Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • 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
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00596—Solid-phase processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00709—Type of synthesis
  • B01J2219/00711—Light-directed synthesis

Definitions

• the subject invention relates to polynucleotide hybridization assays and provides polynucleotide probes attached to solid supports through photo-labile linkages.
• Polynucleotide hybridization assays are used as research and diagnostic tools for the detection and identification of unique or specific polynucleotide sequences in samples of complete, fragmented, or mixed nucleic acid.
• Various hybridization formats have been developed.
• Southern J. Mo I. Biol., 3J3:503 (1975), discloses a polynucleo ⁇ tide hybridization technique employing radiolabeled nucleic acid probes. This procedure permits autoradiographic detection of probe/analyte hybrids and identification of the polynucleotide se ⁇ quence of the analyte.
• the Southern procedure as well as the other diagnostic procedures employing radiolabeled nucleic acid probes, are very complex, time consuming, and have the additional problems and expenses generally associated with radioactive materials such as personnel monitoring and disposal. Thus, such assays have remained a tool of basic research and are not generally employed in applied or commercial areas such as clinical diagnosis.
• compositions useful as probes in bio- medical research and recombinant DNA technology wherein said probes comprise purine, 7-deazapurine or pyrimidine covalently coupled to a moiety capable of forming a detectable complex with a polypeptide, said moiety being coupled to purine bases at the 8-posi- tion, to deazapurine bases at the 7-position, or to pyrimidine bases at the 5-position to form a modified nucleotide.
• the resulting modified nucleotides are incorporated into DNA by nick-translation techniques.
• U.S. Patent 4,486,539 disclose a tech ⁇ nique for. the sandwich hybridization of nucleic acids, said technique co prising contacting single-stranded nucleic acid from a microor ⁇ ganism with a pair of different nucleic acid reagents, both reagents of the pair being single-stranded and complementary with the microor ⁇ ganism-derived nucleic acid and one of the pair being a nucleic acid fragment attached to a solid carrier , such as a nitrocellulose filter , while the other is a nucleic acid fragment labeled with a radioactive marker , whereby a labeled hybrid is formed attached to the solid carrier , for the identification of a microorganism or group of microor ⁇ ganisms present in a sample. The correctness of the identification is tested by detection of the extent of formation of a labeled hybrid attached to the solid carrier.
• nucleic acid probe compositions which have been chemically modified by the covalent attachment of at least one N-2-acetylaminofIuorene group to one of the bases of the nucleic acid. After hybridization with the target homologous nucleic acid sequence, such hybridization may be detected by the use of enzyme-labeled antibodies .
• Falkow et al . U. S. Patent 4,358 ,535 issued November 9 , 1982 , disclose a method for detecting the presence of a pathogen in a clinical sample by depositing and fixing said sample on an inert support and hybridizing the genetic material of the target pathogen to a labeled-nucleic acid probe.
• the label may be a radioisotope , a ligand , a fluorescer , a chemiluminescer, an enzyme , or an antibody.
• Kourilsky et al . , U . K . Patent Application No. 7913031 , published October 31 , 1 979 disclose a method for detecting the possible presence of a DNA fragment in a sample comprising the hybridization of the sought fragment with an RNA probe which is coupled to an en ⁇ zyme either prior to or subsequent to the hybridization reaction .
• the possible presence of the target nucleic acid sequence is revealed by the action of the enzyme-labeled hybridization product on a chromogen substrate.
• Ohtsuka , et al . disclose 2'-0-(o-nitrobenzyl ) derivatives of uridine , adenosine and cytidine useful in the synthesis of ribo- oligonucleotides .
• the 2'-ether provides 2' protection and such 2'-ether linkage is photo-labile so that the resulting ribo- oligonucleotide can be collected .
• the instant invention provides polynucleotide probes , both RNA and DNA , bound to a solid support through photo-labile linkages .
• Such support-bound probes may be utilized in conventional sandwich hybridization assays and subsequently cleaved by photolysis for measurement of sandwich pairs .
• the unhybridized probes may be cleaved by photolysis for subsequent use in any of the well known hybridization assay formats .
• a support-bound photo-labile primer is provided for attachment of polynucleotide probes thereto for subsequent use in sandwich hybridization assays.
• solid-support primers can be used in "building" polynucleotide probes either for use in sandwich hybridization assays or for subsequent photo-cleavage thereof for use in any of the well known hybridization assay formats.
• the photo-labile l inkages of this invention have the advantage of being stable to normal hybridization conditions , as well as to con ⁇ ditions necessary for the synthesis of polynucleotides , e. g. , the conventional "blocking" and “de-blocking” steps associated with the protection of exocyclic a ino groups and 5'-hydroxyl groups during DNA probe synthesis.
