WO1997019192A1 - Procede et sonde servant a detecter une sequence cible d'acide nucleique - Google Patents

Procede et sonde servant a detecter une sequence cible d'acide nucleique Download PDF

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Publication number
WO1997019192A1
WO1997019192A1 PCT/US1996/018615 US9618615W WO9719192A1 WO 1997019192 A1 WO1997019192 A1 WO 1997019192A1 US 9618615 W US9618615 W US 9618615W WO 9719192 A1 WO9719192 A1 WO 9719192A1
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probe
oligonucleotide
sequence
sample
complementary
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PCT/US1996/018615
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English (en)
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Shelley J. Russek
David H. Farb
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Trustees Of Boston University
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Priority to AU10208/97A priority Critical patent/AU1020897A/en
Publication of WO1997019192A1 publication Critical patent/WO1997019192A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • This invention is directed to methods of detecting alterations in the levels of gene expression and quantitating specific nucleotide sequences in analytical samples.
  • this invention provides a rapid analytical method based on recovery of intact nucleic acid segments that are protected from enzymatic hydrolysis through formation of hybridization dimers.
  • RNA extraction and gel electrophoresis Most methods for monitoring alterations in specific gene expression rely on RNA extraction and gel electrophoresis.
  • Northern Blot Analysis, SI Nuclease Protection and RNase Protection assay provide accurate data about both the size and expression of specific transcripts in question, but they are difficult to perform on multiple sample populations.
  • a major drawback of these approaches is that before performing the assay one must usually obtain a cloned fragment of DNA that is specific for the gene in question. This long sequence fragment is usually isolated by either cDNA library screening or polymerase chain reaction (PCR), and it must then be verified by DNA sequencing.
  • the fragment of DNA must contain restriction sites in the appropriate places such that the radiolabeled antisense RNAs can be made appropriate to the nucleotide sequence that is to be monitored.
  • both Northern Blot Analysis and Nuclease Protection assays rely on time consuming gel electrophoresis to distinguish and display the results of the assay.
  • the quality of the results often vary from one experiment to another depending upon the target sequence, making it difficult to distinguish specific protection on a gel from a background of hybridization signals that often include protected fragments due to self-hybridization of the probe.
  • the method of this invention takes advantage of the high specificity of nuclease protection assays using labeled oligonucleotide probes.
  • nucleic acid probes of this invention bind to their target sequence during a hybridization reaction, and regions of unbound single stranded nucleic acid in the template and probe are subsequently digested by the addition of nuclease.
  • An importance difference, however, in the method of this invention is that specially- modified small synthetic oligonucleotides are used as probes instead of large RNA molecules produced from cloned templates using, e.g., bacterial RNA polymerases, and the hybridization signal can be detected without the use of gel electrophoresis.
  • modified oligonucleotides contain a label at the 5 ! end and a biotin capture group at the 3' end.
  • oligonucleotide probes can be detected by streptavidin capture of the 3' biotin group on the probe. Any one cleavage event of the modified oligonucleotide will cause a total loss of detection for the molecule, thereby enhancing signal-to-noise and providing the greatest specificity of sequence detection with the protection technique.
  • oligonucleotide sequences for the probes of this invention can be easily designed based on published sequences, and they can be produced in large quantities that are inactive and therefore RNase non-susceptible, due to the presence of a 2' t-butyldimethylsilyl protective group.
  • the oligonucleotide can be desilyated with tetrabutylammomum fluoride and desalted by thin layer chromatography. Small aliquots of the synthetic ribonucleotide can be stored at -80 until use. Gene expression studies using these labeled nucleotides are performed in cell lysates to minimize on RNA losses from extraction. Using this approach, gene expression can be monitored in a tissue slice of less than 500 ⁇ M.
  • the subsequent capture and quantitation of synthetic ribonucleotide probes circumvents the necessity of using gel electrophoresis for detection while providing high throughput and specificity. Furthermore, the method of the invention has the potential for taking a genome-based approach to mapping gene expression that can be automated to facilitate large scale analysis.
  • the label is a fluorescence label and the capture group is replaced by a second fluorescence label where the distance between the two fluorescence labels attached to the oligonucleotide probe is such that energy will be transferred from one to the other prior to fluorescence.
  • the energy transfer will only occur when the two labels are sufficiently close to each other, usually 20 base pairs or less apart.
  • the necessary proximity is maintained by the nucleic acid segment stretching between the labels, and the integrity of the oligonucleotide segment is therefore critical for fluorescence energy transfer. Digestion by nuclease will eliminate the energy transfer.
  • the probes of this embodiment can hybridize to target sequences on nucleic acids in the sample, and this hybridization will protect the probe from digestion by subsequently-added nuclease.
  • the protected probes (and the presence of corresponding target sequence in the sample) can be detected by their ability to maintain fluorescence energy transfer.
  • Suitable fluorescent labels and suitable methods for detecting transfer of energy are well known. See, for instance, CLEGG RM, Curr. Opm.
  • a capture group is positioned on the 5' end of the oligonucleotide and a fluorescence label (Fl) is attached to the most 3' complementary base, with additional non-complementary nucleic acid sequence 3' to the end of the complementary sequence, the non-complementary sequence connecting an additional fluorescence label (F2) to the probe.
