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WO1999035288A1 - Substrat polynucleotidique clivable specifique d'enzymes et procede d'analyse associe - Google Patents

Substrat polynucleotidique clivable specifique d'enzymes et procede d'analyse associe Download PDF

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Publication number
WO1999035288A1
WO1999035288A1 PCT/US1998/017311 US9817311W WO9935288A1 WO 1999035288 A1 WO1999035288 A1 WO 1999035288A1 US 9817311 W US9817311 W US 9817311W WO 9935288 A1 WO9935288 A1 WO 9935288A1
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Prior art keywords
polynucleotide
fluorescent
enzyme
moieties
reagent according
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PCT/US1998/017311
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English (en)
Inventor
Ai-Ping Wei
Patrick A. Mach
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Minnesota Mining And Manufacturing Company
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Publication date
Application filed by Minnesota Mining And Manufacturing Company filed Critical Minnesota Mining And Manufacturing Company
Priority to AU91110/98A priority Critical patent/AU9111098A/en
Priority to KR1020007007574A priority patent/KR100575407B1/ko
Priority to EP98943281A priority patent/EP1045925A1/fr
Priority to JP2000527669A priority patent/JP2002508935A/ja
Publication of WO1999035288A1 publication Critical patent/WO1999035288A1/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
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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

Definitions

  • This invention relates to a reagent comprising an enzyme-specific cleavable polynucleotide substrate bearing quenched fluorescent moieties, a method of making the same, and an assay method of using the same.
  • Nucleases are enzymes that hydrolyze, and cleave, polynucleotides, i.e., nucleic acids, such as DNA and RNA. Such enzymes are present in every living cell to perform many essential functions in nucleic-acid metabolism, replication, recombination, repair, and modification. Nucleases can be differentiated by their substrate specificity: some cleave both DNA and RNA, others act on one type of nucleic acid only; some are sequence specific and others are not; some act on single-stranded nucleic acid, others require double stranded DNA to function.
  • Nucleases may also be selective in, among other things, the location of the bond they cleave; their cellular location, i.e., intra- or extracellular; ionic and pH requirements; and resistance to heat treatment. Nucleases are further explained in Linn, S.M., Lloyd, R.S., Roberts, R.J., Nucleases (2 nd ed.), Cold Spring Harbor Laboratory Press, Plainview, NY, 1994.
  • Antibiotic resistant bacteria strains and food-borne illnesses are of significant concern to the public.
  • the management ofmicrobial risks in healthcare, cosmetics, food and beverage industries is a serious health and safety issue.
  • Bacterial testing is an integral part of managing microbial risks.
  • Enzymes produced by bacteria are frequently used to test for the presence of that bacteria.
  • the ability of microorganisms to hydrolyze and cleave nucleic acids through production of nuclease has long been recognized. Enzymes are especially useful for detecting non-proliferating bacteria cells because the enzymes can catalyze a cleavage reaction with up to millions of substrate molecules per second, which allows for detecting a small number of bacteria cells.
  • Certain nucleases produced by bacteria constitute widely used means for identification and characterization of certain pathogenic species.
  • nuclease activity includes UN absorption and electrophorectic or chromatographic separation of digested D ⁇ A fragments followed by fluorescent staining. Additional approaches have been developed that employ natural D ⁇ A, synthetic substrates and D ⁇ A-binding molecules:
  • Colometric DNA-binding molecules Colometric DNA-binding molecules.
  • Toluidine blue (TB) or methyl green (MG) can be added to a mixture of natural D ⁇ A and an appropriate growth medium.
  • the dye forms a complex with D ⁇ A that is either blue (TB) or green
  • Fluorescent DNA-binding molecules (ii) Fluorescent DNA-binding molecules.
  • Acridine orange (AO), or other suitable dye such as Hoescht dye 33258 or dimeric cyanine nucleic acid stains such as those available under the trade marks TOTOTM or YOYOTM from Molecular Probes, Inc., Eugene, OR, can be added to a mixture of natural D ⁇ A and growth medium. These compounds are weakly fluorescent in aqueous solution, but become strongly fluorescent when bound to D ⁇ A. Hydrolysis of D ⁇ A by D ⁇ ase causes complete removal of fluorescence, which can be seen as non-fluorescent halos around any bacterial colonies.
  • Chromogenic synthetic substrates Two types of synthetic substrates are used to detect nuclease activities per this method. The first is Thymidine 5'- monophosphate p-nitrophenyl ester ammonium salt. It releases p-nitrophenol upon hydrolysis by a purified extracellular nuclease from a Bacillus species, as indicated by the increase in absorbance at 410 nm. The second is 5-bromo-4-chloro-indolyl- thymidine-3'-phosphate which, upon hydrolysis, releases the indoxyl group which produces a blue-green indigo color by autooxidation.
  • fluorescein 5-isothiocyanate Upon hybridization with an unlabeled complementary strand, 50% of the fluorescence is quenched due to interactions between DNA and fluorophores. Cleavage, i.e., chain scission, of the hybridized species by a restriction endonuclease produces a 2-fold increase in fluorescence intensity due to "dequenching.”
