WO2002084271A2 - Methode et capteurs de detection et d'analyse luminescente et optoelectronique de polynucleotides - Google Patents

Methode et capteurs de detection et d'analyse luminescente et optoelectronique de polynucleotides Download PDF

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WO2002084271A2
WO2002084271A2 PCT/US2002/012176 US0212176W WO02084271A2 WO 2002084271 A2 WO2002084271 A2 WO 2002084271A2 US 0212176 W US0212176 W US 0212176W WO 02084271 A2 WO02084271 A2 WO 02084271A2
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polynucleotide
sensor
stranded
substrate
chromophore
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PCT/US2002/012176
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WO2002084271A3 (fr
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Daniel E. Morse
Galen D. Stucky
Jennifer N. Cha
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The Regents Of The University Of California
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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
    • 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
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • This invention relates to methods, articles and compositions for the detection and analysis of polynucleotides in a sample.
  • Methods, compositions and articles of manufacture for detecting and assaying a target polynucleotide in a sample are provided.
  • a sample suspected of containing the target polynucleotide is contacted with a single-stranded sensor polynucleotide complementary to the target polynucleotide and an agent that allows the sensor polynucleotide, when present in single-stranded form, to fluoresce upon excitation.
  • the sensor polynucleotide is optionally conjugated to a substrate, which may be an optoelectronic sensing device, and can be micro- or nanoaddressable.
  • a chromophore may be used to absorb energy from the excited sensor polynucleotide and emit light which can be detected.
  • the methods can be used in multiplex form.
  • the agent can be a solvent or solution thereof which permits the single-stranded sensor polynucleotide to fluoresce;
  • exemplary solvents include dimethylformamide and aqueous ethylene glycol.
  • the sensor polynucleotide can be attached to a wide variety of substrates, including biosensors, photodiodes, optoelectronic semiconductor chips or semiconductor thin-films, chips, and can be used in microarray form. Where a chromophore is used, it may be present in the solution in which the assay is performed, or may be incorporated into a substrate.
  • Exemplary chromophores include l,l-dichloro-2,3-diphenyl-4-isopentenyl-l-silacyclobut-2- ene (SCB), coumarin and rhodamine.
  • Sensing devices incorporating the sensor polynucleotide and optionally the chromophore are also provided.
  • Kits comprising reagents useful for performing the methods of the invention are also provided.
  • FIG. 1 Photoluminescence spectra of single- and double-stranded DNA dissolved in dry DMF and irradiated at 300 ran. Single strands of DNA in DMF show intense blue fluorescence at 368 nm with UN excitation. A sample of single-stranded D ⁇ A was annealed with its complementary strand by first heating the DMF-dissolved sample to 70°C in a sand bath followed by slow cooling to room temperature. The original fluorescence is completely quenched upon hybridization to form the double stranded structure.
  • FIG. 3 Color changes and "on/off fluorescence of single- and double-stranded D ⁇ A in the presence of DMF and SCB.
  • A Color seen in visible light. SCB in DMF is orange-red (left). Addition of single-stranded DNA changes the color to light yellow (center). Hybridization to form double-stranded DNA restores the original color (right).
  • B Fluorescence of the same samples as in (A), when irradiated at 302 nm. Spectra corresponding to these samples are shown in Figure 4.
  • Figure 6 depicts the fluorescence shift from green to blue wavelengths when single- stranded DNA is added to a solution of coumarin 535 in DMF.
  • Figure 7 shows a schematic representation of an integrated optoelectronic biosensor for DNA or RNA, or for analytes detected by any of a wide range of protein-based "antennas' such as (but not limited to) antibodies, receptors, enzymes. Analyte binding produces a change in the signal to the electronic output from a microaddressable domain on a semiconductor (chip, wafer or polymeric thin-film).
  • the invention described here is much simpler, utilizing the intrinsic fluorescence of the single-stranded polynucleotides themselves, without the requirement of covalently attaching lumiphores or quenchers to the polynucleotide(s).
  • the quencher is simply the complementary strand of DNA or RNA, and requires no modification.
  • the fluorescence of the single-stranded polynucleotide can be detected directly, or an acceptor can be used, which can optionally be attached to a substrate and/or to a polynucleotide.
  • the acceptor can be, for example, a photodiode, an optoelectonic crystalline or polymeric semiconductor, or a "reporter" dye.
  • the ability of single-stranded polynucleotides to fluoresce strongly in response to UN excitation in particular solvents has not been exploited until now in the scientific or industrial community.
  • Sensitivity of this system is high, permitting the use of very small quantities of D ⁇ A both as the sensor and the analyte. Under the conditions described herein, a significant FRET signal has been detected from as little as 1.8 x 10 "5 nanomoles of single stranded oligonucleotide. Specificity also is high, with the ability to detect even small mismatches between the sensor strand and the analyte strand.
  • the intrinsic blue luminescence of D ⁇ A amplified by an agent such as DMF makes possible the development of extremely simple sensors for target polynucleotides. There is no requirement to label or modify the D ⁇ A strands or use complex methods such as genetic engineering.