• polynucleotide probe of this invention bound to a solid support through a photo-labile linkage is of the formula
• SS is the solid support
• 0 represents the 3' oxygen of a nucleotide moiety in the polynucleotide probe.
• the phenyl ring may be naphalene provided that the nitro group is on the ring attached to the 3' oxygen of the nucleotide moiety.
• the sol id support can be of a number of polymer supports.
• examples of such supports are fluoropolymer resins , polystyrene resins , silica , nylon and graft copolymers; and preferred resins include chloromethylated and bromomethylated resins. These supports are preferably utilized in the form of beads of about 0. 1 to 200 microns in diameter.
• the support-bound photo-labile primer of this invention is of the formula
• SS is the solid support bound to the phenyl ring as discussed above, and X is a halogen such as Cl or Br.
• This primer is useful for the synthesis of polynucleotide probes (as exemplified below) , or for the attachment of previously synthesized (or natural ) polynucleotide probes .
• the polynucleotide probes are bound to the " support through the photo-labile linkage , they can be cleaved by application of UV light having a wavelength of between about 280 nm and 350 nm. As seen in the example below , cleavage takes place between the 3' oxygen and the adjacent benzylic group of the linkage. Of course , when ribonucleotides are utilized , the 2' oxygen is similarly available for such linkage. This cleavage can be carried out simply to collect the polynucleotide probes for later use, or can be carried out follow ⁇ ing use of the probe as the "capture" probe in a typical sandwich hybridization assay - in which case the "sandwich" is collected for counting .
• This invention also allows for a diagnostic kit useful in the detection of a target polynucleotide analyte in a physiological sample wherein the kit comprises a first polynucleotide probe bound to a solid support through a photo-labile linkage , and a second polynucleotide probe , both the first and second probes being substantially complementary to substantially mutually exclusive single-stranded regions of the analyte.
• target polynucleotide analyte refers to a segment of single-stranded polynucleotide having a nucleotide base sequence corresponding to a genetic element whose presence in a -6-
• physiological sample is to be detected and/or identified; the term “substantially complementary” refers to sufficient nucleotide base sequence homology between a polynucleotide probe and its target analyte to permit formation of stable probe-analtye hybrids; the term “physiological sample” means a sample of blood, urine or other biolog ⁇ ical tissue, either unprocessed or processed, containing the DNA or RNA of interest; and the term “substantially mutually exclusive” means that upon hybridization by the first and second probes with each target analyte, the two probes should not compete for the same nucleotide base sequence on the analyte to the extent that hybridization is prevented.
• polynucleotide refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which can be single- or double-stranded, optionally incorporated or comprising synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA polymers.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• probes can be conveniently isolated in useful quantities by cloning and amplification of polynucleotide sequences complemen ⁇ tary to target polynucleotides in plasmid or phage vectors, using techniques that are now conventional to those skilled in the art.
• a useful reference covering most aspects of DNA manipulation is Maniatis et al., Molecular Cloning, A Laboratory Manual. (Cold Spring Harbor Laboratory, 1982), the disclosure of which is incor ⁇ porated herein by reference.
• An exemplary cloning vehicle for production of useful quantities of probe polynucleotides is plasmid pBR322 (ATCC 37017), which is described in detail by Rodriquez, et al., in Scott, ed.. Molecular Cloning of Recombinant DNA, (Aca ⁇ demic Press, New York, 1977), p. 72.
• Plasmid DNA can be amplified by growth in the presence of chloramphenicol (170 ug/ml) according to the method of Clewell, J. Bacteriol. J1_0:667 (1972); and purified by the cleared lysate procedure of Guerry et al., J. Bacteriol. 116:1064 (1973), prior to digestion with an appropriate endonuclease.
• digestion with Pstl inactivates the ampicillin resistance marker and generates "sticky ends" suitable for ligation to a probe polynucleotide similarly cleaved with Pstl.
• the resulting recombinant plasmid can then be employed to transform a suitable host bacterium, e.g., E. coli K12 HB101.
• a suitable host bacterium e.g., E. coli K12 HB101.
• high plasmid copy numbers can be attained and the recombinant plasmid DNA isolated and purified as previously de ⁇ scribed.
• a particularly preferred vector for production of probe polynucleotides is a coiiphage, M13, (ATCC 15669-BI) which, like pBR322, is now commercially available (New England Nuclear Corporation, Boston, Massachusetts, USA).
• DNA fragments obtained by digestion of phage DNA and DNA complementary to a target DNA of interest can be joined, amplified, and subsequently purified in single-stranded form prior to conjugation with a reporter molecule, e.g., an enzymatic activator polypeptide such as peroxidase.
