WO2010059944A1 - Enzyme à adn divisé pour le typage visuel du polymorphisme d'un nucléotide unique - Google Patents

Enzyme à adn divisé pour le typage visuel du polymorphisme d'un nucléotide unique Download PDF

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WO2010059944A1
WO2010059944A1 PCT/US2009/065341 US2009065341W WO2010059944A1 WO 2010059944 A1 WO2010059944 A1 WO 2010059944A1 US 2009065341 W US2009065341 W US 2009065341W WO 2010059944 A1 WO2010059944 A1 WO 2010059944A1
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oligonucleotide
analyte
probe
hemin
bopp
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PCT/US2009/065341
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English (en)
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Dmitry Kolpashchikov
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Columbia University
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Priority to US13/130,536 priority Critical patent/US8551768B2/en
Priority to BRPI0920985-9A priority patent/BRPI0920985A2/pt
Priority to EP09828292A priority patent/EP2367838A4/fr
Publication of WO2010059944A1 publication Critical patent/WO2010059944A1/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

Definitions

  • the present invention relates to binary probes for detecting nucleic acids.
  • SNPs Single nucleotide polymorphisms
  • Large-scale sequence analysis is needed for a population-based genetic risk assessment and diagnostic tests once a mutation has been identified.
  • most of the methods for SNP screening require enzymatic manipulations such as endonuclease digestion, ligation or primer extension, and often separation of the resultant products. 1
  • GNP gold nanoparticle
  • FIG. 1 Design of the binary DNA peroxidase for SNP analysis.
  • A Parent peroxidase-like DNA enzyme.
  • B Binary DNA peroxidase probe.
  • C The probe forms active peroxidase upon hybridizing to the abutting positions of the analyte. The enzyme catalyzes oxidation of a colorless substrate to colored products.
  • the triethylenglycol linkers are shown as dashed lines in panel C.
  • the SNP site in the analyte sequence is underlined.
  • FIG. 2 Binary DNA peroxidase probe reports single nucleotide substitution in tau protein coding DNA.
  • A Visual detection.
  • Sample 0 reaction buffer (50 mM HEPES, pH 7.4, 50 mM MgCl 2 , 20 mM KCl, 120 mM NaCl, 0.03% Triton X-100, 1% DMSO, hemin (125 nM), H 2 O 2 (1 mM), and DAB (1 mM));
  • Sample 1 reaction buffer in the presence of 1 ⁇ M peroxidase- like DNA enzyme (positive control).
  • Lane 1 Peroxidase-like DNA enzyme (1 ⁇ M); lane 2: Binary DNA peroxidase (1 ⁇ M both strands); lane 3: Binary DNA peroxidase (1 ⁇ M both strands) and rs242557-A (1 ⁇ M); lane 4: Binary DNA peroxidase (1 ⁇ M both strands) and rs242557-G (1 ⁇ M); lane 5: Strand ⁇ (1 ⁇ M) only; lane 6: Strand ⁇ (1 ⁇ M) and rs242557-A (1 ⁇ M); lane 7: Strand ⁇ (1 ⁇ M) and rs242557-G (1 ⁇ M); 8: 10 base pairs (10 bp) DNA ladder.
  • the low mobility band in lane 4 is indicated by an arrow.
  • FIG. 4 Binary peroxidase-like DNA enzyme reports SNP in the presence of ABTS.
  • A Strands ⁇ and ⁇ in the absence (2), or presence of rs242557-A (3) and rs242557-G (4); Sample 0 contained reaction buffer only; Sample 1 : reaction buffer and peroxidase-like DNA enzyme. The samples were incubated 30 min at room temperature.
  • B Electronic absorption spectra; sample 0 was used as a reference.
  • C Average optical densities of five independent measurements at 419 nm.
  • FIG. 5 Binary DNA peroxidase probe containing various linker combinations.
  • A Sequences of the probe (strands ⁇ tt and ⁇ tt ) containing dithymidine linkers (shown in low cases).
  • B Light absorption of the probes containing different strand combinations in the absence (first group) or presence of rs242557-A (second group), or rs242557-G (third group).
  • the samples contained combinations of 1 ⁇ M strands ⁇ , ⁇ , ⁇ tt , and ⁇ tt (as indicated for each bar type) were incubated 30 min at room temperature in the presence of DAB as an oxidizable substrate followed by the registration of electronic absorption spectrum.
  • the optical densities at 500 nm are represented as average values of four independent measurements.
  • base pair is generally used to describe a partnership of adenine (A) with thymine (T) or uracil (U), or of cytosine (C) with guanine (G), although it should be appreciated that less-common analogs of the bases A, T, C, and G (as well as U) may occasionally participate in base pairings. Nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration may also be referred to herein as "complementary bases”.
  • Complementary nucleotide sequence here generally refers to a sequence of nucleotides in a single-stranded molecule or segment of DNA or RNA that is sufficiently complementary to that on another single oligonucleotide strand to specifically hybridize to it with consequent hydrogen bonding.