  • the DNA-binding molecules are usually not sequence specific, and signals may be affected by any endogenous DNA present in samples. In some cases, they may also be affected by the presence of surfactants, EDTA, or cell debris. Chromogenic synthetic substrates are usually required in large quantities (millimolar amounts), which may increase assay costs and in some cases lead to insolubility. Also, absorption-based methods are inherently several orders of magnitude less sensitive than fluorescence-based methods. As to fluorogenic substrates, there are few reagents available for nucleases. Additionally, reported methods either have moderate performance or are limited to specific enzymes, e.g., exonuclease.
  • a fluorescence detection method is among the most sensitive detection techniques available today. Zeptomolar (10 ⁇ 21 M) amounts of enzyme molecules have been studied using a fluorescence microassay. Single enzyme molecules have been detected in an oil-dispersed droplet by fluorescence microscopy. Individual molecules of an enzyme have been manipulated electrophoretically in a capillary tube and monitored by fluorescence spectroscopy. See Xue, Q., Yeung, E.S., Differences in Chemical Reactivity of Individual Molecules of an Enzyme, Nature, 1995, 373. 681-683. Assays for detection of coliform bacteria using ⁇ - galactosidase as a marker enzyme have been developed.
  • Fluorometry was found to have a 250-fold increase in sensitivity and 5 hour reduction in the time of detection, relative to colorimetric methods. See Van Poucke, S.O., Neils, H.J., Development of a Sensitive Chemiluminometric Assays for the Detection o/ ⁇ - Galactosidase in Permeabilized Coliform Bacteria and Comparison with Fluorometry and Colorimetry, Appl. Env. Microbiol, 1995, 61, 4505-4509.
  • this invention provides a reagent comprising at least one enzyme- specific cleavable polynucleotide substrate bearing at least one fluorescent moiety in close proximity to at least one other adjacent fluorescent moiety, the adjacent fluorescent moieties quenching fluorescence in each other, the fluorescent moieties being readily detectable by fluorometric techniques upon cleavage of the substrate.
  • the reagent may comprise an enzyme-specific cleavable polynucleotide substrate comprising a self-complementary single strand polynucleotide having 3 'and 5' termini each bearing a fluorescent moiety; a polynucleotide substrate comprising two complementary hybridized polynucleotide strands each having 3 ' and 5 ' termini wherein each terminus of one or both 375 ' pair(s) bears a fluorescent moiety; or a polynucleotide substrate comprising a single strand polynucleotide bearing along its length at least two pendent adjacent fluorescent moieties.
  • This invention also provides a method of biological assay comprising the step of: combining (1) a reagent comprising at least one enzyme-specific cleavable polynucleotide substrate bearing at least one fluorescent moiety in close proximity to at least one other adjacent fluorescent moiety, said adjacent fluorescent moieties quenching fluorescence in each other, but being readily detectable by fluorometric techniques upon separation, and (2) a test sample potentiality containing the specific enzyme being assayed, wherein the presence of the specific enzyme being assayed will result in cleavage of the polynucleotide, separation of the fluorescent moieties, and an increase in fluorescence intensity.
  • a further embodiment of this method comprises the additional step of measuring the increase in fluorescence intensity.
  • the fluorescence may readily be observed by conventional techniques such as radiant energy illumination, a fluorescent microscope, a 96-well plate reader, or a flow cytometer.
  • the invention further provides a method for making the enzyme-specific cleavable polynucleotide reagent bearing quenched fluorescent moieties.
  • This method comprises making a reagent comprising an enzyme-specific cleavable polynucleotide bearing fluorescent moieites that quench each other through dye dimerization and that are detectable by fluorometric techniques upon separation, said method comprising the step of: combining one or more fluorescent compounds bearing one or more fluorescent moieties and one or more reactive groups with enzyme-specific cleavable polynucleotides selected from the group consisting of (1) self-complementary single strand polynucleotides having one or more end groups reactive with one or more of the fluorescent compounds, (2) single strand polynucleotides having within the polynucleotide, at locations other than the termini, two or more moieties having pendent groups reactive with one or more of the fluorescent compounds, and (3) complementary polynucleotides each strand having at least one reactive end group at the 3'
  • dimerization means formation of a non-covalently-bonded complex between two dye groups
  • dye stacking means formation of a non-covalently-bonded complex between two or more dye groups, two complexed dye groups are referred to as a "dimer” and three complexed dye groups are referred to as a "trirner";
  • enzyme-specific cleavable polynucleotide means a linear sequence of nucleotides comprising specific bonds that are subject to cleavage by specific enzymes;
  • fluorescence means light emission by a substance at a given wavelength upon absorbing light of a different wavelength, wherein light emission occurs only during light absorption;
  • fluorescent quantum yield means the ratio of the number of fluorescent photons emitted by an emitting substance to the total number of photons absorbed by the substance;
  • fluorogenic substrate means an essentially non-fluorescent material acted upon by an enzyme to produce a fluorescent compound
  • fluorophore means a molecule or portion of a molecule that emits light at a given wavelength when stimulated by absorption of light of a different (usually shorter) wavelength.
  • This invention provides advantages over conventional assay methods for detection and identification of microorganisms. It provides a rapid and convenient approach that employs enzyme-specific cleavable polynucleotide substrates having fluorescent moieties, also referred to herein as fluorogenic nuclease substrates, for measuring activities of and/or detecting the presence of extracellular and intracellular enzymes. It can also be adapted to sequence-dependent or sequence- independent tests. The method and reagents of the invention have led to improved accuracy, faster detection, and overall lower cost in detection and identification of microorganisms.
