly these approaches are not compatible with full-length (FL) cDNA library generation 3) In addition, the number of additional manipulations are time-consuming and labor intensive
There thus remains a need to develop a method or procedure, which when implemented during cDNA library construction, reduces the abundance of a target gene or genes in the cDNA library There also remains the need to devise such a method which could be used to generate cDNA libraries in which clones corresponding to high abundance mRNAs are under-represented, and hence the rate of new gene discovery in such libraries would be greatly enhanced and facilitated Furthermore there remains a need to provide a method which could be used to suppress the synthesis of any cDNA of any abundance class More broadly, there remains a needto provide a simple and versatile method enabling a customization of an mRNA population that is net strictly based on mRNA abundance
The present invention seeks to meet these and other needs The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety
SUMMARY OF THE INVENTION
The invention therefore seeks to overcome the drawbacks of the methods and procedures of the prior art
In general, the invention relates to a method which enables a versatile customization of an mRNA population In a preferred embodiment, the invention relates to a method of customizing an mRNA population which comprises a targeting of a chosen mRNA or mRNAs with at least one oligonucleotide, under conditions which favor the formation of a duplex between the targeted mRNA or mRNAs and at least one oligonucleotide The method then comprises a separation of the duplexed mRNA from non-duplexed mRNA, and/or the use of the properties of duplexed versus non-duplexed mRNA (e g RT processivity inhibition, differential substrate forcleavage/degradation) thereby yielding a customized mRNA population
In general, the invention also relates to methodsbased on the use of specific o gonucleotides to customize an mRNA population
When such a method targets high abundance mRNAs, and is coupled to a method of generating cDNA libraries, the frequency of re-isolatiσi of cDNAs can then be reduced and, as a corollary, favor the isolation of novel cDNAs In general, the invention also concerns methods to subtract targeted cDNAs by suppressing their synthesis
In one particular embodiment, the present invention also relates to a simple method for normalizing mRNAs which does not compromise the generation of a full length cDNA library therefrom The present invention also concerns methods of controlling the time of annealing in order to remove abundant mRNAs or cDNAs from libraries while maintaining less abundant mRNAs or cDNAs in the library
In another embodiment, the invention relates to a procedure of suppressing the synthesis of target transcripts during the first strand cDNA synthesis, based on the ability of a duplex specific RNAse to specifically target and degrade the RNA moiety of a RNA/DNA duplex In a preferred embodiment the RNAse is RNAse H
In one favored embodiment, the mRNA population is customized using an annealing of a complementary DNA oligonucleotide to a given RNA to obtain a RNA/DNA duplex, treating the duplex with RNAse H, thereby resulting in a specific cleavage of only the target RNA at the site of hybridization
In accordance with the present invention, there is therefore provided a method of customizing an mRNA population comprising selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation ofa duplex between the oligonucleotide and the mRNA molecule and submitting the mRNA-o gonucleotide mixture to a purification step which enables a separation
of duplexed mRNA from non-duplexed mRNA thereby yielding a customized mRNA population
There is also provided a method for suppressing the synthesε of target transcripts during first strand cDNA synthesis, comprising the steps of selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation of a duplex between the oligonucleotide and the mRNA molecule and submitting the mRNA-oligonucleotide mixture to a duplex specific RNAse which specifically targets the RNA moiety of the duplex and performing first strand cDNA synthess on the RNAse-treated duplexes of the previous step
The present invention also provides a method of subtracting cDNAs by suppressing their synthesis comprising the steps of annealing a set of complementary DNA oligonucleotides to a given mRNA sample under conditions enabling a formation of mRNA-oligonucleotide duplexes, and performing first strand cDNA synthesis on non-duplexed mRNAs
There is also provided a method of targeting a chosen mRNA or mRNA population for degradation using ohgos which are specific for the chosen mRNA or mRNA population, to generate duplexes of oligos and mRNA, submitting the duplex to a duplex-directed degradation or cleavage which leaves non-duplexed mRNA intact and purifying the non-duplexed mRNAs
