KR20010033983A - Enzyme-specific cleavable polynucleotide substrate and assay method - Google Patents

Abstract

The present invention provides a reagent comprising an enzyme specific cleavable polynucleotide (30, 31) substrate having a quenched fluorescent portion (32, 33), and also provides a method for preparing the same. The polynucleotides 30 and 31 include at least one fluorescent part 32 which is sufficiently close to the other fluorescent part 33 to substantially quench the fluorescence of the fluorescent parts 32 and 33. 32, 33) can be easily detected by fluorescence techniques once separated by cleavage of the polynucleotide. Also provided is a biological assay comprising combining the reagents with a test sample that is likely to contain the enzyme 36 to be analyzed, wherein the enzyme 36 cleaves the polynucleotides to provide fluorescent sites 32,33. ) And yield an increase in fluorescence intensity. This assay can be used for microbial detection and identification, bactericidal identification, drug discovery, enzyme analysis, immunoassay and other biological analyses.

Description

Enzyme-specific cleavable polynucleotide substrates and methods of analysis thereof {ENZYME-SPECIFIC CLEAVABLE POLYNUCLEOTIDE SUBSTRATE AND ASSAY METHOD}

Nucleases (DNases and RNases) are enzymes that hydrolyze and cleave polynucleotides, ie nucleic acids such as DNA and RNA. Such enzymes are present in all biological cells and perform many major functions in nucleic acid metabolism, replication, recombination, repair and modification. Nucleases can be distinguished by their substrate specificity, some nucleases cleave both DNA and RNA, some other nucleases act only on one type of nucleic acid, and some other nucleases are sequence specific And others are not specific, some nucleases act on single-stranded nucleic acids, and others nucleases act only on double-stranded DNA. In addition, nucleases, among other things, include the location of the cleaving bond; Location on the cell, ie intracellular or extracellular location; Ionic and pH conditions; And depending on resistance to heat treatment. For the nuclease are described in detail in the literature [Linn, SM, Lloyd, RS , Roberts, RJ, Nucleases (2 nd ed.), Cold Spring Harbor Laboratory Press, Plainview, NY, 1994].

Antibiotic resistant bacterial strains and foodborne diseases are of considerable importance to the public. In addition, the management of the risk of microbial contamination in the healthcare, cosmetics, food and beverage industries is an important issue in terms of health and stability. Bacterial testing is an essential part of measuring microbial risk. Enzymes produced by bacteria are commonly used to test for the presence of bacteria. The action of microorganisms to hydrolyze and cleave nucleic acids through the production of nucleases has long been known. Enzymes are particularly useful for the detection of non-proliferative bacterial cells because they can detect a small number of bacterial cells by catalyzing a cleavage reaction that cleaves up to millions of substrate molecules per second. Certain nucleases produced by bacteria constitute a widely used means of identifying and characterizing specific pathogenic species.

Known methods of measuring nuclease activity include UV absorption and electrophoresis or chromatographic separation of cleaved DNA fragments followed by fluorescence staining. As another method, the following methods using natural DNA, synthetic substrates and DNA binding molecules have been developed.

(i) colorimetric DNA binding molecules

Toluidine blue (TB) or methyl green (MG) can be added to the mixture of natural DNA and appropriate growth medium. The dye forms a complex with DNA that shows blue (TB) or green (MG). When the complex is hydrolyzed by DNase, it releases the dye, resulting in a color difference zone: rosy pink (TB) or colorless (MG).

(ii) fluorescent DNA binding molecules

Acridine orange (AO) or other suitable dyes such as hex dye 33258 or dimer cyanine nucleic acid dyes (e.g., Molecular Probes, Inc., Eugene, Oregon, Inc.) TOTO ™ or YOYO ™ commercially available) may be added. The compound shows weak fluorescence in aqueous solution but turns to strong fluorescence when bound to DNA. When DNA is hydrolyzed by DNase, the fluorescence is completely removed and can be identified as a non-fluorescent ring around all bacterial colonies.

(iii) chromogenic synthetic substrates

When detecting nuclease activity by this method, two kinds of synthetic substrates are used. One is thymidine 5'- monophosphate p-nitrophenyl ester ammonium salt. It releases p-nitrophenol upon hydrolysis by purified extracellular nucleases derived from Bacillus spp., Which is confirmed by an increase in absorbance at 410 nm. The other is 5-bromo-4-chloro-indolylthymidine-3'-phosphate, which releases a doxyl group upon hydrolysis and yields a bluish green indigo color by automatic oxidation.

(iv) fluorogenic substrates

There are two ways to do this. First, fluorometry using poly (deoxy (etheno) adenylate), which is a derivative of poly (deoxyadenylate). This polynucleotide is inherently weak fluorescent. However, upon DNase hydrolysis, fluorescent etenomononucleotides are released and an increase in fluorescence is observed (excitation = 320 nm and emission = 410 nm). This signal can be correlated with the amount of mononucleotides that are continuously monitored and released. As another method of fluorescence measurement, it is known to use the fluorescent "dequenching" technique. 14-mer polynucleotides may be labeled with fluorescein 5-isothiocyanate (Ex = 490 nm and Em = 520 nm). When hybridized with unlabeled complementary strands, 50% of the fluorescence is quenched due to the interaction of the fluorophores with DNA. Cleavage of hybridized species by restriction enzymes, ie chain cleavage, results in a twofold increase in fluorescence intensity due to “dequenching”.

DNA binding molecules are generally not sequence specific and thus the signal is affected by any endogenous DNA present in the sample. In some cases, it is also affected by the presence of surfactants, EDTA or cell debris. Chromatic synthetic substrates generally increase the cost of analysis because they require large amounts (millimolar), and in some cases may not dissolve. In addition, adsorption-based methods are several times less sensitive than those based on fluorescence. With regard to fluorogenic substrates, few reagents are available for nucleases. In addition, the reported method is limited in performance to normal or specific enzymes such as exonucleases.