  • a wide variety of signal transducers can be used. Thus, direct optoelectronic converters such as photodiodes can be used to transduce the fluorescent signal directly to an electrical signal. Fluorescent transducers such as SCB can be used as intermediate signal amplifiers and/or for wavelength shifting.
  • inventions for assaying for particular polynucleotide sequences have use in a wide variety of different applications, including pharmacogenetic testing and forensic testing to identify the species or individual which was the source of a forensic specimen.
  • Polynucleotide analysis methods can also be used in an antliropological setting.
  • Paternity testing is another area in which such inventions can be used, as is testing for compatibility between prospective tissue or blood donors and patients in need thereof, and in screening for hereditary disorders.
  • the inventions taught herein can be used to study alterations of gene expression in response to a stimulus, disease, drug or medication.
  • Other applications include human population genetics, analyses of human evolutionary history, and characterization of human haplotype diversity.
  • the inventions can also be used to detect polynucleotide sequences from contaminants or pathogens including bacteria, yeast and viruses, and to detect single nucleotide polymorphisms, which may be associated with particular alleles or subsets of alleles. Over 1.4 million different single nucleotide polymorphisms (SNPs) in the human population have been identified (Nature 2001 409:928-933).
  • SNPs single nucleotide polymorphisms
  • the inventions can be used for detection of mutations. Any type of mutation can be detected, including without limitation SNPs, insertions, deletions, transitions, transversions, inversions, frame shifts, triplet repeat expansions, and chromosome rearrangements.
  • the invention can be used to detect nucleotide sequences associated with increased risk of diseases or disorders, including cystic fibrosis, Tay-Sachs, sickle-cell anemia, etc.
  • the inventions described herein are useful for any assay in which a sample can be interrogated regarding a target polynucleotide.
  • Typical assays involve determining the presence of the target polynucleotide in the sample or its relative amount, or the assays may be quantitative or semi-quantitative.
  • the methods of the invention can all be performed in multiplex formats.
  • a plurality of different sensor polynucleotides which preferentially bind to corresponding target polynucleotides can be conjugated to the same substrate, or to a plurality of different distinguishable substrates.
  • Multiplex methods are provided employing 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000 or more different sensor polynucleotides which can be used simultaneously to assay for corresponding different target polynucleotides.
  • a target polynucleotide includes a plurality of target polynucleotides
  • a substrate includes a plurality of such substrates
  • a sensor polynucleotide includes a plurality of sensor polynucleotides
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • nucleic acid molecule polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA").
  • DNA triple-, double- and single-stranded deoxyribonucleic acid
  • RNA triple-, double- and single-stranded ribonucleic acid
  • polynucleotide oligonucleotide
  • nucleic acid oligonucleotide
  • nucleic acid molecule include polydeoxyribonucleotides (containing 2-deoxy-D-r ⁇ bose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidme base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (“PNAs”)) and polymorpholino
  • PNAs peptide nucleic acids
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • these terms refer only to the primary structure of the molecule.
  • these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, and hybrids thereof including for example hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, amino
  • nucleases nucleases
  • toxins antibodies
  • signal peptides poly-L-lysine, etc.
  • intercalators e.g., acridine, psoralen, etc.
  • chelates of, e.g., metals, radioactive metals, boron, oxidative metals, etc.
  • alkylators those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
  • nucleoside and nucleotide will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like.
  • the term “nucleotidic unit” is intended to encompass nucleosides and nucleotides.
  • modifications to nucleotidic units include rearranging, appending, substituting for or otherwise altering functional groups on the purine or pyrimidine base which form hydrogen bonds to a respective complementary pyrimidine or purine.
  • the resultant modified nucleotidic unit optionally may form a base pair with other such modified nucleotidic units but not with A, T, C, G or U.
  • Abasic sites may be incorporated which do not prevent the function of the polynucleotide, i.e. do not cause a change in fluorescence in the assay being performed that obscures the signal or change in signal which is desired to be detected; preferably the polynucleotide does not comprise abasic sites.
  • Some or all of the residues in the polynucleotide can optionally be modified in one or more ways.
  • Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the Nl and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2, of cytidine and the C2- NH2, N'-H and C6 ⁇ oxy, respectively, of guanosine.
  • guanosine (2-amino- 6-oxy-9- ⁇ -D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9- ⁇ -D-ribofuranosyl-purine).
  • Nucleic acid probe and “probe” are used interchangeably and refer to a structure comprising a polynucleotide, as defined above, that contains a nucleic acid sequence that can bind to a corresponding target.
  • the polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
  • Complementary or “substantially complementary” refers to the ability to hybridize or base pair between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between a polynucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%o, and more preferably from about 98 to 100%.
  • RNA or DNA strand will hybridize under selective hybridization conditions to its complement.
  • selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary.
  • Preferential binding or “preferential hybridization” refers to the increased propensity of one polynucleotide to bind to a complementary target polynucleotide in a sample as compared to noncomplementary polynucleotides in the sample.