  • the invention employs the use of measurable differences in fluorescence to allow detection of nuclease.
  • the present invention provides fluorogenic indicators that show a higher signal level when cleaved compared to the much lower signal level when quenched and that operate preferably in the visible wavelength range to minimize interference due to growth medium background fluorescence.
  • the present invention allows more accurate and sensitive measurement of the presence of nuclease by, among other factors, red-shifting the emission wavelength from far UN region (about 350 to 400 nanometer) to the 500-600 nm region of the electromagnetic spectrum and reducing the effect of background signal levels of intact reagents.
  • Another advantage of the present invention is that it is a homogeneous biological assay method, which requires no developing agent. Therefore, it does not require time-consuming separation steps as in assay formats such as enzyme-linked immunosorbent assays (ELISA) and bioluminescence. Detection and identification of microorganisms can be performed by using the primary isolation medium. As can be seen, the present assay method features more efficient use of reagents, labor, time, and equipment.
  • Fig. 1 is a schematic illustration of a conceptual mechanism of a fluorogenic nuclease substrate being cleaved by a bacteria-produced enzyme wherein the fluorogenic nuclease substrate is a self-complementary single strand polynucleotide exhibiting intramolecular dimerization and quenching of fluorescent moieties attached at the 3' and 5 1 termini of the polynucleotide, which fluorescent moieties become separated and readily detectable upon cleavage of the polynucleotide.
  • Fig. 2 is a schematic illustration of a conceptual mechanism of a fluorogenic nuclease substrate being cleaved by a bacteria-produced enzyme wherein the fluorogenic nuclease substate is a single strand polynucleotide having pendent fluorescent moieties in sufficiently close proximity to quench each other through dye stacking, which fluorescent moieties become separated and readily detectable upon cleavage of the polynucleotide.
  • Fig. 3 is a schematic illustration of a conceptual mechanism of a fluorogenic nuclease substrate being cleaved by a bacteria-produced enzyme wherein the fluorogenic nuclease substrate comprises two complementary hybridized polynucleotide strands exhibiting intermolecular dimerization and quenching of two fluorescent moieties labeled at the 3' terminus and 5' terminus, respectively, of the two complementary polynucleotides, which fluorescent moieties become separated and readily detectable upon cleavage of the polynucleotide.
  • Fig. 4 is a graph of the relative fluorescence intensity of a tetramethylrhodamine-labeled
  • Fig. 5 is a graph of the absorption spectra of a tetramethylrhodamine- labeled 24-mer self-complementary single strand polynucleotide before (line c) and after (line d) incubation with micrococcal nuclease in 100 millimolar pH 8.9 bicarbonate buffer solution at 37°C.
  • the substrate provides for separation of quenched fluorescent moieties upon exposure to the nuclease being assayed thereby causing a detectable increase in fluorescence.
  • the nuclease substrate is made by first specifying a polynucleotide to be used. This is done by selecting the bases or base pairs (A/T or G/C) to be included in the polynucleotide. Any configuration that will bring the fluorescent moieties into close proximity will work. Hybridization may be used to achieve a suitable configuration. Hybridization occurs as complementary base pairs of polynucleotide(s) are attracted to each other.
  • Possible configurations include, for example, a self-complementary single-strand polynucleotide having fluorescent dye moieties attached at both the 3' and 5' ends that will self-hybridize, i.e., fold over, to bring fluorescent moieties attached at each end of the polynucleotide into close proximity.
  • An alternative configuration comprises two mutually complementary single strand polynucleotides each having 3 'and 5 ' termini wherein each terminus of one or both 375' pairs bears a fluorescent moiety which polynucleotides hybridize to form a double stranded polynucleotide thereby bringing fluorescent moieties attached at one or both ends of each single strand polynucleotide into close proximity.
  • Another method involves a single strand polynucleotide wherein two or more fluorescent dye moieties are pendent from the polynucleotide chain and are spaced sufficiently closely along the chain at appropriate lengths to cause the fluorescent moieties to come into close proximity and quench fluorescence.
  • hybridization For a hybridized configuration, the extent of hybridization need only be sufficient to bring the attached dye moieties into close proximity. Typically, hybridization of approximately the five base pairs closest to the 375 ' termini to which the fluorescent moieties are attached will suffice. The number may vary depending on factors such as the type of base pairs in the polynucleotide and the dimerization constant of the fluorescent moieties.
  • the polynucleotide is further specified to contain one or more bonds that are cleavable by the nuclease sought to be assayed.
  • the bond(s) are strategically located within the polynucleotide so that cleavage will cause separation of the fluorescent moieties.
  • the cleavable bond(s) should typically be located near the end or ends of the polynucleotide where the fluorescent moieties are attached.
  • the cleavable bonds should typically be located between the dye moieties.
  • Bonding sites for the selected fluorescent moieties are also incorporated into the polynucleotide at the specific locations where the fluorescent moieties are desired. These sites may be at the ends (termini) of the polynucleotide as in the case of hybridized polynucleotides, or along the polynucleotide strand, i.e., backbone, as in the case of a single strand polynucleotide to which pendent dyes will be attached. These bonding sites comprise reactive groups such as aliphatic primary amines that will readily react with the fluorescent compounds comprising fluorescent moieties to be attached to the polynucleotide.
  • the polynucleotide can be synthesized, for example, by known manual or automated DNA synthesis methods.