  • Fl fluorescence label
  • the nucleotide distance between Fl and F2 is adjusted such that, unless the intervening, non- complementary sequence is digested, the presence of F2 in oligonucleotide probes quenches the signal from Fl.
  • the use of two capture groups can increase signal detection by eliminating the interference of probe which remains undigested due to enzyme inefficiency.
  • a label is fixed to the 5' end of the oligonucleotide and a capture group (Cl), preferably biotin, is attached to the most 3' complementary base. Additional nucleic acids are added to the end of the oligonucleotide, the last of which is coupled to an additional capture group (C2) that is different from Cl . Reaction mixtures are first purified by removing undigested oligonucleotides that contain the C2 capture group.
  • Remaining oligonucleotides (protected by hybridization) are then purified away from the reaction mixture by capttiring the nucleic acids via the C 1 capture group. Detection is then completed via the label on the 5' end of the oligonucleotide.
  • Figure 1 shows percent binding to beads of an RNA oligonucleotide with and without 3' biotin label.
  • Figure 2 shows percent binding of probe in the presence and absence of biotin.
  • Figure 3 shows specificity of genome expression momtoring using a ⁇ -Actin antisense RNA probe with control sense RNAs.
  • Figure 4 shows detection of specific RNAs in lysates from brain cell cultures that contain GABA-A receptor ⁇ l subunit transcripts.
  • Figure 5 shows differential expression of GABRG2 subunit mRNAs in the hippocampus of 47 day old prenatally malnourished rats.
  • Figure 6A-6D shows, in schematic form, probe design for the detection of specific nucleic acids.
  • oligonucleotide probes which may be either RNA or DNA, are produced with an attaching group by which the probe may be captured, attached at some distance from the label.
  • all probes have both a detection label and a capture group which are linked together by the oligonucleotide.
  • the probe may have a detection label at the 5' end and a biotin capture group at the 3' end.
  • the probe is incubated with the sample to be analyzed so that the oligonucleotide hybridizes with any complementary sequence present in the sample, and then unhybridized probes are digested by nucleases specific for single-stranded nucleic acids, so that the detection label is separated from the capture group except where the probe is protected by hybridization.
  • the intact probes are collected.
  • this can be accomplished by passing the reaction mixtures through a filter manifold contaiiiing a filter matrix with covalently-coupled streptavidin or adding streptavidin-coated beads to the reaction mixtures and then collecting the beads.
  • filters or beads do not bind nucleic acids if suitably pretreated, and therefore, when streptavidin captures the biotin of the probe, only full length probe, undigested because of protection by the RNA target of interest, is detected from the label on the probe's 5' end.
  • FIG. 6A-6D Alternative assay configurations are shown in diagrammatic form in Figure 6A-6D.
  • a label group is attached to the 5' end of the oligonucleotide and a capture group is attached to the '3 end. Sequence between these groups is complementary to the gene of interest. Enzyme digestion destroys the majority of probe, leaving behind probe that is protected by hybridization or probe that is left after due to inefficient digestion. Molecules containing either label group or capture group are not recovered.
  • the configuration in Figure 6B shows an additional 3' capture group added to the oligonucleotide to aid in removal of all probe that is not protected by hybridization to the target sequence.
  • Non-complementary sequence separates the two capture groups from one another.
  • Probe molecules resistant to digestion that have not hybridized to target are identified and removed via the first capture group.
  • Probe molecules that have been protected by hybridization will not contain the second capture group. These molecules are purified via capture group 1 as in Figure 6A.
  • a label group is attached to the 5' end and a different label group is attached to the 3' end.
  • the distance between the label groups is adjusted to provide for maximum energy transfer between the two groups.
  • Digested probes are not detected.
  • enzyme digestion destroys the majority of probe, leaving behind probe molecules that are protected by hybridization (or background label that is left due to inefficient digestion).
  • a capture group is attached to the 5' end of the oligonucleotide and two label groups (Fl and F2) are attached to nucleotides at the 3' end of the molecule.
  • the distance between Fl and F2 is adjusted such that Fl signal is quenched in the presence of F2.
  • Non-complementary sequence separates the Fl and F2 groups. Intact probe molecules that remain due to inefficient digestion will not be detected. Detection is specific only to the oligonucleotides that contain a 5' capture group and one label (Fl).
  • Probes according to this invention are single stranded oligonucleotides in which at least a portion of their sequence is complementary to the sequence of some segment of a nucleic acid of interest.
  • the oligonucleotides may be ribooligonucleotides, deoxyribooligonucleotides, or mixed DNA/RNA oligomers.
  • modified deoxynucleotides such as 5-methyl-deoxycytidine or 5-bromo-deoxyuridine
  • Suitable oligonucleotides can be designed and produced for use as hybridization probes by a variety of means.
  • the probes are synthesized chemically, preferably based upon known nucleic acid sequences.
  • those using nuclease protection assays found it desirable to prepare probes that were fairly long and or encompassed regions ofthe amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. While it is generally recognized in the art that probes from about 14 to about 20 base pairs can be effective for hybridization, longer probe sequences were often found necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished.
  • probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.
  • shorter oligonucleotide probes will be designed so that the 5' end is of relatively high G+C content. High G+C content on the 5' end reduces "breathing" in the hybrid that might expose the probe to hydrolysis and reduce a specific signal.