  • Stringent hybridization conditions will typically include salt concentrations of less than about IM, more usually less than about 500 mM and preferably less than about 200 mM.
  • Hybridization temperatures can be as low as 5°C, but are typically greater than 22 °C, more typically greater than about 30°C, and preferably in excess of about 37°C. Longer fragments may require higher hybridization temperatures for specific hybridization. Other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, and the combination of parameters used is more important than the absolute measure of any one alone.
  • hybridization conditions which may be controlled include buffer type and concentration, solution pH, presence and concentration of blocking reagents to decrease background binding such as repeat sequences or blocking protein solutions, detergent type(s) and concentrations, molecules such as polymers which increase the relative concentration of the polynucleotides, metal ion(s) and their concentration(s), chelator(s) and their concentrations, and other conditions known in the art.
  • aptamer (or “nucleic acid antibody”) is used herein to refer to a single- or double-stranded polynucleotide that recognizes and binds to a desired target molecule by virtue of its shape. See, e.g., PCT Publication No s. WO 92/14843, WO 91/19813, and WO
  • Polypeptide and “protein” are used interchangeably herein and include a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” “oligopeptides,” and “proteins” are included within the definition of polypeptide. The terms include polypeptides contain [post-translational] modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and sulphations. In addition, protein fragments, analogs (including amino acids not encoded by the genetic code, e.g.
  • homocysteine, ornithine, D-amino acids, and creatine natural or artificial mutants or variants or combinations thereof, fusion proteins, derivatized residues (e.g. alkylation of amine groups, acetylations or esterifications of carboxyl groups) and the like are included within the meaning of polypeptide.
  • substrate and “support” are used interchangeably and refer to a material having a rigid or semi-rigid surface.
  • Alkyl refers to a branched, unbranched or cyclic saturated hydrocarbon group of 1 to 24 carbon atoms optionally substituted at one or more positions, and includes polycyclic compounds.
  • alkyl groups include optionally substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-amyl, isoamyl, n-hexyl, n-heptyl, n-octyl, n-decyl, hexyloctyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like, as well as cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and norbornyl.
  • lower alkyl refers to an alkyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.
  • Alkylaryl refers to an alkyl group that is covalently joined to an aryl group.
  • the alkyl is a lower alkyl.
  • Exemplary alkylaryl groups include benzyl, phenethyl, phenopropyl, 1-benzylethyl, phenobutyl, 2-benzylpropyl and the like.
  • Amide refers to -C(O)NR'R", where R' and R" are independently selected from hydrogen, alkyl, aryl, and alkylaryl.
  • “Amine” refers to an -N(R')R" group, where R' and R" are independently selected from hydrogen, alkyl, aryl, and alkylaryl.
  • Aryl refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocyciic, heterocychc and polycyclic aryl groups, and can be optionally substituted at one or more positions. Typical aryl groups contain 1 to 5 aromatic rings, which may be fused and/or linked.
  • Exemplary aryl groups include phenyl, furanyl, azolyl, thiofuranyl, pyridyl, pyrimidyl, pyrazinyl, triazinyl, indenyl, benzofuranyl, indolyl, naphthyl, quinolinyl, isoquinolinyl, quinazolinyl, pyridopyridinyl, pyrrolopyridinyl, purinyl, tetralinyl and the like.
  • substituents on optionally substituted aryl groups include alkyl, alkoxy, alkylcarboxy, alkenyl, alkenyloxy, alkenylcarboxy, aryl, aryloxy, alkylaryl, alkylaryloxy, fused saturated or unsaturated optionally substituted rings, halogen, haloalkyl, heteroalkyl, -S(O)R, sulfonyl, -SO 3 R, -SR, -NO 2 , -NRR', -OH, -CN, -C(O)R, -OC(O)R, - NHC(0)R, -(CH2) n CO 2 R or -(CH2) n CONRR' where n is 0-4, and wherein R and R' are independently H, alkyl, aryl or alkylaryl.
  • Carbocyciic refers to an optionally substituted compound containing at least one ring and wherein all ring atoms are carbon,
  • Carbocyciic aryl refers to an optionally substituted aryl group wherein the ring atoms are carbon.
  • Halo or “halogen” refers to fluoro, chloro, bromo or iodo. Of the halogens, chloro and fluoro are generally preferred.
  • Haloalkyl refers to an alkyl group substituted at one or more positions with a halogen, and includes alkyl groups substituted with only one type of halogen atom as well as. alkyl groups substituted with a mixture of different types of halogen atoms. Exemplary haloalkyl groups include trihalomethyl groups, for example trifluoromethyl.
  • Heteroalkyl refers to an alkyl group wherein one or more carbon atoms and associated hydrogen atom(s) are replaced by an optionally substituted heteroatom, and includes alkyl groups substituted with only one type of heteroatom as well as alkyl groups substituted with a mixture of different types of heteroatoms. Heteroatoms include oxygen, sulfur, and nitrogen. As used herein, nitrogen heteroatoms and sulfur heteroatoms include any oxidized form of nitrogen and sulfur, and any form of nitrogen having four covalent bonds including protonated forms. An optionally substituted heteroatom refers to replacement of one or more hydrogens attached to a nitrogen atom with alkyl, aryl, alkylaryl or hydroxyl.