  • the polynucleotide is then reacted with one or more compounds comprising fluorescent moieties.
  • the fluorescent compounds typically also comprise reactive groups which will react with the reactive groups on the polynucleotide.
  • the reaction takes place under appropriate concentration, agitation, pH, ionic, and temperature conditions which will cause the fluorescent moieties to attach to the polynucleotide at selected sites.
  • the particular necessary conditions vary based on the reactive groups present in the polynucleotide and fluorescent compound being used, respectively.
  • the pH is controlled by using a buffer solution such as a carbonate or bicarbonate buffer solution.
  • the fluorescent moieties used in this invention possess the capability to dimerize or stack to cause quenching of fluorescence.
  • This invention also provides a method of biological assay comprising the step of: combining (1) a reagent comprising at least one enzyme-specific cleavable polynucleotide substrate bearing at least one fluorescent moiety in close proximity . to at least one other adjacent fluorescent moiety, said adjacent fluorescent moieties quenching fluorescence in each other, but being readily detectable by fluorometric techniques upon separation, and (2) a test sample potentially containing the specific enzyme being assayed, wherein the presence of the specific enzyme being assayed will result in cleavage of the polynucleotide, separation of the fluorescent moieties, and an increase in fluorescence intensity.
  • a further embodiment of this method comprises the additional step of measuring the increase in fluorescence intensity. This can be done with radiant energy illumination, a fluorescent microscope, a 96-well plate reader, or a flow cytometer.
  • Figure 1 depicts a preferred embodiment of the invention.
  • polynucleotide 10 can be synthesized so as to be self-complementary, that is, it undergoes intrastrand self-hybridization.
  • fluorescent dye moieties 11 and 12 are attached at the 3'- and 5'- termini, only weak fluorescence is observed because the dye moieties are in sufficiently close proximity to cause mutual quenching.
  • nuclease 13 generated by bacteria 14
  • polynucleotide 10 may be cleaved at sites such as, e.g., 15, 16, and 17, which can destroy the self-hybridized structure to produce nucleotide fragments 18 and non-dimerized dye moieties 11 ' and 12', which disassociate and fluoresce freely.
  • the polynucleotide chain must be of sufficient length to allow the polynucleotide to fold over and bring the fluorescent dye moieties into close proximity. Typically, approximately fifteen base pairs is sufficient.
  • Figure 2 depicts a second preferred embodiment of the invention.
  • a plurality of fluorescent dye moieties 22 are attached to polynucleotide 20 which contains various sites 24 that are susceptible to enzymatic cleavage.
  • the dye moieties 22 are close enough to each other so that fluorescence of dye moieties 22 is quenched.
  • the conjugate (comprising polynucleotide 20 and fluorescent dye moieties 22) can be treated with nuclease 26, generated by bacteria 28, to cleave polynucleotide 20.
  • Cleavage produces nucleotide fragments 29 and allows dye moieties 22' to separate from one another in the medium such that they fluoresce freely and are easily detectable.
  • a configuration as shown in Figure 2 could be used to test for multiple types of bacteria or enzymes by having a series of different types of paired or grouped fluorescent moieties, each type distinguishable from the others using fluorometric techniques, with the individual moieties of each pair or group being joined by, or on opposite sides of, a cleavable bond susceptible to cleavage by a different bacterial enzyme.
  • Figure 3 depicts yet another preferred embodiment of the invention.
  • Complementary hybridized polynucleotides 30 and 31 have fluorescent dye moieties 32 and 33 attached at their 3'- and 5'- termini, respectively.
  • Nuclease 36 generated by bacteria 38, cleaves polynucleotide(s) 30 and/or 31 at cleavage sites 34 to produce nucleotide fragments 39 and non-dimerized dye moieties 32' and 33' that are easily detectable.
  • a further embodiment could comprise dye moieties attached to each 3' and 5' termini of each complementary polynucleotide.
  • a configuration as shown in Figure 3 could be used to test for two types of bacteria or enzymes at the same time by labeling each complementary 375' termini pair with a different type of fluorescent moiety, each type distinguishable from the other using fluorometric techniques, and constructing the double-stranded polynucleotide to have cleavable sites near each terminus of the double-stranded polynucleotide susceptible to cleavage by a different type of bacterial enzyme.
  • Fluorogenic substrates are molecules that change from essentially nonfluorescent to highly fluorescent upon enzymatic hydrolysis. They are widely used as molecular probes for studies and tests of viral and bacterial enzymes such as proteases, nucleases, saccharidases, phosphatases and kinases. The fluorescence of the substrate may be readily observed by conventional techniques such as radiant energy illumination, a fluorescent microscope, a 96-well plate reader, or a flow cytometer.
  • the enzyme-specific cleavable polynucleotide substrates bearing fluorescent moieties, or fluorogenic nuclease substrates, useful in the present invention can be made by chemical reaction of a polynucleotide and fluorescent dyes such as fluorescein and rhodamine that will dimerize or stack.
  • fluorescent dyes such as fluorescein and rhodamine that will dimerize or stack.
  • Useful polynucleotides are commercially available (e.g., from GeneMed Biotechnologies, South San Francisco, CA).
  • two or more fluorescent moieties are bonded to the polynucleotide so that dimerizing or stacking of the fluorescent moieties can occur (e.g. , through substantial hybridization, proximity, interstrand hybridization, intrastrand hybridization, etc.) so as to mutually quench (referred to as "self-quenching" when the fluorescent moieties are identical) the fluorescence of the fluorescent moieties.
