WO2004007768A1 - Procede permettant d'analyser des variations de sequences d'acides nucleiques polymeres - Google Patents

Procede permettant d'analyser des variations de sequences d'acides nucleiques polymeres Download PDF

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WO2004007768A1
WO2004007768A1 PCT/GB2003/003118 GB0303118W WO2004007768A1 WO 2004007768 A1 WO2004007768 A1 WO 2004007768A1 GB 0303118 W GB0303118 W GB 0303118W WO 2004007768 A1 WO2004007768 A1 WO 2004007768A1
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rna
nucleic acid
exons
differentially processed
sample
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PCT/GB2003/003118
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English (en)
Inventor
Maria Concepcion Jimenez
Irene Gascón ESCOBAR
Susana Coca Gallego
Juan Carlos Rodríguez CIMADEVILLA
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Genomica S.A.U.
Ruffles, Graham, Keith
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Priority to AU2003246944A priority Critical patent/AU2003246944A1/en
Priority to EP03764027A priority patent/EP1549765A1/fr
Publication of WO2004007768A1 publication Critical patent/WO2004007768A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the invention relates to a method to analyze polymeric nucleic acid sequence variations, particularly, exon-intron boundaries with a genome-wide range in any organism that utilizes RNA splicing mechanisms.
  • the invention further relates to methods for identifying nucleic acid sequences comprising qualitative differences between RNAs derived from two distinct conditions being compared.
  • the nucleic acid sequences so identified are useful as, and for the development of, screening tools for identifying molecules of therapeutic interest.
  • the invention can be used or applied in biotechnology and medicine.
  • AS alternative splicing
  • mRNA messenger RNA
  • AS causes variations in the expressed protein.
  • Alternative splicing has been implicated in many processes, including sex determination, apoptosis, and acoustic tuning in the ear. Its functional implications can be simple, generating a single alternative form, or can produce remarkable diversity.
  • the DATAS method however has several important drawbacks: 1) This method is inclined to isolate big exonic fragments (bigger than 100 bp) in preference to the smaller ones. Since a great number of exons (around 50% in vertebrates) are smaller than 100 bp, this bias may lead to a huge under-representation of the entire exon population. 2) The method reveals the sequence of variant exons through random priming of RNA fragments; because of that, cDNA individual clones obtained with the DATAS technology, at best, only represent fragmentary information about the 3' exon boundaries. This alone represents a major problem in drawing information from exon libraries.
  • the present invention comprises a method to investigate the exon-intron boundaries with a genome-wide range in any organism that utilizes RNA splicing mechanisms.
  • SAVE is ideally fitted to the comparative study of two or more sources of RNA that harbour differences in splicing, transcription initiation, poly-adenylation or RNA editing. It represents a clear advantage when compared to currently used methods to study alternative transcripts of the same gene.
  • SAVE affords the isolation, for the first time, of a complete range of exon lengths (specially those having less than 100 bp, for example, between 5 and 50 bp, usually between 10 and 25 bp, typically between 15 and 20 bp).
  • SAVE sequences are also indicative of relative abundance of exon expression.
  • the SAVE methods of the invention can be used in methods for analysing or identifying RNA transcripts, alternative exon expression, differential exon expression, differential exon processing, and/or variant exons.
  • SAVE has additional economic advantages since it provides information of multiple exons in a single sequencing event.
  • RNA for example total RNA or mRNA'from the sources to be compared.
  • sources can be, among others, blood, tissue culture, tissues or whole organisms.
  • a typical comparison can be established between a normal tissue and its diseased counterpart (i.e. normal lung versus lung cancer) or between tissue in different developmental states.
  • Typical substrates of the SAVE methods are RNA fragments obtained by RNAse digestion after cross-hybridisation between RNAs and cDNAs derived from distinct conditions, e.g. Rnase H digestion derived fragments after RNA-cDNA hybridisation.
  • RNAse digests of RNA/cDNA hybrids from two different sources have been shown to contain differentially spliced RNA. Since RNAse digests contain reactive 5' (phosphate) and 3' (hydroxyl) groups, they are perfect substrates for RNA ligation.
  • a central step in SAVE methods is the chain ligation of pools of RNA fragments from the RNAse, e.g. Rnase H, digestion of RNA/cDNA hybrids.
  • these fragments can be ligated in the presence of a hinge RNA linker that is complementary to the consensus sequence for an endonuclease, such as a low frequency endonuclease (i.e., Not I).
  • a hinge RNA linker that is complementary to the consensus sequence for an endonuclease, such as a low frequency endonuclease (i.e., Not I).
  • the latter version of intercalated chain can be later used for the isolation, purification and sequencing of individual exonic fragments (see below). Either type of chain are later used in a second ligation reaction with 5' and 3' RNA linkers. Addition of a linkers is essential in order to amplify the pool of strings, e.g. by PCR.
  • the amplification reaction product can be sequenced or, prior to sequencing, the amplified exon strings can be cloned into a suitable cloning vector.
  • this invention also provides a method for isolating and purifying individual exons from clonal strings.
  • strings intercalated with restriction hinges sites
  • sites can be digested with the corresponding specific restriction enzyme and the different fragments, each with an individual exon or exons can be isolated, e.g. by agarose electrophoresis and gel purification.
  • Individual fragments can be typically spotted on solid surfaces (i.e. micro-arrays for differential display studies).
  • the invention relates to a method to experimentally analyse boundaries within polymeric deoxy ribonucleic acid (DNA) or ribonucleic acid (RNA) molecules based on a previously mentioned SAVE method.
  • the invention in another aspect, relates to a method for identifying nucleic acids comprising sequences corresponding to portions of genes that are differentially processed in two biological samples containing nucleic acids, said method being based on the previously defined SAVE method.
  • a nucleic acid consisting of or comprising a sequence corresponding to a portion of a gene that is differentially processed between two biological samples, consisting of or containing nucleic acid obtained or obtainable according to said method and the applications thereof constitute further aspects of the instant invention.
  • a peptide encoded by said nucleic acid sequence, applications thereof, antibodies against said peptide and the applications thereof constitute further aspects of the instant invention.