  • Heterocychc refers to a compound containing at least one saturated or unsaturated ring having at least one heteroatom and optionally substituted at one or more positions.
  • Typical heterocychc groups contain 1 to 5 rings, which may be fused and/or linked, where the rings each contain five or six atoms.
  • Examples of heterocychc groups include piperidinyl, morpholinyl and pyrrolidinyl.
  • Exemplary substituents for optionally substituted heterocychc groups are as for alkyl and aryl at ring carbons and as for heteroalkyl at heteroatoms.
  • Heterocychc aryl refers to an aryl group having at least 1 heteroatom in at least one aromatic rings.
  • exemplary heterocychc aryl groups include furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-lower alkyl-pyrrolo, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl, tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl, phenanthrolinyl, purinyl and the like.
  • Hydrocarbyl refers to hydrocarbyl substituents containing 1 to about 20 carbon atoms, including branched, unbranched and cyclic species as well as saturated and unsaturated species, for example alkyl groups, alkylidenyl groups, alkenyl groups, alkylaryl groups, aryl groups, and the like.
  • the term “lower hydrocarbyl” intends a hydrocarbyl group of one to six carbon atoms, preferably one to four carbon atoms.
  • a “substiruent” refers to a group that replaces one or more hydrogens attached to a carbon or nitrogen.
  • OC(0)R -NHC(O)R, -(CH2) n C0 2 R or -(CH2) n CONRR' where n is 0-4, and wherein R and R' are independently H, alkyl, aryl or alkylaryl. Substituents also include replacement of a carbon atom and one or more associated hydrogen atoms with an optionally substituted heteroatom. "Multiplexing" herein refers to an assay or other analytical method in which multiple analytes can be assayed simultaneously.
  • Thioamide refers to -C(S)NHR, where R is alkyl, aryl, alkylaryl or hydrogen.
  • Thioether refers to -SR, where R is alkyl, aryl, or alkylaryl.
  • RESULTS OF ASSAYING A SAMPLE FOR A COMPLEMENTARY POLYNUCLEOTIDE Single strands of polynucleotides are shown herein to exhibit strong photoluminescence in the presence of certain solvents, and the resulting luminescence can be transferred to excite other dyes that can be used as reporters or directly to photodiodes or semiconductors for quantitation.
  • a wide range of spectral sensitivities can be obtained by tuning the wavelength absorption and emission maxima of the reporter dyes, photodiodes and/or semiconductors by chemical modification.
  • Intercalation of the aromatic bases of the single-stranded polynucleotide with aromatic lumiphores additionally facilitates both radiative and non-radiative energy transfer to the reporter dye.
  • Quenching of the fluorescence by hybridization with a complementary single-stranded polynucleotide reduces the output of luminescence.
  • hybridization reduces the fluorescence by at least about 70 percent, and preferably at least about 75, 80, 85, or 90 percent or more.
  • hybridization reduces the fluorescence somewhat less, typically by at least about 60 percent, and preferably at least about 65, 70, 75, or 80 percent or more. This quenching is extremelyly sensitive to even a slight mismatch in base-pairing, and thus provides a sensitive and specific detection method for specific sequences of DNA or RNA in a new type of luminescence-based sensor.
  • detection of the analyte can be reported directly as a change in electronic current from a microaddressable domain on an optoelectronic semiconductor chip or optoelectronic semiconductor polymeric thin-film.
  • the method can be modified to use ssRNA as the detector, especially in applications seeking to detect complementary DNA.
  • the intrinsic and intense luminescence of single strands of polynucleotides upon interaction with such solvents can be used in new ways to create sensors for detecting and/or quantitating complementary (matching) sequences of DNA and/or RNA in a sample.
  • This energy can be transferred directly to a photodiode or optoelectronic semiconductors (including crystalline materials such as indium-doped gallium nitride, or polymeric thin-film semiconductors such as those based on polyanilines) for quantitation, or it can be transferred either radiatively or non-radiatively (via intercalation of the aromatic bases of the DNA with aromatic antenna groups of acceptor molecules to secondary "reporter" dyes (such as 1,1- dichloro-2,3-diphenyl-4-isopentenyl-l-silacyclobut-2-ene, coumarin or rhodamine) with a resulting fluorescent energy transfer leading to the excitation of emission from the reporter.
  • secondary "reporter" dyes such as 1,1- dichloro-2,3-diphenyl-4-isopentenyl-l-silacyclobut-2-ene, coumarin or rhodamine
  • semiconducting polymers which may be used include polyphenylene, poly
  • the luminescence of single stranded polynucleotides thus can be harnessed in a wide range of DNA-based luminescence and optoelectronic biosensors.