  • Fluorescence dye quenching may take place by any of a number of known mechamsms, including energy transfer and dye dimerization.
  • energy transfer when a fluorescent dye molecule or moiety is excited by input of energy, typically by irradiation with a specific wavelength of light, energy is transferred from the dye moiety to another moiety, rather than being dissipated by fluorescence.
  • Energy transfer also referred to as Forster-type dipole-dipole interaction, occurs between a fluorescent donor and an acceptor, which may or may not be fluorescent. Energy transfer generally takes place when the distance between donor and acceptor along the substrate backbone is on the order of 10 nanometers. Through energy transfer, the fluorescence of the donor moiety is quenched.
  • Dye dimerization or dye stacking occurs when two or more fluorescence moieties are close enough to each other to allow their planar aromatic rings to interact to form ground state complexes such as dimers and trimers. Each fluorescent moiety acts toward the other fluorescent moiety(ies) causing mutual quenching. The quenching.
  • the absorbance spectra of dyes in a dimer- or stacked state are substantially different from those of the same dyes in energy transfer pairs.
  • Dye dimer absorption spectra show a characteristic decrease in absorbance of the principal absorption peak as dye concentration increases, while showing a characteristic increase in absorbance of the shoulder peak. This phenomenon is illustrated in Figure 5.
  • the principal absorbance peak occurs at approximately 560 nm and the shoulder peak occurs at approximately 530 nm.
  • the dimerized sample (c) exhibits absorbance peaks of approximately 0.03 absorbance units (AU) for both the principal absorbance peak and the shoulder peak while the undimerized sample (d) shows a principal absorbance peak of approximately 0.06 AU and a flattened shoulder peak of approximately 0.028 AU.
  • the phenomenon occuring in the dimerized sample is commonly referred to as "band splitting.” Dimerization or stacking takes place through the formation of ground-state complexes (i.e., through close physical proximity), whereas energy transfer interactions occur through space. Therefore, spectral changes characteristic of dye dimerization are not seen for dyes that interact by energy transfer mechanisms.
  • the quenching mechanism of the dimerized or stacked dye pairs or dye groups used in the instant invention is distinct from the quenching mechanism of fluorescence energy transfer donors and acceptors.
  • the type of dyes that exhibit dimerization or stacking characteristics when bonded to the polynucleotide within a sufficiently close proximity to one another include those dyes which have a generally planar aromatic structure so as to be capable of forming homo- or heterodimers when in solution at sufficiently high dye concentrations (for example, 10 "2 to lO ⁇ M). For many applications this phenomenon is obviously undesirable, but is used to advantage in the present invention.
  • Concentration increases can be accomplished either by increasing the amount of dye in a unit volume or by physically locating two (or more) dye moieties closely together, for example, on a polynucleotide or other small molecule. When the dye moieties are brought into close proximity this causes an effective increase in the local concentration and the polynucleotide bearing fluorescent moieties will stay quenched regardless of the total concentration.
  • Planar aromatic dyes of the fluorescein and rhodamine families such as fluorescein, tetramethyhhodamine (TMR), Rhodamine B, and X-rhodamine are representative dyes that will dimerize in sufficiently high concentration.
  • fluorescent dyes include, e.g., cyanines, and boron-heterocyclic dyes such as 4,4-difluoro-4-borata-3a-azonia-4a-aza -s-indacene available under the trade mark BODIPYTM from Molecular Probes, Inc. of Eugene, OR.
  • Dyes of the rhodamine family such as tetramethylrhodamine, X-Rhodamine, and Rhodamine B have very high fluorescent quantum yields (approximately 0.85) in the visible wavelength range (400 - 700 nm). For this reason they are frequently used for detection of minute amounts of substances.
  • the dye moieties used in this invention additionally comprise reactive end groups which facilitate the attachment of the fluorescent moiety to the polynucleotide.
  • These end groups may comprise succimidylester, maleimide, esters, thioisocyanates, isocyanates, and iodoacetimide.
  • Dye molecules with these reactive groups already attached are available from, for example, Molecular Probes, Inc. (Eugene, OR)
  • the polynucleotides used in this invention must have one or more enzyme- specific cleavable sites. Upon cleavage of the fluorescence-quenched polynucleotide, which bears two or more fluorescent moieties, fluorescence intensity will be enhanced as a result of dissociation of the dimerized or stacked fluorescent moieties. This allows for easy detection of the fluorescent moieties thus indicating the presence of specific enzymes and, if applicable, the presence of the bacteria that produced the enzyme. Due to the interaction between transition dipoles of the resonating dimeric or stacked structure, the fluorescent quantum yield of the dimeric or stacked structure will be quite low when the polynucleotide is intact.
  • the fluorescent moieties can become separated so that significantly higher fluorescence quantum yield in aqueous solution will be observed due to high intrinsic fluorescence. In this manner, the increase in fluorescence and the presence of the hydrolyzing enzyme can be discerned.
  • a homogenous enzyme assay requiring no developing agent and no separation step, can be designed wherein enzyme-specific cleavable polynucleotides bearing fluorescent moieties are placed in solution and an enzyme analyte is added so that any enzyme present in the analyte cleaves the polynucleotide causing dimerization or stacking to decrease with an attendant increase in fluorescence. Therefore, enzyme activity can be directly related to the net increase in fluorescence intensity. If the enzyme is secreted from actively metabolizing cells, the fluorescence intensity may be related to the metabolic activity of the cells.