  • single nucleotide polymorphisms are the target for detection, then the complementarity between the analyte and analyte -binding arm on the binary probes should be exact, 100%. If less selectivity is required, then routine experimentation will determine the level of complementarity that provides the desired result.
  • Nucleotide generally refers to a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate group, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (I' carbon of the pentose) and that combination of base and sugar is a "nucleoside".
  • nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose, it is referred to as a nucleotide.
  • a sequence of nucleotides is typically referred to herein as a "base sequence” or “nucleotide sequence”, and their grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5 '-terminus to 3 '-terminus, unless otherwise specified.
  • Nucleotide analog generally refers to a purine or pyrimidine nucleotide that differs structurally from A, T, G, C, or U, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
  • nucleotide analog encompasses altered bases, different or unusual sugars (i.e. sugars other than the "usual" pentose), or a combination of the two. Nucleotide analogs of DNA or RNA can be used to make binary probes.
  • nucleotide analogs useful according to the present invention include those listed in the approved listing of modified bases at 37 CFR .sctn.1.822 (which is incorporated herein by reference). Other useful analogs include those described in published international application no. WO 92/20823 (the disclosures of which are incorporated herein by reference), or analogs made according to the methods disclosed therein.
  • Oligonucleotide or polynucleotide generally refers to a polymer of single-stranded nucleotides.
  • oligonucleotide and its grammatical equivalents will include the full range of nucleic acids.
  • An oligonucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of deoxy- and ribonucleotides.
  • Binary probe means two molecules or one molecule containing two distinct parts that recognizes a specific sequence in a DNA or RNA analyte, and which, only upon recognition and binding to the analyte, generates a detectable signal such as fluorescence, luminescescence or a visible color change such as in the case of the present binary oligonucleotide peroxidase probe
  • Hemin (trade name Panhematin) is an iron-containing porphyrin. ⁇ that is used in the management of porphyria attacks, particularly in acute intermittent porphyria. It is sometimes distinguished from "Hematin”, which is hemoglobin with iron in ferric state. However, the terms are sometimes equated P ⁇
  • Aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule.
  • Peroxidase-like activity means that the molecule (DNA enzyme) catalysis hydrogen peroxide decomposition, similar to that of horseradish peroxidase.
  • Peroxidases are a large family of enzymes that typically catalyze a reaction of the form: ROOR' + electron donor (2 e " ) + 2H +
  • the optimal substrate is hydrogen peroxide. Colored reaction products allow detection of the enzyme with high sensitivity.
  • the binary probe of the present invention may combine one or more modifications or mutations including additions, deletions, and substitutions. These mutations may, for example, change the length of, or alter the nucleotide sequence of, a loop, a spacer region or a recognition sequence (or domain). Modification or mutation of the recognition site via well-known methods allows one to alter the sequence specificity of an enzymatic nucleic acid molecule.
  • physiologic conditions is meant to suggest reaction conditions emulating those found in mammalian organisms, particularly humans. While variables such as temperature, availability of cations, and pH ranges may vary as described in greater detail below, "physiologic conditions” generally comprise a temperature of about 35 40 0 C, with 37°C being particularly preferred, as well as a pH of about 7.0 8.0, with 7.5 being particularly preferred, and further comprise the availability of cations, preferably divalent and/or monovalent cations, with a concentration of about 2 15 mM Mg2+ and 0 1.0 M Na+ being particularly preferred .
  • BOPP binary oligonucleotide
  • the BOPP acquires peroxidase-like activity after it hybridizes to a specific target DNA or RNA analyte, which causes the BOPP to form a guanine-quadruplex that binds hemin.
  • Hemin bound to the guanine quadruplex (G-quadruplex) demonstrates hydrogen peroxidase-like activity that is -250 times greater than unbound hemin.
  • the BOPP is a non-naturally occurring binary oligonucleotide peroxidase probe for detecting a single stranded oligonucleotide analyte comprising two antiparallel oligonucleotide strands, wherein
  • a first oligonucleotide strand comprises: a. at its 5 '-terminus an oligonucleotide hemin-binding arm (that is optionally flanked by a linker) b. an optional linker that is flanked by a first oligonucleotide analyte binding arm, and c. at its 3 '-terminus, the first oligonucleotide analyte binding arm that is complementary to and selectively hybridizes with a first region of the oligonucleotide analyte, and
  • a second oligonucleotide strand comprises: a. at its 3 '-terminus an oligonucleotide hemin-binding arm that is flanked by an optional linker, b. an optional linker that is flanked by a first oligonucleotide analyte binding arm, and c. at its 5 '-terminus, a second oligonucleotide analyte binding arm that is complementary to and selectively hybridizes with a second region of the oligonucleotide analyte.