Fluorescent detection methods are one of the most sensitive detection techniques in use today. Zetamol ( ^10-21 M) content of enzyme molecules was analyzed using fluorescent microanalysis. Single enzyme molecules were used for detection in oil dispersion drops in fluorescent microanalysis. Each enzyme molecule was processed by capillary electrophoresis and monitored by fluorescence spectroscopy [Xue, Q., Yeung, ES, Differences in Chemical Reactivity of Individual Molecules of an Enzyme, Nature, 1995, 373, 681-683]. A detection assay for colloidal bacteria using β-galactosidase as a marker enzyme has been developed. Fluorescence was found to be 250 times higher in sensitivity and reduced in detection time by 5 hours [Van Poucke, SO, Neils, HJ, Development of a Sensitive Chemiluminometric Assays for the Detection of β-Galatosidase in Permeabilized Coliform Bacteria and Comparison with Fluorometry and Colorimetry, Appl. Env. Microbiol., 1995, 61, 4505-4509.

The present invention relates to a reagent comprising an enzyme specific cleavable polynucleotide substrate having a quenched fluorescent moiety, a method for preparing the same and an analytical method using the same.

1 is a schematic diagram illustrating the conceptual mechanism by which fluorogenic nuclease substrates are cleaved by bacterium-producing enzymes, where fluorogenic nuclease substrates represent intramolecular dimerization and represent the 3 'end and 5' of the polynucleotide. A fluorescently attached single-stranded polynucleotide having a fluorescent moiety attached to the terminal is quenched. When the polynucleotide is cleaved, the fluorescent moiety is separated and can be easily detected.

FIG. 2 is a schematic diagram illustrating the conceptual mechanism by which the fluorogenic nuclease substrate is cleaved by a bacterial producing enzyme, wherein the fluorogenic nuclease substrate is located in close proximity to each other through the dye deposition to extinguish the branched fluorescent part. Retaining single-stranded polynucleotide, when the polynucleotide is cleaved, the fluorescent part is separated and can be easily detected.

FIG. 3 is a schematic diagram illustrating the conceptual mechanism by which a fluorogenic nuclease substrate is cleaved by a bacterial producing enzyme, wherein the fluorogenic nuclease substrate exhibits intermolecular dimerization, with the 3 'end of two complementary polynucleotides Two complementary hybridized polynucleotide chains are quenched with two fluorescent moieties labeled at each of the 5 'ends, and when the polynucleotide is cleaved, the fluorescent moieties are separated and can be easily detected.

FIG. 4 shows a tetramethylhodamine-labeled 25-mer double-strand, which appears before (line b) and after incubation (line a) of micrococcus nuclease at 100 mmol bicarbonate buffer (pH 8.9) at 37 ° C. It is a graph depicting the relative fluorescence intensity of polynucleotides.

FIG. 5 shows tetramethyltamine-labeled 24-mer autocomplementarity that appears before and after incubation of micrococcus nuclease at 100 mmol bicarbonate buffer (pH 8.9) at 37 ° C. (line b). It is a graph which shows the absorption spectrum of single stranded polynucleotide.

details

The present invention discloses a new method for providing a reagent containing a nuclease substrate. The substrate separates the quenched fluorescence upon exposure to the nuclease to be analyzed, thereby detectably increasing fluorescence.

This nuclease substrate is prepared by first specifying the polynucleotide to be used. This is done by selecting a base or base pair (A / T or G / C) to be included in the polynucleotide. The arrangement may be an arrangement in which the fluorescent portions are arranged close together. Hybridization can be used to obtain a suitable arrangement. Hybridization is formed when the complementary base pairs of polynucleotides are attracted to each other. Possible arrangements are, for example, self-hybridization, ie self-complementary single-stranded polynucleotides with fluorescent dye moieties attached to both overlapping 3 'and 5' ends in order to approximate the fluorescent parts attached to each end of the polynucleotide. In another embodiment, each of the 3 '/ 5' pairs has a fluorescent moiety at each end of either or both ends, and the polynucleotides hybridize to form a double-stranded polynucleotide at one or both ends of each single-stranded polynucleotide. The attached fluorescent moieties comprise two mutually complementary single-stranded polynucleotides each having a 3 'end and a 5' end, which are in close proximity to each other. Another method involves single-stranded polynucleotides in which two or more fluorescent dye moieties are branched from the polynucleotide chain and are spaced very closely along the chain at appropriate lengths so that the fluorescent moieties are in close proximity to each other, resulting in quenching fluorescence. do.

In the case of the hybridized form, the degree of hybridization may be sufficient to bring the attached fluorescent parts into close contact with each other. In general, approximately 5 base pairs out of the base pairs closest to the 3 '/ 5' ends to which the fluorescent part is attached are sufficient to hybridize. This hybridization number depends on factors such as the type of base pair in the polynucleotide and the dimerization constant of the fluorescent part.

In addition, the polynucleotide is specified to include one or more bonds that can be cleaved by the nuclease to be analyzed. Strategically, this bond is placed in the polynucleotide such that the fluorescence is separated upon cleavage. In the case of self-complementary single-stranded polynucleotides or for two mutually complementary polynucleotides forming a double-stranded polynucleotide, the cleavable bond should generally be located at or near the end of the polynucleotide to which the fluorophore is attached. In the case of single-stranded polynucleotides having branching moieties, cleavable bonds should generally be located between the dye moieties.

In addition, the binding site for the selected fluorescent moiety is introduced into the polynucleotide at a specific position desired for the fluorescent moiety. Such sites may be the ends (ends) of the polynucleotides as in the case of hybridized polynucleotides or the polynucleotide chains, ie the backbone sites, as in the case of single-stranded polynucleotides to which branched dyes are attached. These binding sites include reactive groups such as aliphatic primary amines that can readily react with fluorescent compounds, including fluorescent moieties to be attached to the polynucleotide.