  • Representative polynucleotides useful to produce fluorogenic nuclease substrates must have the requisite chemical bond attacked by the nuclease being assayed.
  • micrococcal nuclease is known to hydrolyze polynucleotides at phosphodiester bonds, so any polynucleotide with this characteristic can be a micrococcal substrate.
  • nucleotide analogs Chemical modifications of polynucleotides are accomplished using nucleotide analogs and DNA synthesis reagents.
  • phosphoramidites containing aliphatic primary amines may be introduced into a polynucleotide chain at desired positions through automated DNA synthesizers such as the Nucleic Acid Synthesis and Purification System (Model # ABI 3948, Perkin-Elmer Applied Biosystems, Foster City, CA).
  • Terminus modifiers and amino-modified- deoxythymidine (dT) are commercially available to introduce an aliphatic amine with a three-carbon, six-carbon, or nine-carbon linking moiety at the 3' and/or 5' end of polynucleotides.
  • the amine can then react with a variety of labels such as biotin, fluorescent dyes, or alkaline phosphatase to form polynucleotide conjugates.
  • labels such as biotin, fluorescent dyes, or alkaline phosphatase to form polynucleotide conjugates.
  • Common applications of these modified polynucleotides include: (i) nonradioactive hybridization probes; (ii) sequence specific cleavage of DNA, (iii) automated DNA sequencing, and (iv) affinity chromatography.
  • Other moieties that can be added to the ends of a polynucleotide to react with the dye moieties to be attached include those with reactive H groups such as free thiol (SH), carboxyl groups, and OH groups. Amines and thiols are preferred.
  • Enzymatic cleavage is achieved by contacting the fluorogenic nuclease substrate, i.e., the reagent, with the specific enzyme being assayed.
  • Target enzymes suitable for use in the present invention include all enzymes generally classified as nucleases, i.e., those that hydrolyze nucleotide bonds, including, for example, thermonuclease or Staphylococcus nuclease.
  • the enzyme When the enzyme is intracellular, the enzyme may be made available to contact with the fluorogenic nuclease substrate by the use of agents that increase the permeability of the outer membrane (OM) of the enzyme-carrying bacteria. Ethylenediaminetetraacetic acid (EDTA) is commonly used for this purpose by affecting the outer membrane barrier of gram-negative enteric bacteria. Under certain conditions, the OM becomes ruptured and the intracellular enzymes can be released. For rapid and sensitive tests, it is important to maximize the signal-to-noise ratio, i.e., maximize the desired signal over undesired background signals such as, in this case, autofluorescence of bacteria and growth medium.
  • OM outer membrane
  • EDTA Ethylenediaminetetraacetic acid
  • fluorescent dyes that emit at wavelengths much longer than those of background fluorescence
  • the concept described in this invention can be applicable to other dyes that will also red-shift the emission to even longer wavelengths.
  • useful dyes may have the following characteristics: high extinction coefficient (>80,000 cm 'M "1 ), high quantum yields (>0.85 in aqueous solution), spectra that are insensitive to solvent and pH, aqueous solubility, photostability, and a dimerization constant preferably greater than 10 3 .
  • a number of staphylococci strains show DNase activity.
  • An enzyme historically used for identification purposes is the extracellular DNase of Staphylococcus aureus (S. aureus).
  • S. aureus nuclease is excreted in great quantities, is heat-stable, and requires Ca + , but not Mg 2+ , for activity. These characteristics are used for differentiating S. aureus from other staphylococci for detection of staphylococcal contamination in foods.
  • Extracellular DNase is also produced by Streptococcus species, Serratia species, and Bacillus species.
  • Micrococcal nuclease an extracellular enzyme from Staphylococcus aureus, is used as a model system to demonstrate utility of this invention.
  • This enzyme cleaves the 5' phosphodiester bond of a polynucleotide strand, remains stable and active at elevated temperatures (>60°C), and is frequently used as an indicator for S. aureus.
  • elevated temperatures >60°C
  • the presence of nuclease activity at 60°C is a confirmative test for S. aureus.
  • Micrococcal nuclease does not require a cofactor for its function — a feature that can reduce assay complexity and cost.
  • this invention provides novel nuclease reagents, an assay method for using the reagents, and a method for making the reagents.
  • the reagents comprise single or double stranded synthetic polynucleotides bearing two or more fluorescent moieties which may be dye molecules (e.g. teframethylrhodamine).
  • Fluorescence enhancement after nuclease hydrolysis and cleavage is between 2 and 6 fold, depending on the specific configuration of the reagent.
  • These reagents when coupled with the "double cascade" phenomenon in which each enzyme molecule is capable of turning over millions of reagents molecules and more enzyme molecules are released as bacterial cells divide and grow exponentially, offer promising approaches to rapid, sensitive and specific detection of bacteria.
  • This invention finds use in, but is not limited to, detection and identification of microorganisms, sterilization assurance, pharmaceutical discovery, enzyme assays, and immunoassays. Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.