  • Another aspect of the instant invention relates to a method for identifying nucleic acids or nucleic acid domains distinct to a tumour state based on the previously defined SAVE method.
  • Another aspect of the instant invention relates to a method of determining or assessing the therapeutic potential of a test compound with respect to a biological sample based on the previously defined SAVE method.
  • Still another aspect of the instant invention relates to a method of determining or assessing the responsiveness of a patient to a test or treatment based on the previously defined SAVE method.
  • Figure 1 depicts the predicted formation of a single stranded RNA loop when a messenger RNA that contains an alternatively spliced exon hybridises with a cDNA missing such an exon. It shows the predicted outcome of adding Rnase H to RNA/cDNA hybrids. Rnase H digestion of RNA/cDNA hybrids harbouring alternate exons leads to the formation of RNA fragments chemically active at both ends.
  • Figure 2 shows the hinging of RNA strings by intercalating Not I double stranded nucleotide hinges as in the second embodiment of the invention. Not I RNA hinges are ligated to Rnase H digests in order to generate hinged strings.
  • Figure 3 shows the shielding of both hinged or un-hinged RNA strings as in the third embodiment of the invention. Mono-modified linkers (either 3 'OH or 5'P modified RNA linkers) are ligated to the 5' and 3' ends respectively of RNA strings.
  • Figure 4 depicts a technique to convert shielded RNA strings into their complementary DNA (cDNA).
  • the technique dubbed RT-SS or RT-PCR, uses DNA primers homologous or complementary to the 3'OH or 5'P modified RNA linkers, respectively.
  • Figure 5 shows the restriction digestion of the complementary DNA of shielded RNA strings with the appropriate restriction enzyme (for example, EcoRI); these digests are cloned in an equally EcoRI digested suitable vector.
  • EcoRI restriction enzyme
  • Figure 6 shows the restriction digestion of the cloned complementary DNA of shielded RNA strings with the appropriate hinge restriction enzyme (i.e. Not I).
  • Figure 7 shows the biotinylation of the complementary DNA of shielded RNA strings.
  • the Klenow fragment of DNA polymerase is used in the presence of biotinylated dNTPs to fill in restriction nicks.
  • Figure 8 shows one application of the invention consisting in using biotinylated fragments from Figure 7 to spot an streptavidin conjugated surface.
  • Figure 9 shows a typical Blast search of sequences from a single gene fragment.
  • Figure 10 shows a Blast search using an entry sequence from an exon string clone.
  • the string is composed of three different gene fragments.
  • the SAVE method is based on the isolation of full length RNA exonic fragments of a complete spectrum of different lengths, followed by the ligation in chain of such fragments, and the cloning of the resulting libraries. Said method may be applied to the study of differentially processed exons.
  • differentially processed exons refers to RNA fragments that have been subjected to different splicing, transcription initiation, polyadenylation or RNA editing.
  • the invention concerns a method to experimentally analyse boundaries within polymeric deoxy ribonucleic acid (DNA) or ribonucleic acid (RNA) molecules, dubbed String Analysis of (complete) Variant Exons (SAVE).
  • DNA polymeric deoxy ribonucleic acid
  • RNA ribonucleic acid
  • the SAVE method comprises: a) Ligation of RNA fragments with free 5'P and 3'OH groups to form a string, wherein said RNA fragments with free 5'P and 3'OH groups comprise differentially processed exons; b) Ligation of 5' and 3' RNA linkers for subsequent amplification; c) Reverse transcription of the product of reaction (b) ; d) Amplification of the product of reaction (c), preferably by PCR; and optionally, e) Cloning of the product of amplification reaction (d) into a vector.
  • the SAVE method may further comprise (f) sequencing of the amplification product or, after cloning the amplification products into a vector, preferably a recombinant vector, sequencing of the individual clones, and, optionally, (g) comparing the sequences obtained with known sequences, e.g. in gene databases, in order to identify, characterize and/or map each individual exon included in each sequenced string.
  • the SAVE method may further comprise (f ) digesting individual exonic strings with an appropriate restriction enzyme that releases individual exons, and, optionally, (g') purifying recombinant exons from (f ).
  • RNA fragments having free 5'P and 3'OH groups corresponding to differentially processed exons useful for forming a string, consists of differential hybridisation followed by RNAse digestion, e.g. by Rnase H digestion.
  • Differential hybridisation between two situations to be compared consists of cross hybridisation, i.e. mixing RNA from . one situation with cDNA from the other one situation to obtain RNA/cDNA hybrids.
  • the different situations may be normal and abnormal samples of a particular tissue e.g. healthy and diseased tissue e.g. normal tissue and tissue derived from tumours of the tissue, or may be samples of a tissue in different developmental states.
  • RNA/cDNA hybrids can be isolated and treated with an enzyme, for example Rnase H, that eliminates all RNA segments that are complementary to the cDNA. This reaction yields free segments of single stranded RNA (ss RNA) that correspond to differentially processed exons.
  • Rnase H enzyme that eliminates all RNA segments that are complementary to the cDNA.
  • This reaction yields free segments of single stranded RNA (ss RNA) that correspond to differentially processed exons.
  • products of RNAse digestion e.g. Rnase H digestion, contain free reactive 5'P and 3'OH ends
  • T4 RNA ligase enzyme such as the T4 RNA ligase enzyme.
  • T4 RNA ligase welds, i.e. joins, ss RNA fragments through a phosphodiester bond.
  • the above mentioned ligation reaction constitutes a key step in the SAVE technology. It permits generation of random strings of RNA fragments that are representative of the relative abundance of differential exons. In order to rescue and analyse those fragments, we have developed several strategies.
  • RNA linker comprising, or consisting of, a nucleotide sequence which is complementary to the recognition site of a DNA restriction enzyme, preferably, comprising, or consisting of, a nucleotide sequence which is complementary to the recognition site of a low frequency DNA restriction enzyme, and (ii) having free 5'P and 3'OH groups.
  • any DNA restriction enzyme can be used, preferably a low frequency DNA restriction enzyme; nevertheless, in a particular embodiment, said low frequency DNA restriction enzyme is Not I.