The methods of the present invention can provide a certain number of advantages For example, the invention enaues the targeting of any chosen mRNA population, provided that sequence data frαn the chosen mRNA population is available The use of oligos to target an mRNA or mRNA population is a versatile and simple way to apply the customizing method of the present invention to any chosen mRNA or mRNA population Thus, the present invention is not limited to selective elimination of only abundant transcripts
The well-known principles of screening libraries with oligos can be adapted to the herein disclosed methods of customization of mRNA
populations Non-limiting examples of mRNAs which could be targeted by the method of the present invention include mRNAs encoding specific protein signatures (e g kinase domains, zinc fingers RNA binding motifs), housekeeping mRNAs, tissue specific mRNAs and mRNAsexpressed at specific times during the cell cycle Thus, the present invention provides a means of selecting any characteristic of a targeted mRNA which can be used to reduce and/or eliminate same from an mRNA population, provided that oligos whichare specific and complementary thereto are available In combination with a cDNA synthesis method, the method of customizing an mRNA population in accordance with the present invention therefore enables the customizing of the cDNA library
Having targeted a chosen mRNA and enabled the formation of a duplex, the duplexed mRNAs can then be separated from non-duplexed mRNAs in order to yield the customized mRNA population While the present invention is described in more detail using a purification through the cap structuie to selectively purify such flagged mRNAs, the invention is not so limited Indeed a number of methods can be used to separate the oligonucleotide-mRNA duplexes from non-duplexed mRNA, in order to yield the customized mRNA population Non-limiting examples of such methods include hydroxyapatite, which selectively binds double-stranded duplexes, streptavidin/biotin methods, based on the use of biotinylated ohgonucleotides, duplex-dependent mRNA cleavage and/or degradation (enzymatic or non-enzymatic directed cleavage/degradation) In addition, a number of methods known to a skilled artisan can be used to flag mRNAs so as to be able to exercise a separation of desired mRNAs or duplexes comprising same
Of course, it will be understood by the skilled artisan thata targeting of specific mRNAs or RNAs can be done by a number of methods and that the oligos exemplified herein are but a specific embodiment of targeting of specific mRNAs
One preferred utility of the method of customizing an mRNA population is in the generation of customized cDNA libraries Indeed the method of customizing of an mRNA population in accordance with the present invention can be incorporated in an "improved" method to make cDNA libraries As described above, one envisaged application of the invention is during first strand cDNA synthesis to remove abundant genes from libraries This results in a subtraction process and will facilitate the rate of gene discovery More generally, the cDNA of any gene can be specifically inhibited utilizing an approach described herein While the targeting and specific degradation of mRNA has been demonstrated with an RNAse H-dependent (duplex-dependent) degradation of the RNA template, the present invention should not be so limted Indeed, the present invention extends to any method involving enzymatic or non-enzymatic duplex-directed cleavage and/or degradation of known RNA templates Non-limiting examples include oligonucleotide metal complex conjugates containing lanthanide complexes (ref 8)
Similarly, while the specific cleavage and/or degradation of mRNAs has been demonstrated using conventional DNA o gonucleotides to hybridize to the RNA template, any modified oligonucleotide, as known in the art can be used for this purpose, provided that a cleavage of the RNA template occurs, resulting in a physical separation of the 5' and 3' ends of the targeted mRNA template
Furthermore, while in the embodiment described herein, the RNAse H and Reverse Transcπptase activities are separated in two different steps (Steps I and II in Fig 3), the instant invention should not be so limited Tte most common reverse transcπptases in use, MMLV Superscript™, does not contain RNase H activity Hence, in accordance with an embodiment based on an enzymatic-directed cleavage of the mRNA, a separate RNAseH activity needs to be added However, it is known that other reverse transcnptases (RT), such as AMV or HIV reverse transcπptase, contain both activities (reverse
transcriptase and RNAse H) in one complex. Thus, a "one step method" of mRNA cleavage/degradation and cDNA synthesis could be carried out in accordance with the present invention. In such an embodiment, DNA oligonucleotides are annealed to a given RNA template, followed by a direct incubation with the reverse transcriptases containing the RNAse H activity. In ths embodiment, the RT could degrade the complementary RNA strand.