Once the polynucleotide is specified, the polynucleotide can be synthesized, for example, by known manual or automated DNA synthesis methods. Thereafter, the polynucleotide is reacted with at least one compound containing a fluorescent moiety. Fluorescent compounds generally include reactive groups that react with reactive groups on the polynucleotides. The reaction is carried out under suitable concentration, stirring, pH, ionic conditions and temperature conditions to attach the fluorescent moiety to the polynucleotide at a predetermined position. Specific requirements depend on the fluorescent compound used and the reactive groups present in the polynucleotide, respectively. pH is adjusted using a buffer such as carbonate buffer or bicarbonate buffer. The fluorescent portion used in the present invention has dimerization or stacking ability, so that fluorescence may be quenched.

In addition, the present invention

(1) one or more fluorescent parts are held in a position very close to the other one or more adjacent fluorescent parts, and the adjacent fluorescent parts extinguish the fluorescence with each other, but can be easily detected by fluorescence measurement upon separation. Formulating a reagent comprising an enzyme specific cleavable polynucleotide substrate, and (2) a sample that is likely to contain the particular enzyme to be analyzed, wherein the presence of the specific enzyme to be analyzed is cleaved from the polynucleotide It provides a biological assay characterized by separating the parts and consequently increasing the fluorescence intensity.

As another embodiment of this assay, the method may further comprise measuring an increase in fluorescence intensity. This can be done with a radiation energy floodlight, a fluorescence microscope, a 96 well plate reader or a flow cytometer.

1 illustrates a preferred embodiment of the present invention. Polynucleotide 10 may be synthesized to be autocomplementary, ie intrachain in-chain hybridization. When the fluorescent dye portions 11 and 12 are attached to the 3 'end and the 5' end, the dye portions are placed in close contact with each other to quench each other, so that the fluorescence is very weakly observed. When treated with nucleases 13 produced by bacteria 14, the polynucleotide 10 is cleaved at sites such as (15), (16) and (17) to destroy the self-hybridized structure, Generates fragment 18 and non-dimeric dye portions 11 'and 12', which dye portion freely fluoresces upon dissociation. In this embodiment, the length of the polynucleotide chain should be of sufficient length so that the polynucleotides overlap to bring the fluorescent dye moiety close. In general, approximately 15 base pairs are sufficient.

2 shows a second preferred embodiment of the invention. The plurality of fluorescent dye portions 22 are attached to the polynucleotide 20 including various sites 24 that are susceptible to enzymatic cleavage. The dye portions 22 are disposed close enough to each other so that the fluorescent property of the dye portion 22 is extinguished. The conjugate (including the polynucleotide 20 and the fluorescent dye portion 22) is treated with the nuclease 26 produced by the bacteria 28 to cleave the polynucleotide 20. Cleavage produces nucleotide fragments 29 and separates the dye moieties 22 'from each other in the medium, making them freely fluorescent and easily detectable. In this embodiment, the fluorescent portions 22 are preferably separated by a distance of about 10 nm or less between bases present along the backbone of the polynucleotide. This length is determined by an indirect method based on the known or estimated length of the polynucleotide base. The arrangement shown in FIG. 2 can be used to test a plurality of types of bacteria or enzymes with a series of pairs of paired or grouped fluorescent moieties, where each type can be distinguished from each other using fluorometry techniques. Each moiety in each pair or group may be bound by cleavable bonds that are susceptible to cleavage by other bacterial enzymes or may be located on opposite sides of the cleavable bonds.

Figure 3 shows another preferred embodiment of the present invention. Complementary hybridized polynucleotides 30 and 31 have fluorescent dye portions 32 and 33 attached to their 3 'and 5' ends, respectively. Nuclease 36 produced by bacteria 38 cleaves polynucleotides 30 and / or 31 at cleavage site 34 to nucleotide fragment 39 and nondimerized dye moiety 32 '. ) And (33 ') can be easily detected. Another embodiment may include a dye moiety attached to each 3 'end and 5' end of each complementary polynucleotide. The arrangement shown in FIG. 3 can be used to simultaneously test two kinds of bacteria or enzymes. The method labels each complementary 3 '/ 5' end pair with a different type of fluorescent moiety that can be distinguished from each other using fluorometry techniques, and by different types of bacterial enzymes near each end of the double-stranded polynucleotide. A double-stranded polynucleotide is constructed so that a cleavage site that is easy to cleave is located.

Fluorescent substrates are molecules that vary from substantially nonfluorescent to highly fluorescent by enzymatic hydrolysis. This substrate is widely used as a molecular probe in the research and testing of viral and bacterial enzymes such as proteases, nucleases, saccharases, phosphatase and kinases. The fluorescence of the substrate can be readily measured by conventional techniques such as radiation energy projection, fluorescence microscopy, 96 well plate reader or flow cytometer.

Enzyme specific cleavable polynucleotide substrates having a fluorescent moiety or fluorogenic nuclease substrate useful in the present invention may be prepared by chemical reaction of polynucleotides with fluorescent dyes such as fluorescein and rhodamine to dimerize or deposit Can be. Useful polynucleotides are commercially available (eg, GeneMed Biotechnologies, South San Francisco, Calif.). In order to prepare the reagents of the present invention, two or more fluorescent moieties cause dimerization or lamination (eg, through substantially hybridization, proximity, interchain hybridization, intrachain hybridization, etc.) to mutually extinguish the fluorescence of the fluorescent moiety. (If the fluorescent part is the same, it is called "auto quenching.") The said fluorescent part is couple | bonded with a polynucleotide.