  • said hinge RNA linker is not modified in any of its ends 5' and 3' (i.e., it maintains, 5'P and 3'OH ends), said linker is a substrate for a suitable ligase, such as the T4 RNA ligase.
  • a suitable ligase such as the T4 RNA ligase.
  • the RNA fragments having free 5'P and 3'OH, such as Rnase H digests can therefore be ligated to said hinge RNA linkers at one or both ends of the RNA fragment.
  • Strings of hinged RNA fragments can be converted into their complementary DNA (cDNA) and eventually digested into the individual fragments by using the appropriate restriction enzyme (e.g., Not I in the example below).
  • the appropriate restriction enzyme e.g., Not I in the example below.
  • ligation of RNA fragments having free 5'P and 3'OH groups to form a string [step (a)] is accomplished in the absence of said hinge RNA linkers.
  • RNA linkers can be used in conjunction with an appropriate ligase, such as the T4 RNA ligase and pools of RNA strings to create shielded RNA strings.
  • RNA linker comprises or consists of, a nucleotide sequence which is complementary to the recognition site of a DNA restriction enzyme
  • said RNA linkers comprise or consists of, a nucleotide sequence which is complementary to the recognition site of a low frequency DNA restriction enzyme, thereby enabling the convenient cloning of shielded strings cDNAs into vectors containing the same DNA restriction enzyme site, and (ii) is modified only on one end (i.e., it is either 3'OH or 5'P modified).
  • Virtually any DNA restriction enzyme can be used, preferably a low frequency DNA restriction enzyme; nevertheless, in a particular embodiment, said low frequency DNA restriction enzyme is EcoRI.
  • RNA fragments having free 5'P and 3'OH groups are ligated by means of the simultaneous use of said hinge RNA linkers and said RNA linkers, in conjunction with an appropriate ligase, in order to obtain a pool of shielded RNA strings.
  • Shielded RNA strings are converted into their cDNAs by conventional techniques, for example, reverse transcription (RT) in the presence of compatible primers.
  • said primers are oligonucleotides homologous or complementary to the modified 3'OH or 5'P RNA linkers, respectively.
  • cDNAs thus obtained may then be amplified by conventional techniques, e.g. PCR, using the appropriate primers, as illustrated in Figure 4 and in the example included below.
  • Another approach involves a reverse transcription reaction performed upon the shielded RNA strings by using random primers, which will randomly initiate transcription along RNAs.
  • cDNAs thus obtained are then amplified according to conventional molecular biology techniques, for example by PCR using appropriate primers.
  • Amplification products can be subsequently cloned into vectors by conventional techniques.
  • Said vectors can be recombinant vectors such as plasmids, cosmids, phages, YAC, HAC, etc.
  • the nucleic acids inserted therein may be stored as such, or introduced into microorganisms compatible with the vector being used, for replication and/or stored in the form of cultures.
  • plasmids with a multicloning site including a unique DNA restriction enzyme site, for example EcoRI are digested.
  • the cDNA of shielded RNA strings is digested using the same restriction enzyme used to provide insert. Digested vector and insert are subsequently ligated and used to transform a compatible host, for example, a bacteria by conventional methods. . -
  • the SAVE method comprises: a) Ligation of RNA fragments with free 5'P and 3'OH groups, wherein said RNA fragments with free 5'P and 3'OH groups comprise a differentially processed exon or exons, in order to form a string; b) Ligation of RNA fragments as in (a) to hinge RNA linkers intended to intercalate between said RNA fragments; c) Ligation of 5' and 3 ' RNA linkers simultaneously or after the reaction in (a); d) Reverse transcription of the product of reaction (c) using a DNA oligonucleotide primer complementary to the 3 ' RNA linker used in (c); e) Synthesis of a second strand using the product of reaction (d) as a template or, directly; f) Amplification of the product of reaction (e), preferably by PCR; and g) Cloning of the product of reaction (f) into a vector.
  • this second embodiment of the SAVE method may comprise (h) sequencing of the individual clones, and, optionally, (i) comparing the sequences obtained with known sequences e.g. gene databases in order to identify, characterize and/or map each individual exon included in each sequenced string, or alternatively, (h') digesting individual exonic strings with an appropriate restriction enzyme that releases individual exons, and, optionally, (i') purifying recombinant exons from (h').
  • clones expressing cDNAs of individual strings may be, if desired, isolated, plasmid purified and used, for examples, as follows:
  • a) Individual clones can be submitted to a DNA sequencing reaction: sequences obtained with this approach are compared with known sequences e.g. available public gene databases, in order to identify, characterize, and map each individual exon included in each sequenced string; b) Individual clones can be subjected to a restriction digestion using the specific hinge DNA restriction endonuclease (Not I in the example below): this digestion directs the release of individual alternate exonic fragments that can be purified, e.g. by gel purification.
  • Purified alternate exons can be used as probes in multiple applications. For example, they can be individually spotted, at the adequate concentrations, on solid surfaces such as in 384 and 96 well plate formats as well as microarrays and ultimately used in gene profiling studies.
  • the purified product so obtained constitutes a further aspect of the present invention.
  • Probes, oligonucleotides or sequence information obtained using the SAVE methods can be applied in the development of in vitro diagnostic nucleic acid-based tests, such as DNA-based tests. These tests can be based on hybridisation techniques (e.g. on plates, microarrays or other surfaces, or in solution), or on DNA polymerisation techniques (generally speaking, PCR or electrophoretic fragment length analysis, as in STR analysis).
  • hybridisation techniques e.g. on plates, microarrays or other surfaces, or in solution
  • DNA polymerisation techniques generally speaking, PCR or electrophoretic fragment length analysis, as in STR analysis.
  • exonic sequences obtained with the SAVE methods can be analysed for their codon code and thus the amino acid sequence. Alternate exons often code for individual protein domains.
  • a protein domain is a segment of a protein that typically can be separated, synthesized and purified in a functional form.