It will also be recognized that although preferred, a cleavage/degradation of the targeted mRNA is not necessary in all conditions to enable the construction of a customized cDNA library. Indeed, an inhibition of the processivity of the RT would be sufficient to enable the generation of a customized library. In this particular embodiment, a mRhA or mRNAs would be targeted with oligonucleotides and the processivity of the RT would be inhibited by the duplex, thereby reducing or abrogating the generation of cDNAs from the targeted mRNA templates. The cDNAs can then be separated by a variety of means favoring the purification of full-length cDNAs according to physical feature of the mRNAs (cap-dependent purification, size-dependent separation, poly A- dependent purification). In a preferred embodiment, the oligonucleotides are blocked at the 3' end (as commonly known) to block the priming of the RT from the oligo. Since the blocking oligonucleotide could also be used as a primer by the RT, in one embodiment, oligonucleotides which contain a modified 3' end (which does not serve as primer for the RT) are used. Such oligonucleotides which cannot act as primers (6) were used in Fig. 4 (SEQ ID NOS:2 and 3). In one particular embodiment exemplified below, the targeted mRNAs are degraded by RNAse H and full-length mRNAs are selected and purified using oligo dT priming, reverse transcriptase and CAPture™. It will be recognized therefore that the present invention enables a "flagging" of specific mRNAs (e.g. mRNA/oligo duplexes). These duplexes can serve as targets for cleavage or degradation or can block the processivity of a DNA polymerase (e.g.
R T ) For cDNA synthesis in which a selection of full length mRNAs is preferred a method which somehow couples the 3' and 5' ends of an mRNA is preferred (e g oligo dT priming and selection of capped mRNA)
The instant invention further extends to PCR analysis of gene products generated by RT of mRNA into cDNA For example, primers flanking both sides of the duplex could be used
While mRNA is the preferred targeted nucleic acid in accordance with the methods of the present invention, it should be recognized that RNA in general can be targeted wth oligonucleotides in order to customize the RNA population It will be recognized by the skilled artisanthat the methods of the present invention can be adapted to target, for example, uncapped mRNAs (and to generate a cDNA library therefrom)
It shall also be recognized that the present invention could also be adapted to the use of RNA oligonucleotides In one embodiment, full-length cDNAs are selected via a cap structure-dependent purification step using a cap column (CAPture™) It shall be recognized that other cap-dependent purification methods can be used Non- hmiting examples thereof include anti-cap antibodies and direct oxydization of the cap structure (refs 9, 10, 1 1 ) In accordance with one embodiment of the present invention, there is provided a method of customizing an mRNA population comprising selecting an oligonucleotide which is complementary to an mRNA molecule in ai mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation of a duplex between the oligonucleotide and the mRNA molecule, and submitting the mRNA-oligonucleotide mixture to a purification step which enables a separation of duplexed mRNA from non-duplexed mRNA, thereby yielding a customized mRNA population
In another embodiment of the present invention, the mRNA oligonucleotide mixture enables a flagging of specific mRNAs, such that this
flagging enables a selective purification or targeting of chosen mRNAs from the oligonucleotide mRNA mixture
In accordance with another embodiment of the present invention, there is provided a method for suppressing the synthesis of target transcripts during first strand cDNA synthesis, comprising selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture, under conditions which enable a formation of a duplex between the oligonucleotide and the mRNA molecule submitting the mRNA-oligonucleotide mixture to a duplex-specific RNAse which specifically targets the RNA moiety of the duplex, and performing first strand cDNA synthesε on the RNAse-treated duplexes, thereby suppressing the synthesis of RNAse- cleaved duplexes
In accordance with yet another embodiment of the present invention, there is provided a method of subtracting cDNAs by suppressing their synthesis comprising the steps of annealing a set of complementary DNA oligonucleotides to a given mRNA sample under conditions enabling a formation of mRNA-oligonucleotide duplexes, and performing first strand cDNA synthesis on non-duplexed mRNAs In addition, in accordance with another embodiment of the present invention, there is provided a method of customizing an mRNA population comprising selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oiigpnucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation of a duplex betweenthe oligonucleotide and the mRNA molecule, and submitting the mRNA-oligonucleotide mixture to a duplex- dependent cleavage and/or degradation thereby yielding a customized mRNA population