  • TMR tetramethylrhodamine
  • a self-complementary polynucleotide NH 2 CH 2 CH 2 CH 2 -5'-AAC AAA GGA TAA TTA TCC TTT GTT-3'-CH 2 CH CH 2 NH 2 , was obtained from GeneMed Biotechnologies, Inc. (South San Francisco, CA) and its chemical structure was confirmed by DNA sequencing. About 270 nanomoles (nmoles) of the above polynucleotide was dissolved in 600 ⁇ l of deionized (D.I.) water.
  • D.I. deionized
  • TMR tetramethylrhodamine
  • DMSO dimethylsulfoxide
  • mM millimolar pH 8.3 sodium bicarbonate buffer
  • the reaction mixture was introduced from a pipet into a size-exclusion column (Model PD-10, Pharmacia Biotech, Inc., Piscataway, NJ) containing epichlorohydrin-crosslinked (alkaline conditions) dextran gel, a packing material, available under the trademark SEPHADEXTM G25 from Pharmacia Biotech, Inc., to separate labeled polynucleotide from the unreacted dye.
  • the reaction mixture was passed through the column by force of gravity.
  • the DNA fraction was further purified by high performance liquid chromatography (HPLC) on a C18 reversed phase column (Model 600E HPLC, Waters Corp., Milford, MA).
  • the mobile phase in the column was a gradient of acetonitrile (ACN) and 0.1 M triethylamine acetate (TEAA) in water in which the volume ratio of TEAA to ACN started at 92:8 and linearly decreased to 8:92 over 30 minutes.
  • the resultant conjugate had the above polynucleotide structure with the addition of a TMR moiety on each aliphatic amine linking moiety at the 3' and 5' termini.
  • HPLC fractions containing the purified conjugate were pooled, frozen in dry ice, and then vacuum dried at room temperature. The dry conjugate was then dissolved in 5 ml of 100 mM pH 8.9 sodium carbonate buffer solution. As shown in Figure 5, the absorption spectra measured by use of a spectrophotometer (Model MPC- 3100, Shimadzu Scientific Instruments, Columbia, MD) showed band-splitting characteristic of dimerized tetramethylrhodamine. A small amount (about 0.5 mg) of micrococcal nuclease (obtained from Sigma Chemical Co., St. Louis, MO) was added to 3 ml of the above buffered solution.
  • the mixture was incubated overnight at 37°C for enzymatic hydrolysis of the 5 '-phosphodiester bonds of the polynucleotide bearing fluorescent moieties.
  • the solution was then cooled to room temperature.
  • a FLUOROLOGTM spectrofluorometer was then used to measure the fluorescence spectra at room temperature. Enzymatic cleavage resulted in a relative enhancement in fluorescence intensity from 12000 to 28000 at 580 nm and an increase in absorbance from 0.03 absorbance units (AU) to 0.06 AU at 560 nm when excited with light at 520 nm.
  • the length of the aliphatic amine linking moiety at the 5' terminus was increased to allow more efficient stacking between the two dye moieties.
  • the following polynucleotides with longer aliphatic amine linking moieties were obtained from GeneMed, labeled and tested under conditions similar to those previously stated in this example.
  • Example 2 pendent tetramethylrhodamine dye moieties were attached at multiple positions within a polynucleotide sequence. Because of close proximity, the dye moieties stacked together to quench fluorescence. Enzymatic cleavage of the polynucleotide at multiple sites disintegrated the conjugate to liberate dye moieties from a dimeric or stacked state to single moieties, producing a concurrent increase in fluorescence intensity. This concept is schematically illustrated in Figure 2.
  • the polynucleotide strand was designed to contain a total of four amino groups in alternate bases as follows: 5'- TTT TTT Z T Z T Z T Z TTT TTT -3', where Z was an amine-modified C $ derivative of deoxythymidine (dT) (Cat. No. 10-1039-90, Glen Research, Sterling, VA,).
  • the polynucleotide strand was synthesized by GeneMed Biotechnologies, Inc. About 45 nanomoles of the polynucleotide was dissolved in deionized water and reacted at room temperature with 1 mg of tetramethylrhodamine succimidylester pre-dissolved in one drop of DMSO in 100 mM pH 8.3 sodium bicarbonate buffer.
  • the conjugate was purified under the same conditions as in Example 1. Enzymatic hydrolysis was carried out overnight with a small amount (about 0.5 mg) of micrococcal nuclease in 100 mM pH 8.9 sodium bicarbonate buffer at 37°C. Fluorescence spectra were measured at room temperature with excitation wavelength at 520 nm, and an approximately two-fold enhancement in fluorescence intensity at 575 nm was obtained after enzymatic hydrolysis.
  • Example 3 (Comparative) In this example, quenching (of one of the dye moieties) is achieved by energy transfer rather than dye dimerization.
  • X is tetrachlorofluorescien (acceptor)
  • Y is fluorescein (donor)
  • Z is the amino-modified Cg of deoxythymidine (dT) (to allow for attachment of another acceptor, tetramethyhhodamine (TMR), which is pendent from Z).
  • TMR tetramethyhhodamine
  • teframethylrhodamine was attached to an aliphatic amine linking moiety at the 3' and 5' termini, respectively, of two complementary polynucleotides.
  • Inter-strand hybridization brought the dye moieties into close contact with each other and quenched fluorescence.
  • Enzymatic cleavage at multiple sites along the double- stranded polynucleotide disintegrated the ordered structure to liberate the dye moieties from dimers to monomers, producing a significant increase in fluorescence. This concept is schematically illustrated in Figure 3.