  • probes will be preferably at least 40 base pairs.
  • Probes having the desired oligonucleotide sequence can be constructed synthetically, based on known DNA or amino acid sequences, or isolated by one of several approaches using well known methods.
  • the basic strategies for preparing oligonucleotide probes and DNA libraries are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., "Molecular Cloning: a Laboratory Manual” (1989); B. Perbal, "A Practical Guide To Molecular Cloning” (1984), Edge (1981) Nature 292:756; Nambair, et al. (1984) Science 223: 1299; and Jay, et al. (1984) J. Biol. Chem., 259:6311.
  • RNA oligomers for RNase protection assay, or DNA oligomers, for SI analysis, may be prepared on the ABI 392 DNA/RNA synthesizer.
  • Probes according to one embodiment of this invention are connected to both a detectable label and one or more capture groups. By the connections of the label and capture group to the oligonucleotide, the label and capture group remain coupled so long as the oligonucleotide is intact, and immobilization ofthe capture group serves to anchor the label to which it is indirectly coupled.
  • the label upon digestion of the oligonucleotide, the label is no longer coupled to the capture group, and the label may then be readily separated from the capture group once the latter is captured, for example by binding it to a suitable moiety immobilized on a solid substrate.
  • Suitable capture groups are usually one member of a specific binding pair, such as biotin and avidin or streptavidin, an antigen and its antibody, a protein containing at least the F c portion of an immunoglobulin molecule and staphlococcal protein A, a lectin and its corresponding carbohydrate moiety, or similar pairs.
  • Methods for connecting capture groups to nucleotide residues of the oligonucleotide will depend on the specific capture group and the nature ofthe nucleotide residue to which it is being attached. Such methods are well known and selection of a suitable method can be readily made by the skilled artisan. See, e.g., Sambrook, et al.
  • the label is detectable by physical or chemical means, such as colorimetric, chemiluminescent or fluorescent methods or radio detection.
  • Typical labels include radiolabels inco ⁇ orated in one or more of the nucleotide residues of the oligonucleotide or in a moiety attached to one of the residues, a fluorescent group attached to one of the residues, or a member of a specific binding pair different from the capture group, where the second member of the specific binding pair is attached to a detectable moiety such as an enzyme catalyzing a chromogenic or fluorogenic reaction either directly or indirectly. Any method of connecting the label to the oligonucleotide portion ofthe probe may be used, so long as the connection survives under the conditions used in the hybridization step as well as the conditions ofthe digestion if enzyme is omitted.
  • a variety of suitable labels are in routine use for analysis of various nucleic acid materials, and selection of a suitable label and inco ⁇ oration of the label in the probe is within the skill of the art.
  • the detectable label and the capture group(s) may be connected to the oligonucleotide at any point on the oligonucleotide and by any suitable method of connection, so long as coupling between the label and capture group (or between different capture groups) is destroyed by digestion of the oligonucleotide.
  • the label and the capture group cannot be attached to the same nucleotide residue, and usually there will be several residues of the oligonucleotide between the label and the capture group. Increasing the distance between connection points to the oligonucleotide for the capture group and the label will tend to increase sensitivity ofthe assay.
  • oligonucleotides are made to contain a biotin group on the 3' end. This may be accomplished through the use of a CPG Biotin- ON column or CPG Amino-ON column (Clonetech), although the skilled artisan will be aware of numerous other methods of attaching capture groups to a nucleic acid sequence.
  • RNA oligomers are desilated overnight and gel purified. Conjugated DNA oligomers are similarly deprotected overnight and also gel purified.
  • Oligonucleotides conjugated to capture groups at the 3' end can be subsequently labeled at the 5' end with either ⁇ 32 PdATP or ⁇ 35 SdATP in a reaction that is catalyzed by T4 polynucleotide kinase.
  • probes to monitor rare mRNA of specific interest may be labeled with ⁇ 32 P while probes to monitor abundant intemal standards, such as ⁇ -actin, are labeled with ⁇ 35 S.
  • oligonucleotide probes containing a fluorescent tag at the 5' end and a biotin group at the 3' end could also be used in this detection assay.
  • Multiple fluorescent tags on different probes allows the expression of many genes to be monitored together in one sample, in addition to intemal standards.
  • Altemative labels that facilitate enhanced detection are also suitable. See, e.g., U.S. Pat. Nos. 5,312,922 and 5,089,423, inco ⁇ orated herein by reference.
  • the different fluorescence labels are chosen such that the proximity of labels produces either energy transfer or energy depletion.
  • Fluorescent labels suitable for proximity dependent energy transfer or fluorescent quenching can be selected from probes known to provide these characteristics (see Clegg, 1995; Selvin, 1995: Mergny et al., 1994; Gohlke, 1994; Selvin, 1994; Hiyoshi et al., 1994; Ghosh et al., 1994; Wu et al., 1994; and Benson et al., 1993).
  • the method of this invention may be used to detect and/or quantitate the amount of a particular nucleotide sequence in single stranded regions of nucleic acid in any sample.
  • nucleic acid containing the particular sequence complementary to the sequence of the probe is the target nucleic acid
  • the sequence complementary to the ohgonucleotide ofthe probe is the target sequence.
  • the method of this invention may be used to detect the presence of specific nucleotide sequences in any single stranded nucleic acids of a sample
  • the target nucleic acid is usually messenger ribonucleic acid (mRNA).