In accordance with the present invention, there is also provided customized mRNA or RNA populations or customized cDNA libraries obtained using one of the methods of the present invention
In accordance with a related embodiment of the present invention, the RNA or mRNA oligonucleotide mixture instead of being submitted to the duplex-dependent cleavage and/or degradation, is submitted to a purification step enabling the separation of duplexed RNA or duplexed mRNA from non-duplexed RNA or mRNA
In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided hereinbelow
DEFINITIONS
Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission
Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art Such standard techniques can be found in reference manuals such as for example Sambrook et al (1989, Molecular Cloning - A Laboratory Manual Cold Spring Harbor Laboratories) and Ausubel et al (1994, Current Protocols in Molecular Biology, Wiley New York)
The present description refers to a number of rouinely used recombinant DNA (rDNA) technology terms Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency For certainty, it is emphasized that the present invention finds utility with nucleic acids in general Non-limiting examples of nucleic acids which can be used in
accordance with the teachings of the present invention include that from eukaryotic cells such as that of animal cells, plant cells, or microorganisms as well as that from prokaryotic cells and viruses
As used herein, the term "processivity" of a polymerase refers to its property to continue to act on a substrate instead of dissociating therefrom
As used herein, "nucleic acid molecule", refers to a polymer of nucleotides Non-limiting examples thereof include DNA (e g genomic DNA, cDNA) and RNA molecules (e g mRNA) The nucleic acid molecule can be obtained by cloning techniques or synthesized DNA can be double-stranded or single-stranded (coding strand or non-coding strand [anthozoans])
The term recombinant DNA" as known in the art refers to a DNA molecule resulting from the joining of DNA segments This is often eferred to as genetic engineering
The terminology "amplification pair" refers herein to a pair of oligonucleotides (oligos) of the present invention, whch are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below As commonly known in the art, the oligonucleotides are designed to bind to a complementary sequence under selected conditions
The nucleic acid (i e DNA or RNA) for practsing the present invention may be obtained according to well known methods Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assayformat and the particular needs and targeted genomes employed In general, the oligonucleotide probes or primers are at least 10 nucleotides in length, preferably between 12 and 24 molecules and they may be adapted to be especiallysuited to a chosen nucleic acid amplification system As commonly known in the art, the
oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybnzidation thereof with its targeted sequence (see below and in Sambrook et al 1989 Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories, Ausubel et al 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc , N Y )
The term "oligonucleotide" or "DNA" molecule or sequence refers to a molecule comprised of the deoxyπbonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form The term "oligonucleotide" or "DNA" can be found in linear DNA molecules or fragments, viruses, plasmids vectors chromosomes orsynthetically derived DNA As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction Of course, as well-known to the skilled artisan (and as exemplified hereinbelow), oligonucleotides or DNA can also be found as single-stranded molecules As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (πbo or deoxyπbonucleotides) In essence, "oligonucleotides" define at least dimers of nucleotides The size of the oligonucleotide will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill An oligonucleotide can be synthesized chemically or derived by cloning according to well known methods In accordance with preferred embodiments of the present invention, the oligos are single-stranded oligonucleotides
Probes and oligonucleotides of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and α-nucleotides and the like Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann Reports Med Chem 23 295 and Moran et al , 1987, Nucleic acid molecule Acids Res , 14 5019 Probes of the invention can be constructed of either πbonucleic acid (RNA) or deoxyπbonucleic acid (DNA), and preferably of DNA General teachings on the synthesis of oligonucleotides and
substituents and modifications thereof can be found for example in US 5,438, 131 The selection of the best suited synthesis pathway of an oligonucleotide and of the appropriate modifications, and substituentsto be used, may be selected accordingly by the person of ordinary skill to which the instant invention pertains