  • protein domains encoded in some of the SAVE exons can be, for instance synthesized (either in vitro as in the in vitro synthesis of peptides, or in vivo as in the in vivo expression in a microorganism or a cell of a peptide), purified and used as tools in functional studies, as therapeutic targets, as drugs, or as immunogens to raise specific antibodies against the particular domain encoded by the alternate exon.
  • the differentially processed exon nucleic acid sequence identified according to the instant invention may, if desired, be purified.
  • the purified product so obtained constitutes a further aspect of the present invention.
  • Said product, if desired, can be fixed onto a solid surface, such as an array or multi-well plate, for use as a probe for gene expression studies.
  • the peptide encoded by said nucleic acid sequence constitutes a further aspect of the instant invention.
  • Said peptide can be used in generating ligands, for example, peptides and the like, or antibodies against said peptide.
  • the ligands e.g., peptides
  • Antibodies can also be obtained by conventional methods, for example, by immunizing a suitable research animal with said peptides and collecting sera (polyclonal antibodies) or by means of the hybridoma technology (monoclonal antibodies). Said ligands and antibodies constitute further aspects of the present invention. Additionally, said peptide can also be used in a test for therapeutic target discovery and validation. The antibodies so obtained can be used as therapeutics or for in vitro diagnostic tests.
  • the instant invention concerns methods for identifying differences in gene processing occurring between two distinct conditions, for example, between two distinct physiological or pathological conditions.
  • the methods provided by the instant invention can be used, for example, to identify novel targets or therapeutic products, to devise genetic research and/or diagnostic tools, to construct nucleic acid libraries, and to develop methods for determining the toxicological profile or potency of a compound.
  • the instant invention provides a method for identifying nucleic acid regions that are differentially processed in two distinct conditions (test and standard conditions) based on the SAVE methods which comprise isolating RNA fragments consisting of or comprising a differentially processed exon or exons derived from the test condition followed by the ligation in chain of such RNA fragments, conversion into cDNAs, optionally cloning said cDNAs and identifying nucleic acids which correspond to differently processed exons between said two distinct conditions.
  • the isolation of said RNA fragments corresponding to differentially processed exons derived from the test condition is carried out by hybridizing RNAs derived from the test condition with cDNAs originating from the standard condition and RNAse digestion.
  • a further 'aspect of the invention concerns " a method for identifying nucleic acids comprising sequences corresponding to portions of genes that are differentially processed between two biological samples containing nucleic acids.
  • RNA/cDNA hybrids a) Hybridization of a plurality of different RNAs derived from a first sample with a plurality of different cDNAs derived from a second sample, to render a plurality of RNA/cDNA hybrids; b) Obtaning, from said RNA/cDNA hybrids of (a), RNA fragments with free 5'P and 3'OH groups, wherein said RNA fragments with free 5'P and 3'OH groups comprise or consist of differentially processed exons; c) Subjecting said fragments with free 5'P and 3'OH groups obtained in (b) to a SAVE method; and d) Identifying nucleic acids comprising sequences corresponding to portions of genes that are differentially processed between said two biological samples containing nucleic acids.
  • RNA fragments with free 5'P and 3'OH groups wherein said RNA fragments with free 5'P and 3 'OH groups are differently processed exons, for example under distinct conditions, such as physiological or pathological conditions
  • the substrate for SAVE methods can be obtained by any suitable method.
  • said RNA fragments corresponding to a differentially processed exon or exons, for example, derived from the test condition are obtained by hybridizing RNAs derived from the test condition with cDNAs originating from the standard condition. Said hybridization procedure allows one to demonstrate in a convenient manner unpaired regions, i.e.
  • regions present in RNAs in one condition and not in RNAs from another condition essentially correspond to a differentially processed exon or exons, typical of a given condition, and thus form genetic elements or markers of particular use in different fields, such as therapeutics, diagnostics and research.
  • the invention provides a method that allows one to generate a nucleic acid population characteristic of differentially processed events that occur in a test condition as compared to the standard (reference) condition.
  • Said population can be used for the cloning and characterization of nucleic acids, including their use in diagnostics, screening, therapeutics and antibody production or synthesis of whole proteins or protein fragments.
  • This population can also be used to generate libraries that may be used in different application fields (medicine, pharmacogenomics, genopharmacology, genotoxicity, etc.).
  • the biological material can be any cell (including cell lines), organ, tissue, sample, biopsy material, organism, etc. containing nucleic acids.
  • samples derived from organisms such as research animals (e.g., worms, flies, cebra fishes, rats, mouse, etc.), marine species, human beings, plants, etc.
  • Relevant materials include samples from cell types in different physiological or pathological conditions, for example, samples from tumor cells and samples from non-tumor cells of the same subject, samples from cells treated by a test compound and samples from untreated cells, etc.
  • RNAs messenger RNAs
  • RNAs can be prepared by any molecular biology methods, familiar to those skilled in the art. Such methods generally comprise cell, tissue or sample lysis and RNA recovery by means of extraction procedures. This can be done in particular by treatment with chaotropic agents such as guanidinium thiocyanate followed by RNA extraction with solvents (e.g., phenol, chloroform for instance). These methods may be readily implemented using commercially available kits.
  • chaotropic agents such as guanidinium thiocyanate
  • solvents e.g., phenol, chloroform for instance
  • solvents e.g., phenol, chloroform for instance
  • RNA preparations can be carried out using commercially available kits. RNAs can also be obtained directly from libraries or other samples prepared beforehand and/or available from collections, stored in suitable conditions. RNA preparations must contain RNA in a sufficient amount so as to be able to carry out a method of the invention, preferably should comprise at least 0,1 ⁇ g of RNA.
  • the cDNA used in the working of the method provided by the instant invention may be obtained by reverse transcription according to conventional molecular biology techniques, by using a reverse transcriptase and a primer under the appropriate operating conditions.
  • Virtually any reverse transcriptase described in the literature and/or commercially available can be used.
  • the primer(s) used for reverse transcription may be of various types. It might be, in particular, a random oligonucleotide comprising preferably from 4 to 10 nucleotides, advantageously 6 nucleotides. Use of this type of random primer has been described in the literature and allows random initiation of reverse transcription at different sites within the RNA molecules. This technique is especially employed for reverse transcribing total RNA (i.e. comprising mRNA, tRNA and rRNA).