  • mRNA messenger ribonucleic acid
  • the method of this invention is useful for monitoring the appearance and disappearance of mRNA encoding various proteins as a result of embryonic development or cellular signaling in response to hormonal changes.
  • RNA samples may be assayed by the present method
  • the method of the present invention can also be used to detect mRNA in cell lysates from cell culture or tissue samples without the need for extensive prior purification of the nucleic acids. The present method thus permits rapid screening of large numbers of cell samples, which would be unfeasible using earlier methods.
  • mRNA expression by cells in cell culture may be determined by obtaining an aliquot of cells from the culture and making the nucleic acids available for hybridization and enzymatic digestion by simply lysing the cells.
  • a cell culture sample is collected by centrifugation, the supernatant removed from the centrifuge tube, and the tube then vortexed to distribute the cells as a layer on the inside wall. This layer is then dissolved, for example, at a concentration of IO 5 cells/ml in lysis buffer solution (4 M guanidine thiocyanate, 25 mM sodium citrate and 0.5% sarcosyl) and vortexed.
  • lysis buffer solution (4 M guanidine thiocyanate, 25 mM sodium citrate and 0.5% sarcosyl
  • lysis of the tissue section to release RNA may be all that is necessary.
  • the tissue may be placed in lysis buffer, such as the solution described above, at a concentration of 100-250 mg/ml and then homogenized. After lysis of the cells or tissue sections, lysates can be stored at -20 °C or used immediately in the hybridization reaction.
  • the assay method of this invention is carried out by contacting the probe, including a single stranded oligonucleotide connecting detectable label and one or more capture groups, with the target nucleic acid under conditions that promote hybridization, then digesting any probe oligonucleotide that remains in the single stranded state.
  • the assay protocol includes both positive and negative controls.
  • the positive controls are intemal, wherein multiple probes are added to the sample, at least one of which is complementary to a sequence that is known to be present in the sample or is exogenously added to the sample before the hybridization step.
  • the negative controls are extemal, i.e., separate samples which may contain the positive control sequence, but are known not to contain the target sequence.
  • the conditions of temperature, chemical environment, etc., in the sample must be adjusted so that the target nucleic acid material can undergo hybridization with the probe oligonucleotide and also to permit enzymatic degradation by enzymes specific for single stranded nucleic acids.
  • These conditions are well known to the skilled artisan and suitable conditions are exemplified below (see also Ausubel, et al., eds., "Current Protocols in Molecular Biology,” Volume 1, Chapter 4, Sections 4.6 and 4.7, 1989, inco ⁇ orated herein by reference).
  • Incubation to permit hybridization is usually from about one hour to about 16 hours (overnight), although shorter or longer times may be used so long as sufficient hybridization occurs to protect the oligonucleotide portion of a detectable amount of the probes from subsequent digestion.
  • Hybridization and digestion may occur simultaneously, for instance when endogenous RNases are used, so long as a detectable quantity of probe molecules are protected from digestion, and the quantity is proportional to the amount of the target sequence present in the sample. However, the hybridization and digestion are usually carried out sequentially.
  • the sample must be incubated for sufficient time to allow the remaining single stranded oligonucleotide portion of the probe molecules to be hydrolyzed by nucleases which are selective for single stranded nucleotides relative to double stranded nucleotides.
  • nucleases which are selective for single stranded nucleotides relative to double stranded nucleotides. Enzymes of greater selectivity, as reflected in higher relative rates of hydrolysis for single stranded nucleic acids over double stranded nucleic acids, is clearly preferable to preserve a detectable amount of hybridized probe while digesting essentially all single stranded probe (to leave no more than background levels of label coupled to capture group by single stranded oligonucleotide).
  • Typical nucleases are SI nuclease when the probe oligonucleotide is DNA and RNase Tl and/or RNase A when the probe oligonucleotide is RNA.
  • the capture group (along with any intact probes still attached thereto) is separated from the reaction mix.
  • the separation is accomplished by binding the capture group to a solid material which may be porous or non-porous particles, including magnetic beads, a filter, such as a membrane filter, or a solid surface, such as plastic plates, glass plates, or porous plates.
  • the capture group is one member of a specific binding pair, such as biotin-streptavidin, antigen-antibody, antibody-protein A, etc., and the other member of the binding pair is conjugated to a suitable surface.
  • the post-digestion reaction mix is contacted with the surface conjugated with members of the specific binding pair which bind the capture group, immobilizing it on the surface when the rest ofthe reaction mix is removed (e.g., by washing the surface).
  • the amount of intact probes which survived digestion can be determined by measuring the amount of detectable label attached to the surface by coupling through intact oligonucleotides connected to the immobilized capture group, using methods for quantitating detectable label attached to a surface which are well known in the art.
  • two capture groups may be coupled to nucleotides in the ohgomer. The most 3 1 capture group will allow a pre-separation of background probe from relevant hybridization signal. The final capture and detection will be as previously described. Detection
  • Radioactive labels may be detected and/or quantitated using a scintillation counter, Phosphoimager (densitometer) or autoradiography.