  • Luminescence from single-stranded polynucleotides can be transferred either by fluorescence resonance energy transfer (FRET) or by Forster energy transfer to a second chromophore in intimate mixture with the single-stranded polynucleotides in DMF.
  • FRET fluorescence resonance energy transfer
  • Example 4 Another example of this energy transfer from DNA to a second chromophore is set forth in Example 4 ( Figure 6).
  • Samples of different single-stranded oligonucleotides can be solubilized in DMF (or other suitable solvents) and placed in aliquots in microwell titer plates. A chromophore that can obtain energy from the excited single-stranded oligonucleotide and emit light is then added to each well. Test samples to be analyzed for their content of complementary DNA or RNA strands then are added.
  • the presence and amount of DNA or RNA complementary to the original ssDNA in each well is determined either by directly measuring the reduction of fluorescence of the original ssDNA, or by measuring the reduction of fluorescence from the chromophore, or by quantitation of a red shift of the overall fluorescence.
  • single strands of DNA shift the coumarin 535 fluorescence from green to blue; upon the. addition of the complementary DNA or RNA, the overall fluorescence of the solution is shifted back to the green.
  • Another application of these findings to sensors is achieved by anchoring either the single-stranded sensor polynucleotide, the reporter chromophore, or both, to a solid substrate.
  • These substrates may be surfaces of glass, silicon, paper, plastic, or the surfaces of optoelectronic semiconductors (such as, but not confined to, indium-doped gallium nitride or polymeric polyanilines, etc.) employed in the device as optoelectronic transducers.
  • the reporter chromophore can be polymerized and incorporated directly in thin films.
  • Sol-gel materials also can be synthesized using the diethoxy- or dimethoxy- fo ⁇ n of the SCB compound shown in Figure 1, and used for this purpose.
  • transparent sol-gel films with incorporated dye molecules also can easily be made and used for this purpose.
  • the single-stranded polynucleotide detectors can be attached in patterned microscale and nanoscale arrays to these substrates, with corresponding increases in high- throughput capacity and speeds of analysis.
  • the agent can be any solvent or solution thereof in which a single-stranded polynucleotide can dissolve and exhibit detectable fluorescence and/or FRET or Forster energy transfer; the solution must also permit the single-stranded polynucleotide to hybridize to a complementary polynucleotide, when present, and thereby detectably reduce the fluorescence or energy transfer.
  • Single-stranded polynucleotides do not exhibit fluorescence in purely aqueous solvents.
  • At least one solvent other than water is required, and the solution can be entirely nonaqueous; for example, solutions of single-stranded polynucleotides in aqueous ethylene glycol do exhibit such properties, as do solutions of single-stranded polynucleotides in dimethylformamide (DMF).
  • the agent must be provided in a sufficient amount for the fluorescence properties of the single-stranded polynucleotide to be manifested.
  • the solvent preferably does not absorb or emit amounts of relevant wavelengths of light so as to impede the assay being performed.
  • solvent includes solvent systems comprising two or more different solvents.
  • Exemplary solvents which can be tested to determine their applicability to the methods described herein include acetal (1,1-diethoxyethane), acetic acid, acetone, acetonitrile, acetylacetone, acrylonitrile, adiponitrile, allyl alcohol, allylamine, 2- aminoisobutanol, benzal chloride, benzaldehyde, benzene, benzonitrile, benzyl chloride, bromochloromethane, bromoform (tribromomethane), butyl acetate, butyl alcohol, sec-butyl alcohol, t ⁇ ?'t-butyl alcohol, butylamine, tert-butylamine, butyl methyl ketone, p-tert- butyltoluene, Y-butyrolactone, caprolactam, carbon disulfide, carbon tetrachloride, 1-chloro- 1,1-difluoroe
  • the agent is preferably an optionally substituted carboxylic acid amide, and preferably an optionally substituted formic or lower alkyl acid amide.
  • exemplary agents include formamide, acetamide, N-methylacetamide, N-methylformamide, NN- dimethylacetamide, and NN-dimethylformamide.
  • the agent may react with the chromophore.
  • the portion of the sample comprising or suspected of comprising the target polynucleotide can be any source of biological material which comprises polynucleotides that can be obtained from a living organism directly or indirectly, including cells, tissue or fluid, and the deposits left by that organism, including viruses, mycoplasma, and fossils.
  • the sample can also comprise a target polynucleotide prepared through synthetic means, in whole or in part. Typically, the sample is obtained as or dispersed in a predominantly aqueous medium.
  • sample examples include blood, urine, semen, milk, sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, an aspirate, a needle biopsy, a section of tissue obtained for example by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, tumors, organs, samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components), and a recombinant library comprising polynucleotide sequences.
  • in vitro cell culture constituents including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components
  • a recombinant library comprising polynu
  • the sample can be a positive control sample which is known to contain the target polynucleotide or a surrogate therefor.
  • a negative control sample can also be used which, although not expected to contain the target polynucleotide, is suspected of containing it, and is tested in order to confirm the lack of contamination by the target polynucleotide of the reagents used in a given assay, as well as to determine whether a given set of assay conditions produces false positives (a positive signal even in the absence of target polynucleotide in the sample).