  • oligo dT-oligonucleotide As primer, which allows initiation of reverse transcription starting from polyA tails specific to messenger RNAs.
  • the oligo dT-oligonucleotide may comprise from 4 to 20-mers, conveniently about 15- ers.
  • a labelled primer for reverse transcription This allows recognition and/or selection and/or subsequent sorting of RNA by separation from cDNA. This may also allow one to isolate RNA/DNA heteroduplex structures. Labelling of the primer may be carried out by any ligand- receptor based system, i.e. providing affinity mediated separation of molecules bearing the primer.
  • cDNA single stranded complementary DNA
  • reverse transcription is accomplished such that double stranded cDNAs are prepared.
  • This result is achieved by generating, following transcription of the first cDNA strand, the second strand using conventional molecular biology procedures involving enzymes capable of modifying DNA such as DNA polymerase I and T4 phage-derived DNA polymerase.
  • the method comprises the hybridization of a plurality of • - different RNAs derived from a first sample with a plurality of different cDNAs derived from a second sample, to render a plurality of RNA-cDNA hybrids.
  • Each sample may be representative of a particular condition or situation.
  • the method comprises a first hybridization and a second hybridization, conducted in parallel, between RNAs derived from a standard condition and cDNAs derived from the test condition. This variant has great advantage since it allows one to generate two nucleic acid populations, one representing the characteristics of the test condition with respect to the standard condition, while the other representing the characteristics of the standard condition in relation to the test condition.
  • RNAs and cDNAs derived from distinct conditions may be performed by any conventional technique, such as, for example, in a liquid phase, in phenol emulsion, having one of the partners fixed to a support (cDNA is conveniently immobilized), etc. More detailed information concerning this cross hybridization step can be found in US 6,251,590 which is incorporated herein by reference.
  • compositions comprising cDNA/RNA hybrids, representing the qualitative properties of each condition being tested.
  • nucleic acids essentially corresponding to differentially processed exons, specific to each condition, can be identified.
  • the RNA fragments with free 5'P and 3'OH groups can be obtained from said RNA/cDNA hybrids by any conventional method.
  • said RNA fragments with free 5'P and 3'OH groups are obtained by enzymatic digestion with an appropriate enzyme, for example, by Rnase H . digestion of said RNA/cDNA hybrids.
  • Step (c) of the above defined methods may be carried out by any particular embodiment of the SAVE method.
  • step (c) comprises:
  • RNA fragments with free 5'P and 3'OH groups comprise a differentially processed exon or exons; 2) Ligation of 5' and 3' RNA linkers for subsequent amplification, preferably by PCR; 3) Reverse transcription of the product of reaction (2);
  • step (c) comprises:
  • RNA fragments 1) Ligation of said RNA fragments with free 5'P and 3'OH groups obtained in step (b) in order to form a string; 2) Ligation of RNA fragments as in (1) to hinge RNA linkers intended to intercalate between said RNA fragments;
  • the regions characterizing differential processed exons may be identified by any technique known in the art, for example, by sequencing, restriction enzyme digestion, etc.
  • the invention provides a method for identifying nucleic acids representative of a particular condition, for example, a physiological or pathological condition, hi addition, the nucleic acids identified represent the qualitative characteristics of a given condition in that said nucleic acids are generally involved to a great extent in the condition being observed.
  • the method provided by the instant invention affords direct exploration of genetic elements and protein products thereof, playing a functional role in the development of a particular condition. Additionally, such a method allows the identification of nucleic acids characteristic of a particular physiological or pathological condition in relation to a standard (reference) physiological condition, that are to be cloned or used for further applications.
  • the instant invention provides a method for identifying within a biological sample differentially processed nucleic acid regions occurring between two distinct physiological or pathological conditions.
  • nucleic acids that have been identified and/or cloned by the methods of the invention, as well as related products such as peptides, and the applications thereof.
  • the nucleic acids that can be identified and/or cloned by the methods of the invention may be RNAs or cDNAs and may be single or double stranded.
  • the invention concerns a nucleic acid composition, essentially comprising nucleic acids corresponding to differentially processed exons which are distinctive of, i.e. can be used to distinguish between, two distinct conditions. More particularly, these nucleic acids correspond to differentially processed exons identified in a biological test sample and not present in a comparable biological sample under a standard (reference) condition.
  • the invention is equally concerned with the use of the nucleic acids thus cloned as therapeutic or diagnostic products, or as screening tools to identify active molecules.
  • the invention relates to a nucleic acid comprising a sequence corresponding to a portion of a gene that is differentially processed between two biological samples containing nucleic acids obtained according to the above mentioned method for identifying nucleic acids comprising sequences corresponding to portions of genes that are differentially processed between two biological samples ⁇ containing nucleic acids.
  • Said nucleic acid if desired may be fixed onto a solid surface, such as an array or multi-well plate, for use as as a probe for gene expression studies.
  • the invention relates to a nucleic acid sequence obtained according to the above mentioned method for identifying nucleic acids comprising sequences corresponding to portions of genes that are differentially processed between two biological samples containing nucleic acids, wherein said sequence corresponds to a portion of a gene that is differentially processed between two biological samples containing nucleic acids.
  • Said nucleic acid sequence can be used, for example, in diagnostic or prognostic tests, or in a drug evaluation tests in humans or experimental animals.
  • nucleic acids and nucleic acid sequences identified according to the instant invention can be purified.
  • the purified product so obtained constitutes a further aspect of the present invention.
  • Said product, if desired, can be fixed onto a solid surface, such as an array or multi-well plate, for use as a probe for gene expression studies.
  • the peptide encoded by said nucleic acid or, preferably, by said nucleic acid sequence constitutes a further aspect of the instant invention.
  • Said peptide can be used in generating ligands, for example, peptides and the like, or antibodies, against said peptide.