  • a fluorescence detector can be used if a fluorescent tag is attached to the oligonucleotide during synthesis. After separation, detection of flourescent labels may be monitored in 96 well plates using a fluorescent plate reader. Multiple fluorescent tags on different probes allows the expression of many genes to be momtored together in one sample. For other labels, the appropriate detection system will be readily apparent to the skilled worker.
  • the radiolabeled probe with oligomeric RNA for RNase protection or oligomeric DNA for S 1 protection, is diluted in lysis buffer solution at 10 4 -10 5 cpm/5 ⁇ l. 5 ⁇ l of diluted probe is then aliquoted into each of a set of 1.5ml tubes. 45 ⁇ l of cell lysate is added to each tube and mixed by gentle vortexing. Control reactions contain lysate from a tissue source known to not express the message of interest. The reactions are then incubated overnight at 37°C (this temperature varies with the GC content of the probe). Selection of the nuclease will, of course, depend on whether the probe is RNA or DNA.
  • RNase Protection 500 ⁇ l of RNase solution (450 ⁇ l dH 2 0, 50 ⁇ l of 10X RNase buffer (500 mM Tris, pH7.5, 4M NaCl, 100 mM EDTA) and 1 ⁇ l of Tl (RNase Tl, 100 U) are added directly to the hybridization reactions. Separate control reactions for positive controls do not contain nuclease.
  • SI Protection 40 ⁇ l of 2XS 1 digestion buffer (0.56M NaCl, 0. IM NaOAc, 9 mM ZnS0 4 and 250 U of S 1 nuclease are added directly to the hybridization reactions. Positive control reactions do not contain nuclease.
  • reactions are then gently vortexed and incubated at 37° C for 60 min.
  • one of two methods may be used to capture intact probe containing a 5' radiolabel and a 3' biotin group: 25 ⁇ l of equilibrated streptavidin magnetic beads are added to the reaction mixture.
  • protected probe can be recovered by magnetic separation from the reaction mixture followed by immobilization on filter paper, either through a slot blot or filter disk apparatus, and analyzed subsequently by phosphoi aging or scintillation counting.
  • biotinylated probe can be captured directly onto streptavidin containing filters without the use of magnetic beads.
  • Radioactivity on the filter can then be monitored using a Phosphoimager for ⁇ 32 P and 35 S detection.
  • a fluorescence detector can be used if a fluorescent tag is inco ⁇ orated at the 5' end ofthe RNA oligomer during synthesis. After separation, detection can then be monitored in 96 well plates using a fluorescent plate reader. Multiple fluorescent tags on different probes allows the expression of many genes to be monitored together in one sample.
  • a gene transcript common to all cells i.e., a housekeeping gene
  • a housekeeping gene is monitored in addition to the experimentals in order to control for differences across samples and for pipette loading e ⁇ ors.
  • Probes having 5' label and 3' biotin or amino group can be used in a variety of different methods (SI nuclease protection, in solution hybridization, standard RNase protection with large RNA probes) modified to rely on recovering the probe from a reaction mixture (for example by streptavidin filtration) in order to quantitate the specificity of hybridization without gel electrophoresis.
  • SI nuclease protection in solution hybridization, standard RNase protection with large RNA probes
  • This invention also provides a method for characterizing the tertiary structure of a protein or other macromolecule, especially a method for measuring distances between epitopes on the surface of the macromolecule.
  • An antibody specific for one epitope on a macromolecule is conjugated with an oligonucleotide having a capture group distal to the antibody.
  • a second antibody, specific for another epitope distinct from the first epitope, is conjugated to an oligonucleotide complementary to the oligonucleotide conjugated to the first antibody and having a detectable label distal to the second antibody.
  • the conjugated antibodies are used to characterize the macromolecular structure by binding the antibodies to epitopes on the macromolecule and allowing hybridization between the complementary oligonucleotides if the antibodies (and therefore the epitopes they bind to) are positioned in close enough proximity to permit it.
  • the antibody- macromolecule complex is then subjected to nuclease digestion that will digest the oligonucleotides (and release the capture group and label) unless hybridization has occu ⁇ ed. Capture of the antibody-macromolecule complex, and detection of the detectable label in association with the complex (but not with uncomplexed antibodies), confirms the proximity of the two epitopes on the macromolecule under conditions of the assay.
  • Suitable conditions can be readily selected by the skilled artisan, guided by the conditions for hybridization, digestion, and detection provided herein, as can suitable oligonucleotides, capture groups, and detectable labels. It will be readily apparent to the skilled artisan that if the detectable label is a fluorescent label, a second fluorescent label can be substituted for the capture group and detection of hybridization accomplished by detecting fluorescent transfer. Adoption of the method using a second capture group to enhance sensitivity is also easily within the skill of the artisan.
  • This invention also provides improved immunoassay methods for detecting a particular protein or other antigen in a sample comprising mixing a sample containing the antigen with a primary antibody that is conjugated to a first nucleic acid sequence under conditions where antigen- antibody complexes form, then mixing the sample with an oligonucleotide probe according to this invention complementary to the said first nucleotide sequence conjugated to the primary antibody, and incubating for a time sufficient to permit hybridization of complementary nucleic acid sequences to for nucleic acid duplexes; and subsequent to the hybridization, adding to the sample a nuclease which specifically digests the probe unless the probe is part of a nucleic acid duplex. Detection of the probe bound to the complementary oligonucleotide (which is conjugated to the antibody) as described herein, provides a method of detecting the antibody, and therefore the antibody-antigen complex in which it is bound.