Fluorescent dye quenching can occur through many known mechanisms, including energy transfer and dye dimerization. In this case, when energy is input and generally irradiates light of a specific wavelength to excite a fluorescent dye molecule or part, energy is transferred from the dye part to another part rather than dispersed by fluorescence. Energy transfer (also called the posterior dipole interaction) occurs between the fluorescent donor and the receptor, whether or not fluorescent. Energy transfer generally occurs when the distance between the donor and acceptor on the substrate backbone is about 10 nm. Through energy transfer, the fluorescence of the donor portion is quenched. On the other hand, dye dimerization or dye stacking occurs when two or more fluorescent groups are brought close enough to each other to form stable complexes such as dimers and trimers through the interaction of their planar aromatic rings. Each fluorescent part acts on the other fluorescent part to quench each other. The absorbance spectrum of the dye in the dimer state or the stacked state is substantially different from that of the same dye in the energy transfer pair. The dye dimer absorbance spectrum shows a characteristic decrease in absorbance of the absorbance main peak as the dye concentration increases, whereas the increase in characteristic absorbance of the shoulder peak. This phenomenon is illustrated in FIG. 5. For the dye molecules shown in the graph of FIG. 5, the absorbance main peak appears at about 560 nm and the shoulder peak at about 530 nm. The dimeric sample (c) exhibits an absorbance peak with an absorbance unit (AU) of about 0.03 at both the absorbance main peak and the shoulder peak, whereas the nondimeric sample (d) has a main absorbance peak of about 0.06 AU and about 0.028 AU. Leveled shoulder peaks are shown. The phenomenon appearing in the dimeric sample as such is usually referred to as "band segmentation". Dimerization or lamination occurs through the formation of steady state complexes (ie, through physical close proximity), while energy transfer interactions occur through space. Thus, characteristic spectral changes in dye dimerization are not observed in dyes that interact by energy transfer mechanisms.

The quenching mechanism of the dimeric or layered dye pairs or groups of dyes used in the present invention is different from the quenching mechanisms of fluorescent energy transfer donors and acceptors. Types of dyes that exhibit dimerization or stacking properties when bound to polynucleotides in close proximity to each other include homodimers or when in solution at sufficiently high dye concentrations (e.g., 10 ^-2 to 10 ^-4 M). It generally includes dyes having a planar aromatic structure so as to form a heterodimer. This phenomenon is clearly undesirable in many fields, but is advantageously used in the present invention. Concentration can be increased by increasing the amount of dye in a unit dose or by placing two (or more) dye portions physically in close proximity together, such as on a polynucleotide or other small molecule. When the dye portion is placed in close proximity, the local concentration is effectively increased, and the polynucleotide carrying the fluorescent portion is in the quenched state regardless of the total concentration.

Planar aromatic dyes belonging to the fluorescein and rhodamine series, fluorescein, tetramethyltamine (TMR), rhodamine B and X-rhodamine, etc., are representative dyes which dimerize when the concentration is sufficiently high. Other suitable fluorescent dyes include, for example, cyanine, boron-heterocyclic dyes, such as 4,4-difluoro-4-boreta-3a-azonia-4a-aza-s-indacene (in Eugene, Oregon, USA). Molycula Probes, Inc. under the tradename BODIPY ™). Rhodamine-based dyes, such as tetramethyltamine, X-rhodamine and rhodamine B, exhibit very high fluorescence quantum yields (about 0.85) in the visible wavelength range (400-700 nm). Therefore, this dye is often used to detect trace amounts of material.

The dye portion used in the present invention may further include a reactive end group for easily attaching the fluorescent portion to the polynucleotide. This terminal group may include succimidyl ester, maleimide, ester, thioisocyanate, isocyanate and iodoacetamide. Dye molecules to which such reactive groups are already attached are commercially available, for example, from Molecular Probes, Inc., Eugene, Oregon, USA.

Polynucleotides used in the present invention must have at least one enzyme specific cleavable site. Fluorescent quenched polynucleotides having two or more fluorescent moieties will increase in fluorescence intensity upon cleavage as the dimerized fluorescent moiety or stacked fluorescent moieties dissociate. As a result the fluorescence is easily detected, suggesting the presence of specific enzymes and, where appropriate, the bacteria producing enzymes. Due to the interaction between the transition dipoles of the resonant dimer structure or the laminated structure, the fluorescent quantum yield of the dimeric structure or the laminated structure is significantly lower when the polynucleotide is intact. However, if the polynucleotide is enzymatically cleaved to dissociate the dimer or stacked structure, the fluorescent moiety can be separated, resulting in a significantly higher fluorescent quantum yield in aqueous solution due to the inherent high fluorescence. In this way, the increase in fluorescence and the presence of hydrolase can be distinguished. On this basis, homogeneous enzyme assays can be devised which do not require a colorant and a separation step. This assay involves making an enzyme specific cleavable polynucleotide bearing a fluorescent moiety and adding an enzyme analyte, wherein any enzyme present in the analyte cleaves the polynucleotide to dimerize Or reduces lamination and consequently increases fluorescence. Thus, enzyme activity is directly related to the total increase in fluorescence intensity. If the enzyme is isolated from active metabolic cells, the fluorescence intensity correlates with the metabolic activity of the cells.

The ability of one enzyme molecule to convert millions of reagent molecules is an amplification process. This amplification is further enhanced when enzymes are obtained from live cell culture because more enzyme molecules are produced as the cells proliferate. Because of these two factors, use with fluorescence techniques yields a preferred method for the rapid, sensitive and specific detection of bacteria.

Representative polynucleotides useful for generating fluorogenic substrates must possess the necessary chemical bonds that are the target of nucleases to be analyzed. For example, as described below, micrococcus nucleases are known to hydrolyze polynucleotides at phosphodiester bonds, so any polynucleotide having this feature can be a micrococcus substrate.

Chemical modification of polynucleotides is carried out using nucleotide analogs and DNA synthesis reagents. For example, phosphoramidites containing aliphatic primary amines are preferred via automated DNA synthesizers such as the Nucleic Acid Synthesis and Purification System (Perkin Elmer Applied Biosystems, Foster City, Calif., Model # ABI 3948). Can be introduced into the polynucleotide chain. Many terminal modifying agents and amino modified deoxythymidines (dT) are used to introduce aliphatic amines having C ₃ , C ₆ or C ₉ linkages at the 3 ′ and / or 5 ′ ends of the polynucleotide. The amine can then be reacted with various labels, such as biotin, fluorescent dyes or alkaline phosphatase, to form polynucleotide conjugates. Typical uses of such modified polynucleotides include (i) non-radioactive hybridization probes, (ii) sequence specific binding of DNA, (iii) automated DNA sequencing and (iv) affinity chromatography. Other parts that may be added to the ends of the polynucleotide to react with the moiety of dye to be attached include those having reactive H groups such as free thiols (SH), carboxyl groups and OH groups. Preferred are amines and thiols.