  • the ligands e.g., peptides
  • Antibodies can also be obtained by conventional methods, for example, by immunizing a suitable research animal with said peptides and collecting sera (polyclonal antibodies) or by means of the hybridoma technology (monoclonal antibodies). Said ligands and antibodies constitute further aspects of the present invention. Additionally, said peptide can also be used in a test for therapeutic target discovery and validation. The antibodies so obtained can be used as therapeutics or for in vitro diagnostic tests.
  • the instant invention is directed to the preparation of nucleic acid libraries, to the nucleic acids and libraries thus prepared, as well as to the uses of such materials in all fields, for example, biology, biotechnology, medicine, etc.
  • the invention further concerns a method for preparing nucleic acid libraries representative of a given condition of a corresponding biological sample.
  • This method comprises cloning nucleic acids representative of differentially processed exons of a given condition but not present in another condition, to generate libraries specific to qualitative differences existing between the two conditions being investigated.
  • These libraries are constituted by cDNA inserted in plasmid or phage vectors, and can be overlaid on nitrocellulose filters or any other support known to those skilled in the art, such as chips or biochips.
  • the choice of initial RNAs will partly determine the particulars of the resulting libraries as mentioned in US 6,251,590.
  • the nucleic acid library is constructed by cross hybridizing RNA arising from the physiological or pathological condition being tested with cDNA derived from the standard physiological condition and selecting nucleic acids of interest by proceeding as previously described.
  • the library clones may be hybridized with the cDNA populations occurring in both conditions being investigated. The clones effectively hybridizing with both populations would be considered as non specific and optionally discarded. The clones which hybridize with only one out of two populations are considered as specific and could be selected to construct refined or enriched libraries.
  • the instant invention further concerns any nucleic acid library comprising nucleic acids specific of differentially processed exons typical of a physiological or pathological condition.
  • These libraries conveniently comprise cDNAs, generally of double stranded nature, corresponding to RNA regions specific of differentially processed exons.
  • Such libraries may be comprised of nucleic acids, generally incorporarated within a cloning vector, or of cell cultures containing said nucleic acids.
  • nucleic acid libraries provided by the instant invention can be generated by conventional methods, for example, by spreading, on a solid medium, of a cell culture transformed by the cloned nucleic acids, or alternatively, by transfer the nucleic acids onto biochips or any other suitable device. Additional information concerning the generation of nucleic acid libraries can be found in US 6,251,590 which is incorporated herein as reference.
  • a support material membrane, filter, biochip, chip, etc
  • a support material comprising a nucleic acid library as defined above constitutes a further aspect of the instant invention.
  • the instant invention is concerned with the use of the methods, nucleic acids or libraries previously described for identifying molecules of therapeutic or diagnostic value, more specifically with the use of said methods, nucleic acids or libraries for identifying proteins or protein domains that are altered in such a pathology.
  • One of the major strengths of these techniques is, indeed, the identification, within a messenger, and consequently within the corresponding protein, of the functional domains which are affected in a particular disorder. This makes it possible to assess the importance of a given domain in the development and persistence of a pathological state.
  • a direct advantage of restricting to a given protein domain the impact of a pathological disorder is in that the latter can be viewed as a relevant target for screening small molecules for therapeutic purposes.
  • This information further constitute a key for designing therapeutically active compounds that may be delivered e.g. by gene therapy; such compounds can namely be antibodies or ligands (e.g., peptides) against domains identified by techniques herein described, for example, single chain-antibodies derived from neutralizing antibodies against said domains.
  • therapeutically active compounds that may be delivered e.g. by gene therapy; such compounds can namely be antibodies or ligands (e.g., peptides) against domains identified by techniques herein described, for example, single chain-antibodies derived from neutralizing antibodies against said domains.
  • the methods according the invention provide molecules which may be coding sequences derived from alternative exons or which may correspond to non coding sequences born by introns differentially processed between two physiological or pathological conditions. From the foregoing data, different information can be obtained.
  • the differentially processed exons may correspond to non coding regions located 5' or 3' of the coding sequence or to introns occurring between two coding exons.
  • the differential splicings could reflect a modification of the messenger stability or translatability.
  • a search for these phenomena should be conducted based on such information and might qualify the corresponding protein as a candidate target in view of its accumulation or disappearance.
  • Retention of an intron in a coding sequence often results in the truncation of the native protein by introducing a stop codon within the reading frame. Before such a stop codon is read, there generally occurs translation of a number of additional codons whereby a specific sequence is appended to the translated portion which behaves as a protein marker of alternative splicing.
  • the invention is directed to a method for identifying nucleic acids or nucleic acid domains involved in a pathological condition based on a SAVE method which comprises:
  • identification of differentially processed exons which are specific to the pathological condition in relation to the healthy condition comprises obtaining RNA fragments corresponding to said differentially processed exons from said RNA/cDNA hybrids and subjecting said RNA fragments to a SAVE method.
  • SAVE methods may comprise ligating in chain RNA fragments having free 5'P and 3'OH ends to obtain RNA strings, optionally in the presence of hinge RNA linkers, ligating RNA linkers to render shielded RNA strings, converting said shielded RNA strings into cDNAs and optionally cloning said cDNAs in vectors.
  • Any of the particular embodiments of the SAVE method can be used in operating this method.
  • the first embodiment of the SAVE method can be used whereas in another particular case, the second embodiment of the SAVE method can be used.
  • the pathological condition is a tumor
  • the invention provides a method for identifying nucleic acids or nucleic acid domains distinct to a tumor state, comprising:
  • the identification of differentially processed exons which are specific to the tumor state in relation to the healthy condition comprises obtaining RNA fragments corresponding to said differentially processed exons from said RNA/cDNA hybrids and subjecting said RNA fragments to a SAVE method.
  • the invention is directed to a method for identifying proteins or protein domains involved in a pathological condition based on the SAVE methods which comprises:
  • step (c) identifying protein or protein domains corresponding to one or several splicing forms identified in step (b),
  • identification of differentially processed exons which are specific to the pathological condition in relation to the healthy condition comprises obtaining RNA fragments corresponding to said differentially processed exons from said RNA/cDNA hybrids and subjecting said RNA fragments to a SAVE method.