  • the sample can be diluted, dissolved, suspended, extracted or otherwise treated to solubilize and/or purify any target polynucleotide present or to render it accessible to reagents which are used in an amplification scheme or to detection reagents.
  • the cells can be lysed or permeabilized to release the polynucleotides within the cells.
  • One step permeabilization buffers can be used to lyse cells which allow further steps to be performed directly after lysis, for example a polymerase chain reaction.
  • the target polynucleotide is single-stranded as used in the assays described herein, but can originally be obtained in single-stranded, double-stranded, or higher order forms, and can be linear or circular.
  • Exemplary single-stranded sources of the target polynucleotide include mRNA, rRNA, tRNA, l nRNA, ssRNA or ssDNA viral genomes, although these polynucleotides may contain internally complementary sequences and significant secondary structure.
  • Exemplary double-stranded sources of the target polynucleotide include genomic
  • the target polynucleotide is obtained in double-stranded or higher order form, the target polynucleotide is denatured at some point prior to performing the assay to allow access by the sensor polynucleotide; any denaturation method that does not impede the performance of the assay can be used, for example heating, low osmolarity, high pH, and combinations of methods.
  • the target polynucleotide can be prepared synthetically or purified from a biological source.
  • the target polynucleotide may be purified to remove or diminish one or more undesired components of the sample or to concentrate the target polynucleotide prior to amplification. Conversely, where the target polynucleotide is too concentrated for a particular assay, the target polynucleotide may first be diluted.
  • the nucleic acid portion of the sample comprising the target polynucleotide can be subjected to one or more preparative reactions. These preparative reactions can include in vitro transcription (INT), labeling, fragmentation, reverse transcription, amplification and other reactions. Nucleic acid amplification increases the copy number of sequences of interest.
  • amplification methods are suitable for use, including the polymerase chain reaction method (PCR), the ligase chain reaction (LCR), self sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASB A), the use of Q Beta replicase, reverse transcription, nick translation, and the like, and combinations thereof.
  • Amplified target polynucleotides may be subjected to post amplification treatments. For example, in some cases, it may be desirable to fragment the target polynucleotide prior to hybridization with a polynucleotide array, in order to provide segments which are more readily accessible to the target polynucleotides and which avoid looping and/or hybridization to multiple probes. Fragmentation of the nucleic acids can be carried out by any method producing fragments of a size useful in the assay being performed; suitable physical, chemical and enzymatic methods are known in the art.
  • a sensor polynucleotide is provided that is complementary to the target polynucleotide to be assayed, and has a predetermined sequence.
  • the sensor polynucleotide can be provided in solution or conjugated to a substrate; exemplary substrates are described below.
  • the sensor polynucleotide can be branched, multimeric or circular, but is typically linear, and can contain nonnatural bases.
  • the sensor polynucleotide can be synthesized and provided in solution, or can be synthesized directly on the substrate to be used in the assay, or can be synthesized separately from the substrate and then coupled to it.
  • Direct synthesis on the substrate may be accomplished by incorporating a monomer that is coupled to a subunit of the sensor polynucleotide into a polymer that makes up or is deposited on or coupled to the substrate, and then synthesizing the remainder of the sensor polynucleotide to incorporate that subunit.
  • the substrate or its coating may include or be derivatized to include a functional group which can be coupled to a subunit of the sensor polynucleotide for synthesis, or may be coupled directly to the complete sensor polynucleotide. Suitable coupling techniques are known in the art.
  • the substrate can comprise a wide range of material, either biological, nonbiological, organic, inorganic, or a combination of any of these.
  • the substrate maybe a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, Si0 , SiN , modified silicon, or any one of a wide variety of gels or polymers such as
  • polystetrafluoroethylene (poly)vinylidenedifiuoride, polystyrene, cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolic acid, poly(lactide coglycolide), polyanhydrides, poly(methyl methacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymeric silica, latexes, dextran polymers, epoxies, polycarbonates, or combinations thereof.
  • Conducting polymers and photoconductive materials can be used.
  • Substrates can be planar crystalline substrates such as silica based substrates (e.g. glass, quartz, or the like), or crystalline substrates used in, e.g., the semiconductor and microprocessor industries, such as silicon, gallium arsenide, indium doped GaN and the like, and includes semiconductor nanocrystals.
  • silica based substrates e.g. glass, quartz, or the like
  • crystalline substrates used in, e.g., the semiconductor and microprocessor industries such as silicon, gallium arsenide, indium doped GaN and the like, and includes semiconductor nanocrystals.
  • the substrate can take the form of a photodiode, an optoelectronic sensor such as an optoelectronic semiconductor chip or optoelectronic thin-film semiconductor, or a biochip.
  • the location(s) of the individual sensor polynucleotide(s) on the substrate can be addressable; this can be done in highly dense formats, and the location(s) can be microaddressable or nanoaddressable.
  • Silica aerogels can also be used as substrates, and can be prepared by methods known in the art. Aerogel substrates may be used as free standing substrates or as a surface coating for another substrate material.