Enzymatic cleavage is carried out by contacting a fluorogenic nuclease substrate, i.e., a reagent with a particular enzyme to be analyzed. Target enzymes suitable for use in the present invention include all enzymes generally classified as nucleases, ie enzymes that hydrolyze nucleotide bonds, for example thermonucleases or Staphylococcus nucleases.

If the enzyme is an intracellular enzyme, the enzyme can be prepared for easy contact with the fluorogenic nuclease substrate using an agent that increases the permeability of the outer membrane (OM) of the enzyme bearing bacteria. Ethylenediaminetetraacetic acid (EDTA), which affects the outer membrane barrier of Gram-negative intestinal bacteria, is generally used for this purpose. Under certain conditions, OM may be disrupted to release intracellular enzymes.

For rapid sensitive tests, it is important to maximize the desired signal relative to the undesired background signal, such as the signal-to-noise ratio, in this case the autofluorescence of the bacteria and growth medium. Most conventional fluorogenic substrates emit light in the far ultraviolet wavelength region (350-450 nm) where most bacterial growth media exhibit significant autofluorescence. For example, the general culture medium of Staphylococcus aureus shows significant maximal emission at 425 nm and 475 nm when excited at 360 nm. To avoid this problem, substrate concentrations are generally used at high concentrations. However, this increases the cost of analysis, can be highly toxic to the organism, and sometimes precipitates the substrate. Thus, the present invention employs fluorescent dyes (e.g., tetramethyltamine: Ex = 550 nm, Em = 580 nm) that are emitted at wavelengths much longer than the background fluorescent wavelengths to red shift the detection wavelength toward the visible light spectrum and cause autofluorescence. Overcome the disadvantages of interference In addition, the technical idea disclosed in the present invention may be applied to other dyes that shift the emission wavelength to a longer wavelength. In general, useful dyes have the following characteristics: high extinction coefficient (> 80,000 cm ^-1 M ^-1 ), high quantum yield (> 0.85 in aqueous solution), solvent- and pH-insensitive spectrum, aqueous solubility, light Stability and preferably a dimerization constant of at least 10 ⁻³ .

Many Staphylococcus strains exhibit DNase activity. An enzyme that has traditionally been used for identification purposes is the extracellular DNase of S. aureus. S. aureus nucleases are secreted in large quantities, thermostable, and require Ca ²⁺ instead of Mg ^{2+ for their} activity. This feature is used to distinguish S. aureus from other Staphylococcus in detecting Staphylococcus contamination in food. Extracellular DNases are also produced by Streptococcus spp., Serratia spp. And Bacillus spp.

Micrococcus nuclease, an extracellular enzyme of S. aureus, is used as a model system to confirm the utility of the present invention. This enzyme cleaves the 5 'phosphodiester linkage of the polynucleotide chain, remains stable and active at high temperatures (> 60 ° C), and is commonly used as an indicator of S. aureus. The presence of nuclease activity at 60 ° C. is a confirmatory test for S. aureus. Micrococcus nucleases do not require cofactors for their function. This is a feature that can reduce the complexity and cost of analysis.

In summary, the present invention provides novel nuclease reagents, analytical methods using the reagents, and methods of making the reagents. This reagent comprises a single- or two-chain synthetic polynucleotide having two or more fluorescent moieties that may be dye molecules (eg tetramethyltamine). Fluorescence increase after nuclease hydrolysis and cleavage is on the order of 2 to 6 times, depending on the specific form of the reagent. When this reagent is combined with the "double cascade" phenomenon, that is, each enzyme molecule can convert millions of reagent molecules and more enzyme molecules are released when bacterial cells divide and multiply exponentially, Rapid, sensitive and specific bacterial detection methods are provided. The present invention provides, but is not limited to, microbial detection and identification, sterilization confirmation, drug discovery, enzyme assays and immunoassays.

The objects and advantages of the present invention will be described in more detail through the following examples, but the specific materials and amounts and other conditions and details used in these examples should not be understood as limiting the invention.

The present invention schematically provides a reagent comprising at least one enzyme specific cleavable polynucleotide substrate having at least one fluorescent portion in close proximity to the at least one adjacent fluorescent portion, wherein the adjacent fluorescent portions are mutually contiguous. Fluorescence is quenched and can be easily detected by fluorometry techniques upon cleavage of the substrate.

The reagents include enzyme specific cleavable polynucleotide substrates containing autocomplementary single-stranded polynucleotides each having a fluorescent moiety at the 3 'end and the 5' end; A polynucleotide substrate comprising two complementary hybridized polynucleotide chains each containing a 3 'end and a 5' end having a fluorescent moiety at each end of one or both 3 '/ 5' pairs; Or a polynucleotide substrate containing single-stranded polynucleotides having two or more branched adjacent fluorescent portions along the length direction.

In addition, the present invention

(1) one or more fluorescent parts are held in a position very close to the other one or more adjacent fluorescent parts, and the adjacent fluorescent parts extinguish the fluorescence with each other, but can be easily detected by fluorescence measurement upon separation. Combining a reagent comprising an enzyme specific cleavable polynucleotide substrate, and (2) a test sample that is likely to contain the specific enzyme to be analyzed, wherein the presence of the specific enzyme to be analyzed is cleaved It provides a biological assay characterized by isolating the fluorescence and consequently increasing the fluorescence intensity.

As another embodiment of this assay, the method may further comprise measuring an increase in fluorescence intensity.

Fluorescence can be readily measured by conventional techniques such as radiation energy luminescence, fluorescence microscopy, 96 well plate reader or flow cytometer.

The present invention also provides a method of preparing an enzyme specific cleavable polynucleotide reagent having a quenched fluorescent moiety. This method produces an enzyme specific cleavable polynucleotide-containing reagent that quenches each other through dye dimerization but retains the fluorescent moiety that can be detected by fluorescence techniques once separated. This way

(1) a self-complementary single-stranded polynucleotide having at least one terminal group reactive with at least one fluorescent compound, and (2) at least two moieties containing a branch reactive with at least one fluorescent compound other than at the ends in the polynucleotide. Single-stranded polynucleotides held in position and (3) one or more reactive end groups reactive with one or more fluorescent compounds, each chain at the 3 'and / or 5' ends of one or both of the 3 '/ 5' pairs, respectively And combining at least one fluorescent compound having at least one fluorescent moiety and at least one reactive group with an enzyme specific cleavable polynucleotide selected from the group consisting of complementary polynucleotides, the combination comprising reactive conditions Under test to produce the reagents.