  • any of the particular embodiments of the SAVE methods can be used in operating this method.
  • the first embodiment of the SAVE method can be used whereas in another particular case, the second embodiment of the SAVE method can be used.
  • the protein(s) or protein domains may be isolated, sequenced, and used in therapeutic or diagnostic applications, including antibody production.
  • the instant invention further relates to the exploitation of the resulting information in research, development of screening assays for chemical compounds, for example, low molecular weight chemical compounds, and development of gene therapy or diagnostic tools.
  • the invention is concerned with the use of the methods, nucleic acids or libraries described above in dete ⁇ nining or assessing the therapeutic potential of a test compound with respect to a biological sample.
  • two standard libraries are established from a control cell culture or organ and counterparts thereof simulating a pathological model. The therapeutic efficiency of a given product may then be evaluated by monitoring its potential to antagonize gene expression, variations of which are specific of the pathological model being considered.
  • the invention is further directed to a method for determining or assessing the therapeutic efficiency of a test compound upon a given biological sample comprising hybridizing nucleic acid libraries provided by the instant invention typical of said biological sample in a healthy state and in a disease state (or at different developmental stages), with a preparation of nucleic acids originating from the biological sample treated by said test compound, and assessing the therapeutic potential of the test compound by dete ⁇ mning to what extent hybridization occurs with the individual libraries prepared.
  • the invention provides a method of determining or assessing the therapeutic potential of a test compound with respect to a biological sample, comprising:
  • RNA/cDNA hybrid library may contain sequences specific to the treated sample and another RNA/cDNA library may contain differentially processed exons specific to the un-treated samples.
  • the therapeutic potential of the test compound can be readily assessed by looking at the hybridization profile of said, nucleic acid preparation with said libraries as mentioned above.
  • the method of the invention allows one to assess the efficiency of a neuroprotective test compound by carrying out hybridization with a differential library according to the invention derived from a healthy nervous cell and a neurodegenerative model cell.
  • a differential library derived from a healthy nervous cell and a neurodegenerative model cell.
  • one is interested in testing an anti-cancer compound using differential libraries established from tumor and healthy sample cells.
  • the invention is still further directed to the use of the methods, nucleic acids, proteins e.g. peptides or antibodies or libraries described hereinabove in pharmacogenomics, i.e., to assess (predict) the response of a patient to a given test compound or treatment.
  • the procedures described in the instant invention make it possible to establish cDNA libraries that are representative of qualitative differences occurring between a pathological condition which is responsive to a given treatment and another condition which is unresponsive or poorly responsive thereto, and thus may qualify for a different therapeutic strategy.
  • these standard libraries Once these standard libraries are established, they can be hybridized with probes prepared from the patients' mRNAs.
  • the hybridization results allow one to determine which patient has a hybridization profile corresponding to the responsive or non responsive condition and thus refine treatment choice in patient management, h this application, the purpose is on the one hand to suggest, depending on the patient history, the treatment regimen most likely to be successful, and on the other hand, to enroll in a given treatment regimen those patients most likely to benefit therefrom.
  • two qualitative differential screening libraries are prepared: one based on a pathological model or sample known to respond to a given treatment, and another based on a further pathological model or sample which is poorly responsive or unresponsive to therapy. These two libraries are then hybridized with probes arising from mRNAs extracted from biopsy tissues of individual patients. Depending on whether such probes preferentially hybridize with the alternatively spliced forms specific to one particular condition, the patients may be divided into responsive and unresponsive subjects to the standard treatment which initially served to define the models.
  • the invention is also directed to a method for determining or assessing the response of a particular patient to a test compound or treatment comprising hybridizing a library characteristic of a responsive biological sample to said compound/treatment and a library characteristic of an unresponsive or poorly responsive biological sample to said compound/treatment, with, a nucleic acid preparation of a pathological biological sample of the patient, and assessing the responsiveness of the patient by determining the extent of hybridization with those different libraries.
  • the invention provides a method of determining or assessing the responsiveness of a patient to a test compound or treatment, comprising: a) hybridizing a nucleic acid preparation of the biological sample of the patient, with at least a first and second nucleic acid library, wherein said first library comprises nucleic acid molecules comprising sequences corresponding to portions of genes which are differentially processed in a responsive biological sample as compared to a non-responsive or poorly responsive biological sample, and said second library comprises nucleic acid molecules comprising sequences corresponding to portions of genes which are differentially processed in the non-responsive or poorly responsive biological sample as compared to the responsive biological sample, wherein said first and second nucleic acid libraries comprise nucleic acid molecules obtained by the method for identifying nucleic acids comprising sequences corresponding to portions of genes that are differentially processed between two biological samples containing nucleic acids provided by the instant invention; and b) assessing the responsiveness of the patient by examining the extent of hybridization of said nucleic acid preparation with said different libraries.
  • the method of the invention allows one to determine or assess the responsiveness of a patient to a test compound or treatment, wherein said test compound or treatment is a neuroprotective compound or an anti-cancer compound or compounds used to treat any medical condition.
  • SAVE methods of the invention can be applied to the comparative study of variant exons in samples of human lung cancer.
  • This study requires a minimum of two samples (for example, biopsies; e.g., normal and cancer lung tissues from the same patient) that are processed as follows:
  • RNA samples are dounce lysed using a Douncer (1 min) in the presence of 1 ml of RNA Wiz lysis buffer.
  • Total RNA is purified following standard phenol/chloroform/isopropanol purification methods as is well known in the art.
  • RNA 0.5-3 ⁇ g total RNA was incubated with l ⁇ l of 0.5 ⁇ g/ ⁇ l oligo dT(15)-T7 primer at 70°C for 3-4 min, snap cooled on ice and mixed with 12 ⁇ l of a 1 st Strand Master mix (4 ⁇ l 5X First strand buffer; 1 ⁇ l 1 ⁇ g/ ⁇ l CAPSWITCH primer; 2 ⁇ l 0.1M DTT; 1 ⁇ l RNaseIN (Promega Cat# N2111); 2 ⁇ l lOmM dNTP (Pharmacia Cat# 27-2035-02); 2 ⁇ l Superscript II (Gibco BRL Cat# 18064-071). The reaction was carried out at 42°C for 90 min in a thermal cycler.