  • the substrate can take any form and typically is a plate, slide, bead, pellet, disk, particle, microp article, nanoparticle, strand, precipitate, optionally porous gel, sheets, tube, sphere, container, capillary, pad, slice, film, chip, multiwell plate or dish, optical fiber, etc.
  • the substrate can be any form that is rigid or semi-rigid.
  • the substrate may contain raised or depressed regions on which a sensor polynucleotide is located.
  • the surface of the substrate can be etched using well known techniques to provide for desired surface features, for example trenches, v-grooves, mesa structures, or the like.
  • Surfaces on the substrate can be composed of the same material as the substrate or can be made from a different material, and can be coupled to the substrate by chemical or physical means.
  • Such coupled surfaces may be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or any of the above-listed substrate materials.
  • the surface can be optically transparent and can have surface Si-OH functionalities, such as those found on silica surfaces.
  • the substrate and/or its optional surface are chosen to provide appropriate optical characteristics for the synthetic and/or detection methods used.
  • the substrate and or surface can be transparent to allow the exposure of the substrate by light applied from multiple directions.
  • the substrate and/or surface may be provided with reflective "mirror" structures to increase the recovery of light.
  • the substrate and/or its surface is generally resistant to, or is treated to resist, the conditions to which it is to be exposed in use, and can be optionally treated to remove any resistant material after exposure to such conditions.
  • Sensor polynucleotides can be fabricated on or attached to the substrate by any suitable method, for example the methods described in U.S. Pat. No. 5,143,854, PCT Publ.
  • Still further techniques include bead based techniques such as those described in PCT
  • Reagents are delivered to the substrate by either (1) flowing within a channel defined on predefined regions or (2)
  • a protective coating such as a hydrophilic or hydrophobic coating (depending upon the nature of the solvent) can be used over portions of the substrate to be protected, sometimes in combination with materials that facilitate wetting by the reactant solution in other regions. In this manner, the flowing solutions are further prevented from passing outside of their designated flow paths.
  • Typical dispensers include a micropipette optionally robotically controlled, an ink-jet printer, a series of tubes, a manifold, an array of pipettes, or the like so that various reagents can be delivered to the reaction regions sequentially or simultaneously.
  • Chromophores useful in the inventions described herein include any substance which can absorb energy from a single-stranded polynucleotide in an appropriate solution and emit light.
  • the chromophore can be a lumiphore or a fluorophore.
  • Typical fluorophores include fluorescent dyes, semiconductor nanocrystals, and lanthanide chelates.
  • Exemplary fluorescent dyes include coumarin 535, l,l-dichloro-2,3-diphenyl-4- isopentenyl-l-silacyclobut-2-ene (SCB) and dialkoxy- forms thereof such as diethoxy- and dimethoxy-, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamme, a carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy2, JOE, NED, ROX, HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green
  • Alexa Fluor 350 Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br 2 , BODIPY 530/550, BODJJPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, BODIPY R6G, BODIPY TMR and BODIPY
  • fluorescent semiconductor nanocrystals are known in the art; methods of producing and utilizing semiconductor nanocrystals are described in: PCT Publ. No. WO 99/26299 published May 27, 1999, inventors Bawendi et al.; USPN 5,990,479 issued Nov. 23, 1999 to Weiss et al.; and Bruchez et al., Science 281:2013, 1998.
  • Exemplary lanthanide chelates include europium chelates, terbium chelates and samarium chelates.
  • the excitation source can comprise blue or UN wavelengths shorter than the emission wavelength(s) to be detected.
  • the source may be: a broadband UN light source such as a deuterium lamp with an appropriate filter, the output of a white light source such as a xenon lamp or a deuterium lamp after passing through a monochromator to extract out the desired wavelengths, a continuous wave (cw) gas laser, a solid state diode laser with output in the blue, or any of the pulsed lasers with output in the blue.
  • This excitation source is of an energy capable of exciting the single-stranded polynucleotide(s) used in the experiment to emit light.
  • Kits comprising reagents useful for performing the methods of the invention are also provided, h one embodiment, a kit comprises a single-stranded sensor polynucleotide that is complementary to the target polynucleotide of interest and an agent that allows the sensor polynucleotide to fluoresce when present in single-stranded form.
  • the sensor polynucleotide can bind to the target polynucleotide, and a sample may be assayed for the presence or amount of the target polynucleotide using the components of the kit.
  • the sensor polynucleotide is optionally attached to a substrate as described above.
  • a chromophore as described above may also optionally be included in the kit.
  • kit The components of the kit are retained by a housing. Instructions for using the kit to perform a method of the invention are provided with the housing, and may be located inside the housing or outside the housing, and may be printed on the interior or exterior of any surface forming the housing which renders the instructions legible.
  • the kit may be in multiplex form, containing pluralities of one or more different sensor polynucleotides which can hybridize to corresponding different target polynucleotides.