In this specification,

"Dye dimerization" means the formation of a non-covalent complex between two dye groups,

By "dye lamination" is meant the formation of a non-covalent complex between two or more dye groups, wherein the two complexed dye groups are called "dimers" and the three complexed dye groups are called "trimers".

By "enzyme specific cleavable polynucleotide" is meant a linear sequence of nucleotides containing a specific bond that is susceptible to cleavage by a specific enzyme.

The term "fluorescent" means that the material emits light of a certain wavelength when absorbing light of a different wavelength, wherein the emission of light occurs only during light absorption.

"Fluorescent quantum yield" means the ratio of the number of fluorescent photons emitted by a light emitting material to the total number of photons absorbed by the material.

The term "fluorescent substrate" refers to a substance which is substantially non-fluorescent which produces a fluorescent compound upon enzyme action.

The term “fluorophore”, “dye portion” or “dye group” means a molecule or portion of a molecule that emits light of a predetermined wavelength when it is stimulated by absorbing light of different wavelengths (usually short wavelength).

The present invention provides advantages over conventional assays that have been used for the detection and identification of microorganisms. The present invention provides a rapid and rapid method for detecting the presence and / or measuring activity of intracellular and extracellular enzymes using enzyme specific cleavable polynucleotide substrates of fluorescent moiety retention, also referred to herein as fluorogenic nuclease substrates. Provide a convenient way. This method can also be adapted to sequence dependent tests or sequence independent tests. The methods and reagents of the present invention have resulted in improved accuracy, faster detection and lower total cost in detecting and identifying microorganisms. In various embodiments, the invention utilizes measurable differences in fluorescence capable of detecting nucleases. As a preferred embodiment, the present invention exhibits higher signal intensity upon cleavage as compared to lower signal intensity upon quenching, and preferably fluorescein acting in the visible range which minimizes interference by basic fluorescence of the propagation medium. Provide an indicator.

The invention first of all redistributes the emission wavelength from the far ultraviolet region (about 350-400 nm) to the 500-600 nm region of the electromagnetic spectrum and reduces the effect of the reference signal intensity of the original reagent to detect the presence of nucleases. This provides a more accurate and sensitive measurement method. Another advantage of the present invention is that it is a homogeneous biological assay that does not require a colorant. Thus, it does not require time-consuming separation steps such as in enzyme-linked immunosorbent assays (ELISA) and assay formats such as bioluminescence. Detection and identification of microorganisms can be performed using a basic identification medium.

As will be apparent from the present specification, the assays of the present invention are characterized by more efficient use of reagents, labor, time and devices. In addition, the careful design and selection of polynucleotides and fluorophores attached thereto offer the possibility of simultaneously investigating several bacteria and / or enzymes. The analytical methods described herein are closely related to the detection and identification of microorganisms, sterility confirmation, pharmaceutical drug discovery, clinical analysis, nucleic acid hybridization, sequencing and amplification.

Test Methods

Fluorescence spectrum

All fluorescence measurements were performed with a fluorescence spectrometer commercially available under the trade name FLUOROLOG ™ from Specs Instruments, Edison, NJ. In a typical measurement, 3 ml of the assay solution was placed in a quartz cuvette and placed in the sample fixture of the fluorometer. The fluorescence emission spectrum of the assay solution was measured at fixed excitation wavelengths at room temperature. The width of the excitation and emission grooves of the fluorometer was set to 0.5 millimeters. Fluorescence spectra of assay solutions with or without nucleases were measured under the same instrument conditions.

Example 1

In this example, two tetramethyltamine (TMR) dye moieties were combined at the 3 'end and the 5' end of the autocomplementary 24-mer polynucleotide. The dye portions were brought into close contact with each other by in-chain hybridization, resulting in quenching of fluorescence. Enzymatic cleavage at multiple sites disrupted the rule structure. As a result, the dye moiety is dedimerized, indicating a significant increase in fluorescence. Such a technical idea is schematically illustrated in FIG. 1.

NH ₂ CH ₂ CH ₂ CH ₂ -5'-AAC AAA GGA TAA TTA TCC TTT GTT-3'-CH ₂ CH ₂ CH ₂ NH ₂ , a self-complementary polynucleotide, is available from GeneMed Biotechnologies, Inc. (South San Francisco, Calif.), And its chemical structure was confirmed by DNA sequencing. About 270 nmol of this polynucleotide was dissolved in 600 µl of deionized water. 90 nmol (200 μl) of a polynucleotide solution was added to tetramethyltamine (TMR) succimidyl ester (Morcuric Probes, Eugene, Eugene, Oregon, USA) 4 mg [100 mM sodium bicarbonate buffer (pH 8.3) Pre-dissolved in 1 drop of dimethyl sulfoxide (DMSO) in the [] and reacted at room temperature. This mixture was a sufficient volume (ie, about 2 mL) to maintain a proper pH. In this amidation process, the ester of the TMR moiety reacted with the amine moiety attached to the polynucleotide.

The reaction mixture was pipetted using a size exclusion column containing epichlorohydrin crosslinked (alkali condition) dextran gel (available under the tradename SEPHADEX ™ G25 from Ink, Inc.) as a filler material (Piece, NJ) Charged to Pharmacia Biotech, Inc., Kataway, Model PD-10), the labeled polynucleotide was separated from the unreacted dye. The reaction mixture was passed through the column under gravity. The resulting DNA fractions were further purified by C18 reverse phase column (High Performance Liquid Chromatography (HPLC)) (Waters Corporation, Milford, Mass., Model 600E HPLC). As the mobile phase of the column, a gradient of 0.1M triethylamine acetate (TEAA) and acetonitrile (ACN) in water was used, with the volume ratio of TEAA: ACN decreasing linearly to 8:92 over 30 minutes starting at 92: 8. I was. The final conjugate was the polynucleotide structure with TMR addition on each of the aliphatic amine linkages at the 3 'and 5' ends.