  • a total of 128 ⁇ l of a 2 nd Strand Master Mix (106 ⁇ l DEPC H2O; 15 ⁇ l Advantage PCR buffer; 3 ⁇ l 10 mM dNTP mix; 1 ⁇ l RNase H (2 U/ ⁇ l Gibco BRL Cat# 18021-071); 3 ⁇ Advantage Polymerase (Clontech Cat# 8417-1)) was added to each tube.
  • the samples were then incubated at 37°C for 5 min to digest mRNA, and either 94°C for 2 min to denature, 65°C for 1 min for specific priming and 75°C for 30 min for extension, or Smart PCR with some modifications (essentially, 94°C for 1 min, followed by (94°C 20", 68°C 6') 22 cycles.
  • the reaction was stopped with 7.5 ⁇ l 1M NaOH solution containing 2 mM EDTA and incubated at 65°C for 10 min to inactivate the enzyme.
  • Hybridization Purified samples from the previous step can be used in RNA/cDNA hybridisation reactions.
  • Hybridisation was carried out at RoT values of 5-500, depending on the experiment, in a buffer containing 80% formamide (from a deionized stock), 250 mM NaCl, 25 mM HEPES (pH 7.5), and 5 mMEDTA.
  • Hybridisation was carried out at 37°C in a dry oven with continuous rotation; even volumes as small as 5 ⁇ l did not require mineral-oil overlays.
  • RNA/cDNA hybrids were usually digested with Rnase H as recommended by the manufacturer (Amersham). Further digestion with Dnase I was optional, but was usually carried out at 37°C for 30 min. Dnase I was inactivated after the reaction by heating the sample at 70°C for 10 min.
  • RNA Exon Soup or RES RNA Exon Soup
  • the reaction mastermix contained the following:
  • Total volume was adjusted to 10 ⁇ l/reaction with H 2 O and 6 ⁇ l of master-mix was added to each reaction tube containing the dried RES, and incubated overnight at 16°C.
  • the sample was divided in two halves. One was directly used as a template in RT-Second strand or RT-PCR reactions as below. The other half was purified in an Rneasy column before use in those reactions.
  • the oligoNotLinkRNA (5'AAUCAGAAGGCGGCCGCAAGA-OH), was in vitro phosphorylated in the presence of T4 polynucleotide kinase (incubating in essentially T4 PNK buffer for 1 hr at 37°C; T4 PNK buffer, 50 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, and 10 units of T4 PNK), and purified using a Prepare Bio-6 Chromatograph column (Bio-Rad Cat# 732-6222) as follows: the column washed once with 700 ⁇ l DEPC H2O and spun at 700xg for 2 min at room temperature.
  • pellets- containing the RES were resuspended in an RNA ligation mix containing 1 ⁇ l lOx ligase buffer, 1 ⁇ l RNase inhibitor, 0.5 ⁇ l T4 RNA ligase (6 U/ ⁇ l), 1 ⁇ l NotLinkRNA phosphorylated linker and 6 ⁇ l DEPC H2O.
  • RNA primers (EcoLinkRNA (P-AAUACCGAGAAUUCCCUUGCG-3'), VRNA (5'-ACUGACAUGGACUGAAGGAGUAG-OH)) were used in ligation reactions in order both to shield RNA strings and to allow cloning of their complementary DNAs.
  • EcoLinkRNA and VRNA primers were used in an RNA ligation mix containing the purified hinged strings. This reaction generated shielded strings with a ligated 5' VRNA linker and a 3' EcoLinkRNA linker.
  • reaction was precipitated in the presence of 1 ⁇ l 0.1 ⁇ g/ ⁇ l or 1 ⁇ g/ ⁇ l linear acrylamide and 2.5 M ammonium acetate after addition of 2.5 volumes of 95% room temperature ethanol and the dried pellet used in RT-PCR reactions.
  • the latter reaction was carried out as above except that the primers were EcoLinkPCR instead of the dT-T7 primer and VPCR instead of the CAPSWITCH primer.
  • RT-PCR generated complementary DNAs from RNA shielded strings, as well as a suitable plasmid vector were digested with the appropriate restriction enzyme (i.e. EcoRI).
  • the digested insert library of chained cDNAs
  • vector pGEM-T, Promega Corp.
  • T4 DNA ligase The products of such ligation were used ultimately to transform competent E.coli cells, that were grown on ampicillin selection plates. Individual clones from such transformations were grown and lysed and purified plasmid DNA obtained.
  • This DNA was later sequenced or used in other reactions such as digestion with hinge restriction endonuclease and subsequent agarose gel purification of the yielded DNA fragments (individual exonic fragments). Those fragments could be, for example, spotted on solid surfaces for use as probes in, for instance, gene expression studies.

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Abstract

L'invention concerne une analyse en chaîne (complète) des exons variants (SAVE), permettant de mettre en oeuvre des procédés consistant à évaluer les frontières entre exon et intron, des points de départ de transcription et des sites de polyadénylation, tels que ceux utilisés par une cellule donnée, un tissu ou un organisme à un moment donné ou dans une situation donnée (physiologique ou pathologique). Cette SAVE permet de mettre en oeuvre des procédés consistant à lier entre eux des fragments d'ARN issus de la digestion de la ribonucléase. L'utilisation de lieurs ARN dans la SAVE permet de mettre en oeuvre des procédés consistant à produire des chaînes de fragments d'ARN, à les amplifier, à les cloner, à les séquencer et à les purifier pour pouvoir les utiliser ultérieurement.
PCT/GB2003/003118 2002-07-17 2003-07-17 Procede permettant d'analyser des variations de sequences d'acides nucleiques polymeres WO2004007768A1 (fr)

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US6251590B1 (en) * 1998-03-11 2001-06-26 Exonhit Therapeutics S.A. Differential Qualitative screening
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US4946773A (en) * 1985-12-23 1990-08-07 President And Fellows Of Harvard College Detection of base pair mismatches using RNAase A
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