  • Such assays can altematively employ the process described above, based on hybridization of two oligonucleotides brought into proximity by proximal binding of two antibodies to which the oligonucleotides are attached, and digestion of unhybridized oligonucleotides. It will be readily apparent that a particular embodiment of the general process occurs when one oligonucleotide is conjugated to an antibody that specifically binds the other antibody (thereby ensuring proximity of the two oligonucleotides conjugated to the two antibodies).
  • Use of this process to detect binding of a secondary antibody to a primary antibody specific for a particular analyte by a procedure including hybridization, digestion and detection as described herein is contemplated by the present invention. Such an analytical procedure is particularly attractive to confirm binding of the secondary antibody to the primary antibody, which may be used, for example, to provide an intemal experimental control. Similar assay procedures may, of course, employ specific binding pairs with characteristics analogous to antibody binding.
  • RNA having a sequence complementary to the sequence to be detected is prepared on the ABI 392 DNA/RNA synthesizer. Oligoribonucleotides (>r48bp) are made to contain a biotin group or amino group on the 3' end. This is accomplished through the use of a CPG Biotin column (Clonetech). RNA oligomers are desilylated overnight and gel purified. Oligoribonucleotides are subsequently labeled at the 5' end with either ⁇ 32 PdATP or ⁇ 35 SdATP in a reaction that is catalyzed by T4 polynucleotide kinase.
  • Probes having sequence complementary to rare mRNA of specific interest are labeled with ⁇ 32 P while probes complementary to abundant intemal standards are labeled with ⁇ 35 S.
  • the intemal standards may be RNA common to most cells, such as ⁇ -actin, or exogenously added RNA or single stranded DNA having sequences that are not expected in the sample. Usually the sequences complementary to control probes will be present in substantial excess.
  • Cell lysates may be RNA common to most cells, such as ⁇ -actin, or exogenously added RNA or single stranded DNA having sequences that are not expected in the sample. Usually the sequences complementary to control probes will be present in substantial excess.
  • the cell culture sample is collected by centrifugation and the supernatant is removed.
  • the tube is then vortexed to distribute the cells as a layer on the inside wall.
  • This layer is then dissolved at a concentration of IO 5 cells/ml in lysis buffer solution (4 M guanidine thiocyanate, 25 mM sodium citrate and 0.5% sarcosyl) and vortexed.
  • lysis buffer solution 4 M guanidine thiocyanate, 25 mM sodium citrate and 0.5% sarcosyl
  • the radio labeled RNA probe is diluted in lysis buffer solution at 10 4 -10 5 cpm/5 ⁇ l. 5 ⁇ l of diluted probe is then aliquoted into each 1.5ml tube. 45 ⁇ l of cell lysate is added to each tube and mixed by gentle vortexing. Negative control reactions contain lysate from a cell or tissue source known to not express the message of interest. The reactions are then incubated overnight at 37°C (this temperature varies with the GC content of the probe).
  • RNase Tl 500 ⁇ l of RNase solution (450 ⁇ l dH20, 50 ⁇ l of 1 OX RNase buffer (500 mM Tris, pH7.5, 4M NaCl, 100 mM EDTA) and 1 ⁇ l of Tl (RNase Tl, 100 U) are added directly to the hybridization reactions. Control reactions do not contain nuclease. Reactions are then gently vortexed and incubated at 37°C for 60 min. After the reaction mixture has equilibrated to room temperature, one of two methods is used to capture intact probe containing a 5' radiolabel and a 3' biotin group: 25 ⁇ l of equilibrated streptavidin magnetic beads are added to the reaction mixture.
  • probes captured on the beads can also be separated from the reaction mixture by immobilization on filter paper, either through a slot blot or filter disk apparatus, and analyzed subsequently by phosphoimaging or scintillation counting.
  • protected biotinylated probe can be captured directly onto streptavidin-containing filters without the use of magnetic beads.
  • Figure 1 shows percent binding to beads of an RNA oligonucleotide with and without 3' biotin label.
  • Avidin coupled magnetic beads were incubated in the presence of radiolabeled oligonucleotide that did and did not possess a biotin group at the 3' end. Percent binding was determined by measuring the amount of radioactivity associated with the beads.
  • Figure 2 shows percent binding of probe in the absence and presence of biotin. A molar excess of biotin (5.2 nmol) over magnetic beads coupled to streptavidin successfully competed for the binding of the biotinylated probe. The results of this experiment demonstrate that binding of the probe to streptavidin beads is dependent on the presence of the 3' biotin capture group in the oligonucleotide.
  • Figure 3 shows specificity of genome expression monitoring using a ⁇ -Actin antisense RNA probe with control sense RNAs. Percent protection was monitored using a tenfold excess of probe over target sense RNA. Significant protection was monitored only when the target contained sequence complementary to the probe.
  • Figure 4 shows detection of specific RNAs in lysates from brain cell cultures that contain GABA-A receptor ⁇ l subunit transcripts.
  • Lysates were prepared from one 100mm dish containing cultured cells from either rat hindlimb or rat neocortex. Significant detection of the neutral specific ⁇ 1 subunit transcript was detected in rat neocortex.