  • TMR tetramethylrhodamine
  • T m The melting temperature, T m; was calculated based on the percentage of GC pair content in the polynucleotide chain and the ionic strength of the hybridization solution (Catalog Number F123A, Promega Corp., Madison, WI), using the following formula:
  • T m 81.5 + 16.6(log 1Q [Na + ]) + 0.41 (%GC) - (600/N)
  • N is an integer signifying the number of bases in the polynucleotide chain.
  • This formula predicts the T m reasonably well for polynucleotides between 14 and 60-70 nucleotides in length.
  • the Na + concentration of the Promega hybridization solution is 0.39.
  • T m of the above polynucleotide was calculated to be 63.8°C. Hybrids formed between short polynucleotides at 5-10°C below the T m are reversible and easy to unwind. Therefore, the reaction temperature should be at least 10°C below the T m .
  • hybridizations with short polynucleotides should be carried out under stringent conditions using high concentrations (0.1-1.0 picomole/ml) of polynucleotides.
  • hybridization of the two strands bearing fluorescent dyes was carried out overnight at 45°C (19°C below T m ) in a high stringency hybridization solution (the Promega solution).

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Abstract

L'invention concerne un réactif comprenant un substrat polynucléotidique (30, 31) clivable spécifique aux enzymes comportant des fragments (32, 33) à fluorescence atténuée. L'invention concerne également un procédé de fabrication dudit réactif. Le polynucléotide (30, 31) comprend au moins un fragment fluorescent (32) suffisamment proche d'un autre fragment fluorescent (33) pour atténuer considérablement la fluorescence des fragments (32, 33), qui sont toutefois, facilement détectables par des techniques fluorimétriques à la suite de la séparation par clivage du polynucléotide. L'invention concerne également un procédé d'analyse biologique qui consiste à combiner le réactif à un échantillon de test contenant potentiellement l'enzyme (36) en cours d'analyse. L'enzyme (36) dissocie le polynucléotide de manière qu'il libère les fragments fluorescents (32, 33) et que l'intensité de fluorescence augmente. Le procédé d'analyse peut être appliqué dans la détection et l'identification de micro-organismes, la stérilisation sûre, la recherche pharmaceutique, le dosage d'enzymes, l'immunodosage, et dans d'autres analyses biologiques.
PCT/US1998/017311 1998-01-09 1998-08-20 Substrat polynucleotidique clivable specifique d'enzymes et procede d'analyse associe WO1999035288A1 (fr)

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AU91110/98A AU9111098A (en) 1998-01-09 1998-08-20 Enzyme-specific cleavable polynucleotide substrate and assay method
KR1020007007574A KR100575407B1 (ko) 1998-01-09 1998-08-20 효소 특이적 절단성 폴리뉴클레오티드 기질 및 이의 분석방법
EP98943281A EP1045925A1 (fr) 1998-01-09 1998-08-20 Substrat polynucleotidique clivable specifique d'enzymes et procede d'analyse associe
JP2000527669A JP2002508935A (ja) 1998-01-09 1998-08-20 酵素特異的切断可能ポリヌクレオチド基質およびアッセイ方法

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WO2001079563A3 (fr) * 2000-04-18 2002-09-19 Naxcor Sondes d'acides nucleiques degradables et methodes de detection d'acides nucleiques
WO2003008643A3 (fr) * 2001-07-20 2003-08-21 Cancer Rec Tech Ltd Procedes et materiaux
FR2836926A1 (fr) * 2002-06-14 2003-09-12 Commissariat Energie Atomique Substrat oligonucleotidique et procede permettant d'analyser la presence d'activites de reparation de lesions de l'adn
US6673568B1 (en) * 1999-10-25 2004-01-06 Genprime, Inc. Method and apparatus for prokaryotic and eukaryotic cell quantitation
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EP0699768A1 (fr) * 1994-09-01 1996-03-06 F. Hoffmann-La Roche AG Procédés pour la extinction de la fluorescence en phase solution de sondes oligonucléotidiques marqués avec un fluorophore
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US7153654B2 (en) 1993-07-23 2006-12-26 University Of Utah Research Foundation Assay procedure using fluorogenic tracers
US6673568B1 (en) * 1999-10-25 2004-01-06 Genprime, Inc. Method and apparatus for prokaryotic and eukaryotic cell quantitation
WO2001079563A3 (fr) * 2000-04-18 2002-09-19 Naxcor Sondes d'acides nucleiques degradables et methodes de detection d'acides nucleiques
US6573048B1 (en) 2000-04-18 2003-06-03 Naxcor Degradable nucleic acid probes and nucleic acid detection methods
WO2001085997A1 (fr) * 2000-05-08 2001-11-15 Qtl Biosystems Llc Ameliorations apportees a l'approche qtl-polymere fluorescent de la biodetection
US6743640B2 (en) 2000-05-08 2004-06-01 Qtl Biosystems Llc Fluorescent polymer-QTL approach to biosensing
WO2003008643A3 (fr) * 2001-07-20 2003-08-21 Cancer Rec Tech Ltd Procedes et materiaux
FR2836926A1 (fr) * 2002-06-14 2003-09-12 Commissariat Energie Atomique Substrat oligonucleotidique et procede permettant d'analyser la presence d'activites de reparation de lesions de l'adn
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