HPLC fractions containing the purified conjugate were collected, frozen on anhydrous ice and then vacuum dried at room temperature. The anhydrous conjugate was then dissolved in 5 ml of 100 mM sodium carbonate buffer, pH 8.9. As shown in FIG. 5, an absorption spectrum measured using a spectrophotometer (Shimazu Scientific Instruments, Model MPC-3100, Columbia, MD) shows the characteristic band splitting characteristics of dimerized tetramethyltamine. Indicated. A small amount (about 0.5 mg) of Micrococcus nuclease (obtained from Sigma Chemical Company, St. Louis, MO) was added to 3 ml of the buffer solution. This mixture was incubated overnight at 37 ° C. for enzymatic hydrolysis of the 5′-phosphodiester bond of the polynucleotide bearing fluorescent moiety. This solution was then cooled to room temperature. The fluorescence spectra were then measured at room temperature using a FLUOROLOG ™ fluorescence spectrometer. As a result of enzyme cleavage, the absorbance at 560 nm increased to 0.06 AU at 0.03 absorbance unit (AU) when excited with light at 520 nm, and the fluorescence intensity increased from 12000 to 28000 at 580 nm.

In another test, the length of the aliphatic amine linkage at the 5 'end was increased to allow for more efficient lamination between the two dye sections. The following polynucleotides with longer aliphatic amine linkages were obtained from Gin Med and were labeled and tested under similar conditions as described above.

H ₂ N- (CH ₂ CH ₂ O) _6- (CH ₂ ) ₃ -5'-AAC AAA GGA TTA TAA TCC TTT GTT-3'-CH ₂ CH ₂ CH ₂ -NH ₂

H ₂ N- (CH ₂ CH ₂ O) _9- (CH ₂ ) ₃ -5'-AAC AAA GGA TTA TAA TCC TTT GTT-3'-CH ₂ CH ₂ CH ₂ -NH ₂

A two-fold increase in fluorescence was observed with each test.

Example 2

In this example, a branched tetramethylhodamine dye moiety was attached at multiple positions in the polynucleotide sequence. Due to the proximity, the dye portions were laminated together to quench the fluorescence. Enzymatic cleavage of the polynucleotide at multiple positions disrupted the conjugate, dissociating the dye portion into a single portion in the dimer or stacked state, while simultaneously increasing the fluorescence intensity. This technical idea is schematically illustrated in FIG. 2.

The polynucleotide chain was designed to include a total of four amino groups in the replacement base as follows. 5'-TTT TTT ZTZTZTZ TTT TTT-3 '. Where Z is an amine modified C ₆ derivative of deoxythymidine (dT) (Glen Research, Sterling, Va., Catalog No. 10-1039-90). Synthesis of the polynucleotide chain was synthesized by Gin Med Biotechnology, Inc. Approximately 45 nmol of polynucleotides are dissolved in deionized water and 1 mg of tetramethyltamine (TMR) succimidyl ester pre-dissolved in 1 drop of dimethylsulfoxide (DMSO) in 100 mM sodium bicarbonate buffer (pH 8.3). And reacted at room temperature. The conjugate was purified under the same conditions as in Example 1. Enzymatic hydrolysis was performed by reacting overnight at 37 ° C. using a small amount (about 0.5 mg) of micrococcus nuclease in 100 mM sodium bicarbonate buffer (pH 8.9). The fluorescence spectrum was measured at room temperature with an excitation wavelength of 520 nm, and the fluorescence intensity at 575 nm increased about 2 times after enzymatic hydrolysis.

Example 3 (Comparative Example)

In this example, quenching (quenching of one dye portion of the dye portions) was carried out by energy transfer, not dye dimerization.

DNase activity by fluorescence energy transfer was analyzed using the following two polynucleotides obtained from Ginmed Biotechnology, Inc.

A. 5 'TTA XTA AYT CCG TTT AA 3'

B. 5 'TTA ZTA AYT CCX TTT AA 3'

Wherein X is tetrachlorofluorescein (receptor), Y is fluorescein (donor), Z is another receptor branched from an amino modified C ₆ [Z of deoxythymidine (dT), tetramethyltamine (TMR) attachment. Two different dyes, the donor and the receptor, X and Y, in the A configuration were inserted into the polynucleotide sequence. Unlike Example 2, which is branched from all dye addition polynucleotides, in this example, the donor and one receptor were bottled into the polynucleotide backbone itself. The B configuration is similar to A except that the second receptor (TMR) is branched from Z. When the polynucleotide was intact, the donor and the receptor were located within energy transfer distance of each other. The excitation state energy of fluorescein was non-radioactively transferred to tetrachlorofluorescein (and / or tetramethyltamine in B) to quench donor fluorescence. Receptor fluorescence is not quenched. After DNA is enzymatically hydrolyzed, the donor and receptor are separated, resulting in little energy transfer. The donor fluorescence of Compound A after hydrolysis by micrococcus nuclease increased about 30% at about 530 nm upon excitation at 490 nm. When visually observed, the fluorescence changed from orange blue to dark blue, indicating that the energy transfer efficiency was reduced. Similar results were observed for compound B.

Example 4

In this example, tetramethyltamine was attached to aliphatic amine linkages present at the 3 'end and the 5' end of the two complementary polynucleotides. The dye chains were brought close to each other and fluorescence was quenched through interchain hybridization. By enzymatic cleavage at multiple sites along the double-stranded polynucleotides, disrupting the regular structure, dissociation of the dye portion from the dimer to the monomer yields a significant increase in fluorescence. This technical idea is schematically illustrated in FIG. 3.

Two complementary 25-mer polynucleotides were synthesized by Ginmed Biotechnology, Inc.