  • Figure 5 shows differential expression of GABRG2 subunit mRNAs in the hippocampus of 47 day old prenatally malnourished rats.
  • A41 represents well nourished rats and B41 malnourished
  • detection of ⁇ 2 transcripts in RNAs extracted from hippocampal tissue indicates a significant increase of expression in the brain of malnourished animals.
  • Example 2 The procedure is identical to that of Example 1, except ohgonucleotide probes containing a fluorescent tag at the 5' end and a biotin group at the 3' end are used in this detection assay. Detection of fluorescent probes may be monitored in, for example, 96 well plates using a fluorescent plate reader or CCD camera.
  • Synthetic RNA can also be produced using standard methods for the production of Riboprobes. These Oligonucleotides are then tagged with biotin and labeled at the 5' end with detectable labels as described above.
  • assays that employ fluorescence energy transfer reaction mixtures are subjected to enzymatic digestion. For small probes, the amount of energy present at a particular region of the spectrum is monitored using standard procedures. For the detection of rare mRNAs, the protected probe is recovered after digestion using a capture procedure as previously described and the presence of fluorescence from the Fl label is detected using standard methods. Control assays are performed to measure the integrity of the RNA probe. The fluorescence from a fully intact probe is quenched and therefore will not be detected until the F2 label is removed by digestion.
  • Short DNA oligonucleotides are prepared on the ABI 392 DNA/RNA synthesizer. Oligodeoxyribonucleotides ( 48bp) are made to contain a biotin group on the 3' end. This is accomplished through the use of a CPG Biotin column (Clonetech). DNA oligomers are deprotected overnight and gel purified. Oligodeoxyribonucleotides -ire subsequently labeled at the 5' end with either ⁇ 32 PdATP or ⁇ 5 SdATP in a reaction that is catalyzed by T4 polynucleotide kinase.
  • Probes which hybridize with rare mRNA of specific interest are labeled with ⁇ 32 P while probes which hybridize with intemal standards are labeled with ⁇ 35 S.
  • intemal standards are segments of sequences found in abundance, such as ⁇ -actin.
  • Ohgonucleotide probes containing a fluorescent tag at the 5 ' end and a biotin group at the 3' end can also be used in this detection assay.
  • Hybridization Assay A tenfold molar excess of radiolabeled DNA probe and 2-20 ⁇ g of RNA extracted from the tissue of interest are added to 10 ⁇ l of hybridization buffer (80% deionized Formamide/ lOOmM Sodium Citrate pH. 6.4/300 mM Sodium Acetate pH.
  • Negative control reactions contain RNA from a tissue source known to not express the message of interest. The reactions are then incubated overnight at 37°C (this temperature varies with the GC content of the probe). 10 ⁇ l of 2XS 1 digestion buffer (0.56M NaCl, O. IM NaOAc,
  • Captured beads can also be separated from the reaction mixture by immobilization on filter paper, either through a slot blot or filter disk apparatus, and analyzed subsequently by phosphoimaging or scintillation counting.
  • biotinylated probe can be captured directly onto streptavidin containing filters without the use of magnetic beads.
  • a primary antibody is conjugated to a nucleic acid that contains sequence complementary to the especially prepared probe (prepared by the method of Sano, Smith, and Cantor, Science 258:5079, 1992).
  • the antibody is mixed with either an antigen solution containing lysed cells or a solution containing solubilized cells at 20 - 200 ng ml antigen to 1 -10 ⁇ g/ml antibody. Solution is incubated for 30 min. Antigen-antibody complexes are separated from unbound antibody.
  • Probe complementary to a region ofthe oligonucleotide coupled to the antibody is then added to the reaction mixture containing antigen-antibody complexes and hybridization and digestion takes place as described in the above examples. Hybridization is then detected using a method that is dependent on the probe configuration, i.e. one or more capture groups and fluorescence energy transfer (see Figure
  • Antigen detection can also be carried out using DNA-coupled antibodies that are affixed to a surface, such as a magnetic bead or a microtiter well plate.
  • the oligonucleotide attached to the antibody may contain one or more fluorescence labels that can interact with one or more fluorescence labels on the detection probe. Hybridization after digestion would then be detected by a fluorescence energy transfer or liberation of quenching due to the proximity between the two protected oligonucleotides.
  • Example 5 Allelic discrimination.
  • a probe in a configuration as described in Diagram 1, is generated that contains one or more mutations in the nucleic acid sequence specific to a previously identified variant or suspected variant. Hybridization to DNA or RNA is performed as previously described. Detection ofthe probe indicates the presence of the variant form of nucleic acid sequence. Control reactions include the addition of wildtype DNA oligonucleotide (containing a majority of complementary sequences to the probe) in the presence and absence of the target of interest in order to verify that the probe is only detecting the variant. Variant alleles can altematively be detected by the loss of probe signal when the probe contains only sequence of the wildtype.
  • Example 7 Procedure is identical to Example 1 except that an oligoribonucleotide is synthesized to contain two capture groups that are separated by non-complimentary sequence.
  • the first capture group is biotin and the second is an amino group at 3' end of the oligoribonucleotide.
  • any probe remaining due to inefficient digestion is removed by covalent coupling to tosylactivated magnetic beads (Dynabeads) Hepbum et al., American Biotechnology Laboratory, September 1992: 44-45.