As used herein, a primer' defines an oligonucleotide which is capable of annealing to a target sequence thereby creating a double stranded region or duplex which can serve as an initiation point for DNA synthesis under suitable conditions 'Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al 1989, supra and Ausubel et al , 1997, supra) and are commonly known in the art In the case of a hybridization toa nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution 1 % SDS and 100 μg/ml denatured carrier DNA (e g salmon sperm DNA) The non-specifically binding probe can then be washed off the filter by several washes in 0 2 x SSC/0 1 % SDS at a temperature which is selected in view of the desired stringency room temperature (low stringency), 4ZC (moderate stringency) or 65°C (high stringency) The selected temperature is based on the melting temperature (Tm) of the DNA hybrid Of course, RNA-DNA hybrids can also be formed and detected In such cases the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill Stringent conditions will be preferably used (Sambrook et al 1989 supra)
Although the present invention is not specifically dependent on the use of a label such a label might be beneficial in certain embodiments Probes or oligonucleotides can be labeled according to numerous well known methods (Sambrook et al 1989, supra) Non-limiting examples of labels inclucte
3H,
14C,
32 P
33 P and
35 S Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, andantibodies Other detectable markers for use with oligos which can enable a detection and/or purification and/or separation in accordance with the methods of the invention, include biotin/streptavidin It will become evident to the person of ordinary skill that the choice of a particular label and/or "bait" dictates the manner in which it is bound to the oligonucleotide and the manner in which it is separated through
As commonly known, radioactive nucleotides can be incorporated into oligonucleotides of the invention by several methods Non- limiting examples thereof include kinasing the 5' ends of the probes using v- 32P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E coli in the presence of radioactive dNTP (i e uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like
Similarly, the oligos of the present invention can incorporate a radiolabel by T4 DNA polynucleotide kinase or can be synthesized with fluorescently labelled bases
Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods See generally Kwoh et al , 1990, Am Biotechnol Lab 8 14-25 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), hgase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Qβ
rep case system and NASBA (Kwoh et al 1989 Proc Natl Acad Sci U3A 86, 1173-1 177, ϋzardi et al , 1988, BioTechnologyδ 1197-1202, Malek et al , 1994, Methods Mol Biol , 28 253-260, and Sambrook et al , 1989, supra) Preferably, amplification will be carried out using PCR Polymerase chain reaction (PCR) is carried out in accordance with known techniques See, e g U S Pat Nos 4,683, 195 4,683,202, 4,800, 159, and 4 965, 188 (the disclosures of all three U S Patent are incorporated herein by reference) In general, PCR involves, a treatment ofa nucleic acid sample (e g in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess whether the sequence or sequences to be detected are present Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophoresis, or using a detectable label in accordance with known techniques, and the like For a review on PCR techniques (see PCR Protocols,
A Guide to Methods and Amplifications, Michael et al Eds, Acad Press, 1990)
As used herein, the term "gene" is well known in the art and relates to a nucleic acid sequence defining a single protein or poiypeptide. A "structural gene" defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific ammo acid sequence thereby giving rise the a specific poiypeptide or protein It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art
The term ' vector" is commonly known in the art and defines a plasmid DNA phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned Numerous types of vectors exist and are well known in the art The term "allele" defines an alternative form of a gene which occupies a given locus on a chromosome
As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to adaughter cell As well known, a mutation can be, for example, a detectable change in one or more deoxyπbonucleotide For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position Spontaneous mutatiorε and experimentally induced mutations exist The resϋt of a mutations of nucleic acid molecule is a mutant nucleic acid molecule A mutant poiypeptide can be encoded from this mutant nucleic acid molecule As used herein, the term "purified" refers to a molecule having been separated from a cellular component Thus, for example, a "purified protein" has been purified to a level not found in nature A "substantially pure" molecule is a molecule that is lacking in substantially all other cellular components In accordance with preferred embodiments of the present invention, the term "purified" is used to relate to a non-duplexed nucleic acid molecule having been separated from a duplexed nucleic acid molecule, or vice versa
The present invention also relates to a kit comprising an oligonucleotide or kit thereof of the present invention specifically designed to target (or "flag") a chosen mRNA or mRNAs For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers Such containers include small glass containers, plastic containers or strips of plastic or paper Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-cortam mated and the agents