  • oligodeoxyriboA oligo dA
  • oligodeoxyriboT oligo dT
  • oligodeoxyriboG oligo dG
  • oligodeoxyriboG oligo dC
  • Figure IB The sequences of oligonucleotides used were as follows: oligo dG-5'GGGGGGGGGGGGG3'; oligo dA- 5AAAAAAAAAAAAAAA3'; oligo dC-5'CCCCCCCCCCCCCC3'; and oligo dT- 5TTTTTTTTTTTTTTTTTT3'.
  • the spectra of the purine-containing oligo dA and oligo dG showed the same peak emission maxima and shape but differed distinctly from the similar emission spectra of the pyrimidine-containing oligo dC and oligo dT ( Figure IB), demonstrating that the luminescence wavelength profiles are strongly dependent on the aromatic structures of the specific bases.
  • Representative quantum yield and fluorescence lifetime measurements were performed with oligodeoxyribonucleotide A, yielding values of ca. 5%, and two lifetime components of 13ns and 195ns.
  • the samples were excited with the 337 nm line of a pulsed (4 ns pulse width) nitrogen laser.
  • the emission was dispersed by a monochromator and analyzed using a digital oscilloscope.
  • the observed decay of photoluminescence was stimulated using a two exponential fitting procedure. Sequence specific differences in the quantum efficiency values are expected.
  • This transduced signal can further be used as a reporter for D ⁇ A hybridization.
  • the complementary strand B was added to a DMF solution containing both the ssD ⁇ A A and SCB and annealing was allowed to occur, the resulting Watson-Crick hybridization completely quenched the UN-excited fluorescence resulting from energy transfer between the D ⁇ A and SCB (Figs. 3B, 4).
  • the annealing conditions alone caused no quenching of the ssD ⁇ A fluorescence nor of the FRET signal.
  • Hybridization between the complementary strands also caused the visible color of the SCB solution to return to its original orange-red (Fig. 3 A).
  • the requisite energy transfer between the ssD ⁇ A and the SCB may depend on the direct intercalation of the aromatic bases of the ssD ⁇ A with the phenyl groups of the SCB, facilitating ⁇ - ⁇ interaction between the energy donor and acceptor species, or on some similar electronic or spatial coupling.
  • This suggestion is further supported by the observation that the interaction with single-stranded D ⁇ A alters the color of the SCB in DMF, while interaction with double-stranded D ⁇ A (in which the aromatic bases are unavailable) leaves the color of
  • This system also allows for the detection of D ⁇ A mismatches through fluorescence shifts.
  • oligodeoxyribonucleotide C (5'CCCATGATGCAAAT3'), containing a 2 base-pair mismatch with strand A, was hybridized with A, a significant blue shift in fluorescence was observed (Fig. 5).
  • Fig. 5 a significant blue shift in fluorescence was observed.
  • the commercially available dye, coumarin 535 was dissolved in DMF and exposed to UN excitation at 280 nm; it displayed a fluorescent peak at -520.
  • Two samples were prepared, one containing ssD ⁇ A, and one lacking ssD ⁇ A; each was dissolved in DMF along with coumarin 535.
  • Addition of single-stranded D ⁇ A shifted the fluorescence emission spectrum of the coumarin-containing solution to blue wavelengths, with a strong emission at 380 nm and a weaker emission at 494 nm (see Figure 6).
  • the samples of coumarin 535 alone and in the presence of ssD ⁇ A were excited with a hand-held UN lamp, the green and blue colors of the two samples were readily detectable with the naked eye.

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Abstract

L'invention concerne des méthodes, des compositions et des articles d'analyse d'un échantillon à la recherche d'un polynucléotide cible. Un échantillon susceptible de contenir le polynucléotide cible est mis en contact avec un polynucléotide capteur monocaténaire complémentaire du polynucléotide cible et un agent permettant au polynucléotide capteur lui-même, lorsqu'il est présent sous forme monocaténaire, de devenir fluorescent avec l'excitation. Le polynucléotide capteur est éventuellement conjugué à un substrat, qui peut être un dispositif de détection optoélectronique, et peut être micro ou nano adressable. Un chromophore peut être utilisé pour absorber l'énergie du polynucléotide capteur excité et émettre de la lumière. Les méthodes de l'invention peuvent être utilisées sous forme multiplexe. L'invention concerne également des dispositifs de détection incorporant le polynucléotide capteur et éventuellement le chromophore. L'invention concerne enfin des kits comprenant des réactifs permettant de mettre en oeuvre lesdites méthodes.
PCT/US2002/012176 2001-04-16 2002-04-16 Methode et capteurs de detection et d'analyse luminescente et optoelectronique de polynucleotides WO2002084271A2 (fr)

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USRE47874E1 (en) 2002-08-26 2020-02-25 The Regents Of The University Of California Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores
EP1549772A4 (fr) * 2002-08-26 2007-10-24 Univ California Procedes et compositions de detection et d'analyse de polynucleotides au moyen de multichromophores collecteurs de lumiere
US8841072B2 (en) 2002-08-26 2014-09-23 The Regents Of The University Of California Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores
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