NH _2- (CH ₂ ) ₃ -5'-AAC AAA GGA TTA TAG TGG GAA ATC A-3 '

NH _2- (CH ₂ ) ₃ -3'-TTG TTT CCT AAT ATC ACC CTT TAG T-5 '

About 22.5 nmol of each polynucleotide was reacted with 1 mg of tetramethyltamine (TMR) isothiocyanate in 100 mM sodium carbonate buffer (pH 9.0), respectively, overnight at 23 ° C. under stirring (shaking). The dye was first dissolved in 1 drop of DMSO and then mixed with the polynucleotide. A thio-urea bond was formed between the amine group attached to the polynucleotide and the isothiocyanate of the TMR moiety. Each reaction mixture was passed through a size exclusion column described in Example 1 (Pharmacia Biotech, Inc., Piscataway, NJ, PD-10) to separate labeled species from unreacted dye molecules. Melting point T _m was calculated based on the ratio of the ionic strength of the hybridization solution (Promega Corp., Madison, Cat. No. F123A, Catalog No. F123A) to the ratio of the GC pair content in the polynucleotide chain using the following equation.

T _m = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% GC)-(600 / N)

Where N is an integer representing the number of bases in the polynucleotide chain. Through this equation, one can predict whether T _m is suitable for polynucleotides having a length between 14 and 60 to 70 nucleotides in length. The Na ⁺ concentration of the promega hybridization solution is 0.39. Based on the above formula, the T _m of the polynucleotide was calculated to be 63.8 ° C. Hybrids formed between polynucleotides short at temperatures below 5 to 10 ° C. below T _{m were} likely to dissociate reversibly. Therefore, the reaction temperature should be at least 10 ° C. lower than T _m . In addition, to produce stable hybrids, hybridization with short polynucleotides should be performed under stringent conditions using high concentrations (0.1-1.0 picomolar / ml) of polynucleotides. In this example, hybridization of two chains with fluorescent dyes was performed overnight at 45 ° C. (19 ° C. lower than T _m ) in a highly stringent hybridization solution (Promega solution). The conjugate solution was observed to exhibit fluorescence intensity at 575 nm (Ex = 550 nm), which was observed at 37 ° C. in 100 mM bicarbonate buffer (pH 8.9) than before treatment with nuclease (US). After an overnight incubation with Sigma Chemical Company, catalog number N-3755, St. Louis, Missouri, increased approximately 6 times. This is shown in FIG.

Various modifications and modifications of the present invention will be well understood by those skilled in the art without departing from the scope and spirit of the present invention. Accordingly, the present invention should not be understood as limited only to the above-described exemplary embodiments.

Claims (20)

One or more enzyme specific cleavable polynucleotide substrates having at least one fluorescent site in close proximity to the other at least one adjacent fluorescent site, wherein the adjacent fluorescent sections are fluorescence quenched with each other, A reagent characterized by being easily detectable by a measurement technique. The reagent according to claim 1, wherein the fluorescent part comprises a fluorescent dye group. The reagent according to claim 1 or 2, wherein the fluorescent part is the same. The reagent of claim 1 or 2, wherein the polynucleotide substrate comprises about 24 base pairs. The reagent according to claim 1 or 2, wherein the polynucleotide substrate comprises self-complementary single-stranded polynucleotides having a 3 'end and a 5' end each having a fluorescent moiety. The two complementary hybridizations according to claim 1 or 2, wherein the polynucleotide substrate has a fluorescent moiety at each end of one or both of the 3 '/ 5' pairs, respectively, having a 3 'end and a 5' end. A reagent characterized by comprising a polynucleotide chain. The reagent of claim 1 or 2, wherein the polynucleotide substrate comprises a single stranded polynucleotide having two or more contiguous branched fluorescent portions along its length. The reagent according to claim 7, wherein the adjacent fluorescent parts are separated from each other by an interval of 10 nm or less when the distance between bases is measured along the polynucleotide backbone. The reagent according to claim 1 or 2, wherein the enzyme specific cleavable polynucleotide substrate can be cleaved by an enzyme selected from the group consisting of DNase and RNase. The reagent of claim 9, wherein the DNase is a bacterial DNase. The reagent of claim 2, wherein the fluorescent dye group comprises at least two dimerized or stacked fluorescent dye groups. The reagent of claim 2, wherein each dye group comprises a planar portion. The reagent according to claim 2, wherein each dye group is selected from the group consisting of fluorescein, rhodamine, cyanine and boron heterocyclic dye groups. The reagent according to claim 13, wherein each rhodamine dye is selected from the group consisting of tetramethyltamine, X- rhodamine and rhodamine B. (1) the reagent according to claim 1 or 2, and (2) combining a test sample that is likely to contain the particular enzyme to be analyzed, wherein the presence of the specific enzyme to be analyzed yields polynucleotide cleavage, fluorescence separation and increase in fluorescence intensity . The biological assay of claim 15 further comprising measuring an increase in fluorescence intensity. The biological assay of claim 16, wherein the increase in fluorescence intensity is measured by means selected from radiation energy flooding, fluorescence microscopy, 96 well plate reader, and flow cytometer. The biological assay of claim 15, wherein the enzyme is derived from bacteria. (1) a self-complementary single-stranded polynucleotide having at least one terminal group reactive with at least one fluorescent compound, and (2) at least two moieties containing a branch reactive with at least one fluorescent compound other than the polynucleotide terminus Single-chain polynucleotides retained at and (3) one or more reactive end groups reactive with at least one fluorescent compound at each of the 3 'and / or 5' ends of one or both of the 3 '/ 5' pairs; Combining an enzyme specific cleavable polynucleotide selected from the group consisting of complementary polynucleotides and at least one fluorescent compound having at least one fluorescent moiety and at least one reactive group, the combination being under reactive conditions Characterized in that they are quenched from each other through dye dimerization, which is characterized by the formation of reagents, A method for preparing an enzyme specific cleavable polynucleotide containing reagent having a fluorescent moiety that can be detected by 20. The method of claim 19, wherein the compound having a fluorescent moiety is selected from the group consisting of fluorescein, rhodamine, cyanine and boron heterocyclic dye groups.