or solutions of each container can be added in a quantitative fashion from one compartment to another In a particular embodiment such containers will include for example a container which wil accept the test sample (e g mRNA), a container which contains the oligonucleotides used in the assay and containeis which contain reagents used to separate o go-mRNA duplexes from non- duplexed mRNA In another embodiment, such containers will include, for example, a container which will accept the test sample (e g mRNA), a container which contains the oligonucleotides used in the assay (to "flag" a chosen mRNA/mRNAs) RNAse H (to degrade duplexes), containers which contain oligo d(T) and reverse transcriptase (to synthesize cDNA from the treated mRNA sample), containers which contain reagents enabling the selection of full length cDNAs
BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which
Figure 1 shows an example of the steps involved in generating cDNA libraries from mRNA Although a number of strategies can be used for cDNA library generation, of which only one is shown above, all libraries require as a first step, a primer which Reverse Transcriptase (RT) can prime from In general, representation of mRNA abundance is maintained overall during construction, such that abundant mRNA species end up-being the most abundant cDNAs in the libraries, this is an impediment to novel gene discovery since random isolation of cDNAs from libraries will often lead to re-isolation of more abundantly expressed genes
Figure 2 is a schematic diagram illustrating the specificity of cleavage of targeted RNA/DNA duplex by RNAse H
Figure 3 is a schematic outline which shows an incorporation of a subtraction of cDNAs by suppression of cDNA synthesis with an affinity
selection step to remove cDNAs which correspond to RNAse H-targeted mRNAs using affinity selection of mRNA/cDNA duplexes with 5' m7G structures (5,7) The designation of mRNAs as species l-lll relate to their classification with respect to their frequency of distribution (see text for further details) Figure 4 shows an embodiment of the subtraction by suppression of synthesis according to one aspect of the present invention using the WT1 and D75 mRNAs A) is a schematic diagram illustrating WT1 and D75 mRNAs, showing the sizes of the expected cDNA products resulting from pπmiηg with oligo d(T) For WT1 one product of 1500 nucleotides (nts) is expected whereas for D75 two products are expected, one resulting from priming from tie poly (A) tail ( 2300 nts) and one resulting from internal priming from an A-πch strech (2000 nts) The region on the WT1 targeted by the oligonucleotides is shown and is expected to generate a cDNA product of -800 nts B) shows the sequence of oligonucleotides used in the targeting reactions, it must be noted that for oligos having the nucleic acid sequences defined in SEQ ID NOS 2 and 3, the ribose of the last residue is in tie L configuration and this is denoted by the symbol "L" C) shows complementary DNA products (cDNAs) from RNA templates targeted for subtraction by RNAse H The nature of the templates ae illustrated above the panel The presence or absence of RNAse H during pretreatment is also indicated above the panel When targeting, oligonucleotides were pre-incubated with the test transcripts, this is mdicatedon the bottom of the panel The arrow indicates the cDNA product produced from 3' truncated WT1 template generated by RNAse H digestion of WT1
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention
DESCRIPTION OF THE PREFERRED EMBODIMENT
In accordance with one preferred embodiment of the present invention an approach in which suppression of the synthesis of targeted transcripts occurs during the first strand reaction (see Fig 1 for summary of first strand synthesis) is favored Such an embodiment is based on the fact that RNAse H is an enzyme which specifically targets and degrades the RNA moiety of a RNA/DNA duplex Therefore annealing of a complementary DNA oligonucleotide to a given RNA followed by treatment with RNAse H, results in a specific cleavage of only the target RNA at the site of hybridization (Fig 2) This has the effect of physically separating the RNA targets into two portions, one half containing the 3' end, and another half containing the 5' m7G cap structure As outlined in Fig 3, if cDNAs are generated from these twoportions, utilizing for example reverse transcriptase and an oligo d(T) primer, a cDNA molecule originating from the targeted mRNA (mRNA species I, in Figs 2 and 3) would extend from the poly (A) tail to the site of cleavage However, a cDNA molecule originating from mRNA species II (which is not targeted by an oligonucleotide) would extend from the poly (A) tail to the m7G cap structure at the 5' end of the molecule Following cDNA synthesis (Step II in Fig 3), there are essentially two types of RNA/DNA hybrids in the mixture - one in v\hιch the RNA moiety of the hybrid contains a m7G cap structure at the 5' end, and one in which the RNA moiety of the duplex contains a 5' phosphate (i e truncated cDNAs) The products of this reaction can then be separated using a 5' affinity capture method specific for mRNA having m7G ends (5) This affinity procedure utilizes an immobilized protein (elF-4E, a 24kDa cap-binding protein) which specifically recognizes m7G groups to isolate mRNA with m7G ends When the mixture of reaction products from Step II is subjected to 5' m7G selection using elF-4E, cDNAs whose mRNAs have not been targeted for degradation by RNAse H will be retained (since these contain a cap structure) whereas cDNAs whose mRNAs have been targeted for degradation will not be retained (since the RNA moiety of the incomplete cDNAs have a 5' phosphate group) (Fig 3) Since the