  • the unbound mixture containing the hybridization is recovered using streptavidan purification as described above.
  • Example 8 Detection with Fluorescent Labels
  • Example 3 The procedure is identical to that of Example 3, except ohgonucleotide probes containing a fluorescent tag at the 5' end and a biotin group at the 3' end are used in this detection assay. Detection of fluorescent probes may be momtored in, for example, 96 well plates using a fluorescent plate reader or CCD camera.
  • Synthetic DNA can also be produced using standard methods for the production of DNA probes. These oligonucleotides are then tagged with biotin and labeled at the 5' end with detectable labels as described above.
  • assays that employ fluorescence energy transfer reaction mixtures are subjected to enzymatic digestion. For small probes, the amount of energy present at a particular region ofthe spectrum is monitored using standard procedures. For the detection of rare mRNAs, the protected probe is recovered after digestion using a capture procedure as previously described and the presence of fluorescence from the Fl label is detected using standard methods. Control assays are performed to measure the integrity of the probe. The fluorescence from a fully intact probe is quenched and therefore will not be detected until the F2 label is removed by digestion.

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Abstract

L'invention concerne un procédé consistant à rendre monobrin l'acide nucléique d'un échantillon et à l'exposer à une sonde contenant une séquence complémentaire d'une séquence cible dans des conditions d'hybridation. Tout acide nucléique monobrin présent après l'hybridation est digéré par apport de nucléase. La séquence cible est présente si les sondes sont intactes après la digestion des brins uniques de l'échantillon. Ces sondes possèdent soit un élément dans une paire de fixation spécifique (telle que biotine-avidine), qui est fixé à la sonde à distance d'un marqueur détectable; après digestion, la sonde est immobilisée par l'autre élément de la paire de fixation, soit deux marqueurs fluorescents exerçant un effet réciproque s'ils se trouvent à proximité l'un de l'autre.
PCT/US1996/018615 1995-11-20 1996-11-20 Procede et sonde servant a detecter une sequence cible d'acide nucleique WO1997019192A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO1999036571A2 (fr) * 1998-01-13 1999-07-22 Biochip Technologies Gmbh Procede de detection de sequences nucleotidiques
GB2346694A (en) * 1998-10-12 2000-08-16 Stuart Harbron Hybridisation assay in which excess probe is destroyed
US7560232B2 (en) * 2002-12-19 2009-07-14 Promega Corporation Methods of capturing, detecting and quantifying RNA:DNA hybrids and a modified RNase H useful therein
AU2007231625B2 (en) * 1997-12-19 2011-04-07 Stephen Felder High Throughput Assay System

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WO1991017264A1 (fr) * 1990-05-02 1991-11-14 Imperial Chemical Industries Plc Analyse de sequences specifiques d'acide nucleique
JPH05123195A (ja) * 1991-10-30 1993-05-21 Hitachi Ltd 核酸のホモジニアス検定法
EP0601889A2 (fr) * 1992-12-10 1994-06-15 Maine Medical Center Research Institute Sondes d'acides nucléiques
WO1995025538A1 (fr) * 1994-03-18 1995-09-28 The General Hospital Corporation Methodes de detection de polymorphismes amplifies et clives des sites de restriction
US5466578A (en) * 1992-04-09 1995-11-14 The United States Of America As Represented By The Secretary Of The Navy Surfactant-enhanced light emission- or absorbance-based binding assays for polynucleic acids

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Publication number Priority date Publication date Assignee Title
WO1991017264A1 (fr) * 1990-05-02 1991-11-14 Imperial Chemical Industries Plc Analyse de sequences specifiques d'acide nucleique
JPH05123195A (ja) * 1991-10-30 1993-05-21 Hitachi Ltd 核酸のホモジニアス検定法
US5466578A (en) * 1992-04-09 1995-11-14 The United States Of America As Represented By The Secretary Of The Navy Surfactant-enhanced light emission- or absorbance-based binding assays for polynucleic acids
EP0601889A2 (fr) * 1992-12-10 1994-06-15 Maine Medical Center Research Institute Sondes d'acides nucléiques
WO1995025538A1 (fr) * 1994-03-18 1995-09-28 The General Hospital Corporation Methodes de detection de polymorphismes amplifies et clives des sites de restriction

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JIN ZENG ET AL,: "Differential cDNA cloning by enzymatic degrading subtraction (EDS)", NUCLEIC ACIDS RESEARCH, vol. 22, no. 21, 1994, XP002039836 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2007231625B2 (en) * 1997-12-19 2011-04-07 Stephen Felder High Throughput Assay System
WO1999036571A2 (fr) * 1998-01-13 1999-07-22 Biochip Technologies Gmbh Procede de detection de sequences nucleotidiques
WO1999036571A3 (fr) * 1998-01-13 1999-10-21 Biochip Technologies Gmbh Procede de detection de sequences nucleotidiques
GB2346694A (en) * 1998-10-12 2000-08-16 Stuart Harbron Hybridisation assay in which excess probe is destroyed
GB2346694B (en) * 1998-10-12 2001-01-24 Stuart Harbron Improved hybridisation assay in which excess probe is destroyed
US7560232B2 (en) * 2002-12-19 2009-07-14 Promega Corporation Methods of capturing, detecting and quantifying RNA:DNA hybrids and a modified RNase H useful therein

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