• a reporter molecule e.g., an enzymatic activator polypeptide such as peroxidase.
• M13 phage as a cloning vehicle has been described by Messing, Recombinant DNA Tech. Bull. 2:43, (1979), the disclosure of which is hereby incorporated by reference.
• the base residue can be any purine, modified purine, pyrimidine, or modified pyrimidine base capable of stable incorporation into a single-stranded polynucleotide without significantly affecting the capacity of the polynucleotide to form hybrids with target polynucleotides having substantial complementarity.
• a common feature of all base residue useful in the present invention is a point or points suitable for attachment, preferably covalent, of any of the well known reporting mechanisms.
• nucleotides can optionally comprise various substituents, which can be linked to either base or sugar portions, and which do not deleterious- ly affect the capability of the resulting polynucleotide to form hybrids with complementary target polynucleotides.
• Polymer "tails" comprising a number of nucleotides appropriate for conjugation to a reporting system can be added to probe polynucleotides by use of calf-thymus terminal deoxynucleotidyl transferase (TdT), which catalyzes the addition of deoxynucleotides to the 3'-hydroxyl ends of single- or double-stranded DNA, as disclosed by Roychoudhury et al., Nucleic Acid Res. 3:101 (1976).
• TdT calf-thymus terminal deoxynucleotidyl transferase
• the resin is filtered through sintered glass funnel and washed with water (2 x 500 mis.), dioxane (2 x 300 mis.), and methanol (2 x 200 mis.).
• the resin is dried under vacuum for 2 hours and stored over Drierite in freezer. Nitrogen analysis of the nitrated resin is done to confirm appropriate nitration of the chloromethylated resin.
• the suspension of sodium hydride/dimethylformamide is withdrawn with another syringe and transferred to a three-necked flask containing 5'-0-blocked 2'-deoxy nucleoside in dry dimethylformamide (2 mis . ) under Argon atmosphere.
• o-nitrochloromethyl resin (84 mg/440 umoles ) is added in small incre ⁇ ments .
• the mixture is allowed to stir at room temperature for 3 hours and then is left standing at room temperature overnight.
• the resin is filtered through a sintered glass filter funnel and washed successively with dimethylformamide (2 mis . x 2) , 90% dimethyl ⁇ formamide in water (2 mis. x 1 ) , and acetonitri le (2 ml . x 2 ) .
• the resin is then dried in a desiccator under vacuum.
• Resin ( 1 -2 mg ) is treated with cone, ammonium hydroxide ( 1 ml . ) and allowed to react in a sealed glass ampule in a heating block overnight at 55°C.
• the resin is centrifuged , and the supernatant (ammonium hydroxide) transferred to another tube. Both the resin and the supernatant are analyzed for the presence of dimethoxytrityl nucleoside based on the liberation of dimethoxytrityl cation when treated without any processing with trichloroacetic in methylene chloride , (supernatant is first evaporated to dryness before treating it with trichloracetic acid in methylene chloride) .
• O-nitrochloromethylated resin is used to synthesize oligodeoxy- nucleotides either manually or on a DNA synthesizer using phosphora- midite or phosphotriester chemistry. In either case, synthesis begins with the attachment of the first deoxynucleoside (at the 3'-end) to o-nitrochloromethylated resin fol lowing the procedure described earlier. The chain is then extended stepwise to the desired length through the 5'-end of the of the oligomer by coupling subsequent nucleoside phosphodiesters or nucleoside phosphoramidites to the 5'-OH end of the preceding nucleoside.
• the desired oligomer Once the desired oligomer is synthesized , it can be subjected to the alkaline treatment (at 55°C) to deblock amino protecting groups of the purine/pyrimidine bases. Under these conditions , the oligomer stays immobilized to support.
• the cleavage of the finished oligonucleotide or the hybridized double stranded oligomer from the photo-labile support can be done by irradiating the sample to 320nm lamp for 4 hours in an RPR-100 apparatus ( Ryanote , the Southern Co. , Ha den , Connecticut) .

Landscapes

• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Nanotechnology (AREA)
• Genetics & Genomics (AREA)
• General Health & Medical Sciences (AREA)
• Zoology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Wood Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Condensed Matter Physics & Semiconductors (AREA)
• Microbiology (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Materials Engineering (AREA)
• Biophysics (AREA)
• Composite Materials (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Analytical Chemistry (AREA)
• Crystallography & Structural Chemistry (AREA)
• Saccharide Compounds (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=25300278

Family Applications (1)

Country Status (3)

Families Citing this family (3)

Citations (1)

Family Cites Families (2)

• 1987
  • 1987-03-31 EP EP19870902955 patent/EP0302881A4/en not_active Withdrawn
  • 1987-03-31 JP JP50228987A patent/JPH01500003A/ja active Pending
  • 1987-03-31 WO PCT/US1987/000659 patent/WO1987005942A1/en not_active Application Discontinuation

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events