purified material from step III can now be used for second strand synthesis (see Fig 1 ), the instant procedure eliminates cDNAs corresponding to the specific RNAse H targeted transcript, from the resulting cDNA library By hybπdrang the mRNA to be used as a template for cDNA library construction with a cocktail of oligonucleotides complementary to known targets of abundant transcripts (non- hmiting examples include α-tubulin, elongation factorαl and α-actm ), the cDNA synthesis of these known abundant genes can be prematurely terminated during first strand synthesis These truncated cDNAs are then removed following 5' affinity CAPture™ with GST/melF-4E (5) (Step III in Fig 3) The suppression procedure can even be made isoform-specific by directing the blocking (e g targeting of specific motifs or differentially spliced exons) oligonucleotide to the unique region of a particular transcript
The present invention is illustrated in further detail by the following non-limiting example
EXAMPLE 1 Validation of one embodiment of the method of subtracting cDNAs using WT1 or D75 mRNAs. In the experiment presented in Fig 4, two mRNA species, WT1 and D75, were pretreated with 4 complementary DNA oligonucleotides specific to WT1 (l-IV) and one control oligonucleotide (SP6) which does not hybridize to either WT1 or D75 (Fig 4B) Following hybridization of the oligonucleotides to WT1 or D75, the RNAs were incubated with RNAse H Following RNAseH treatment, cDNA reactions were performed with MMLV Superscript II™ and oligo d(T) (Fig 4C) As shown in lane 1 , a cDNA product originating from WT1 has the expected size of 1 ,500 nts As well, cDNA synthesized from D75 mRNA generates products of 2,000 and 2,300 rts, due to priming from the poly (A) tail and mispπming from an internal A-πch tract by the oligo d(T) primer (lane 2) Pretreatment of either WT1 (lane 3) or D75 (lane 9) with RNAse H, in the absence of a targeting DNA oligonucleotide does not
change the profile of the cDNA products (this shows the duplex-dependent activity of RNAseH) Pretreatment of WT1 with 4 different complementary oligonucleotides (shown in Fig 4B) and RNAse H, produced mainly two effects - a novel cDNA product corresponding to -800 nts appears (lanes 4-7) and there is an apparent reduction in the amount of full-length WT1 cDNA generated in this experiment 4 oligonucleotides containing different 3' ends were utilized In oligo #1 (SEQ ID NO 1 ), the last nucleotide is a cytosme residue which is not complementary to the targeted WT1 sequence (containing a terminal cytosme residue instead of a guanosme residue) In oligo # 2 (SEQ ID NO 2), the nbose of the last nucleotide is in the L configuration instead of the D configuration In oligo #3 (SEQ ID NO 3), the nbose of the last nucleotide is in the Lconfiguration and the linkage of the nucleotide to the rest of the oligo ismverted to give a 3'-3' linkage Oligo # 4 (SEQ ID NO 4) is perfectly complementary to the WT1 target sequence Oligonucleotides with different 3' ends were used in order to show a) that modified nucleotides can be used in accordance with one embodiment of the present invention and, b) to determine if carryover of the targeting primer to the RT reaction would result in cDNA molecules priming not only from the oligo d(T) primer, but also from the targeting oligossince oligos #2 and #3 cannot act as primers for the reverse transcriptase (RT) enzyme
Figure 4 shows that indeed, oligonucleotides with different 3' ends can be used to target a chosen mRNA for degradation In addition, since no novel cDNA products specific to oligos #1 and 4 (which can prime cDNA synthesis) are observed in Figure 4 (compare lanes 4 and 7 to lanes 5 and 6), it appears no carryover of the targeting primer to the RT reaction enables new cDNA primings
Suppression of cDNA synthesis is specific to the WT1 oligos which target a known region of the WT1 gene since an SP6 oligonucleotide (SEQ ID NO 5), which is not complementary to any region of WT1 , did notresult in suppression of cDNA synthesis (compare lane 8 to lanes 4-7) Furthermore,
the results presented herein are specific to the WT1 transcript since targeting D75 with these oligos did not alter the cDNA profile obtained with this transcript (lanes 10-14)
Taken together, these results demonstrate tie feasibihbity of blocking RT processivity by first degrading the mRNA template In addition, it validates the duplex-dependent method of customizing an mRNA population
Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims
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