  • the hemin- and analyte-binding arms of the oligonucleotide strands of the probe so that a linker between the two is not needed in order for the probes to form a four- way junction with target analyte.
  • the BOPP was based on a peroxidase-like hemin binding DNA aptamer shown in FIG. IA that was obtained earlier by in vitro selection. 3 When the aptamer binds to hemin, it forms a guanine quartet (herein a guanine- quadruplex or G-quadruplex) that demonstrates hydrogen peroxidase-like activity that is -250 times greater than hemin alone.
  • Hemin is a porphyrin, a small molecule, that binds to G-quadruplex of the BOPP noncovalently, by stacking and hydrophobic interactions.
  • the covalent binding of one or both of the antiparallel oligonucleotide strands of the probe to hemin will increase the sensitivity of the assay.
  • the mechanism by which the association of hemin with the G- quadruplex increases the peroxidase activity of the hemin is unknown. Without being bound by theory, the hydrophobic environment of G-quadruplex may increase the affinity of hemin for hydrogen peroxide.
  • the hemin arms can be configured differently as follows: the oligonucleotide hemin-binding arm on the first oligonucleotide strand can comprise 3 ' gggatggg 5 (SEQ ID NO: 4), and the oligonucleotide hemin-binding arm on the second oligonucleotide strand can comprises 5' gggttggg 3'; or the oligonucleotide hemin-binding arm on the first oligonucleotide strand can comprise 3' ggg 5, and the oligonucleotide hemin-binding arm on the second oligonucleotide strand can include 5' gggcgggttggg 3'; or the oligonucleotide hemin-binding arm on the first oligonucleotide strand can comprise 3' gggcgggatggg 5, and the oligonucleotide
  • Analyte binding arms were added to each of the two antiparallel oligonucleotide strands (to the 3' end of one strand and to the 5' end of the other strand) via a flexible linker such as triethylenglycol (FIG. 1).
  • the analyte can be DNA or RNA with the analyte binding arms being designed accordingly.
  • the two oligonucleotide strands of the BOPP exist predominantly in the dissociated form (at certain concentrations and buffer conditions).
  • the free end of the strand (made from the DNA hemin-binding aptamer) forms a guanine quadruplex (hereafter "G-quadruplex") structure that acquires peroxidase activity when bound to hemin (FIG. 1C).
  • G-quadruplex guanine quadruplex
  • the BOPP bound to hemin is an active peroxidase that catalyzes the oxidation of a colorless substrate to a colored product that can be detected both visually and spectrophotometrically.
  • FIG. 2 demonstrates the change of light absorption of the solution containing the BOPP when 3-3'-diaminobenzidine tetrahydrochloride (DAB) was used as an oxidizable substrate.
  • DAB 3-3'-diaminobenzidine tetrahydrochloride
  • the solution turned brown in the presence of 1 ⁇ M rs242557-G, but not in the presence of rs242557-A SEQ ID NO: 1 that contained just a single nucleotide mismatch (compare 4 with 3 in FIG. 2A).
  • the light absorption in the presence of the mismatched target was as low as in the absence of any analyte (compare 3 with 2 in panels B and C).
  • high absorption was observed in the presence of complementary target (sample 4, B and C).
  • the signal-to-background ratio (S/B) was ⁇ 10 after 30 min of incubation. This signal was only -38% lower than that of the solution containing the parent peroxida
  • DAB can be substituted with another peroxidase substrate, ABTS (2, 2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid)) (FIG. 4).
  • ABTS 2, 2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid)
  • FIG. 4 the S/B ratio was reduced to ⁇ 6 in this case, making the color difference less contrasting.
  • Substitution of the triethylene glycol linkers in the structure of strands ⁇ and ⁇ with dithymidine linkers substantially reduced the intensity of the positive signal in the presence of the specific complementary target (FIG. 4).
  • one of the strands contained dithymidine, while another triethylene glycol linker, the staining was intensive enough to be visualized.
  • At least one strand of the probe can be composed of purely natural deoxyribonucleotides.
  • Those skilled in the art will be able to make variations of the probe in the linker and analyte binding arms, as described below. For example, more oligonucleotides can be added to the analyte binding arms to form stabilizing stem loop structures.
  • the basic binary probe of the present invention is made of two synthetic, non- naturally occurring, anti -parallel oligonucleotide strands that can be made of DNA or RNA or a combination of both.
  • Each strand of the DNA or RNA probe has a customized fragment that is complementary to a selected target nucleic acid analyte (analyte -binding arm), and a customized fragment complementary to hemin (hemin-binding arms).
  • the analyte- and hemin-binding arms are optionally connected to each other by linker molecules.
  • SSA structure stabilization arms
  • the newly discovered binary probes have two separate, antiparallel DNA or RNA strands.
  • the probe has several distinct regions on each strand: an analyte - binding arm flanked by an optional flexible linker that is flanked by a hemin-binding arm that binds to hemin conferring on the probe peroxidase-like activity that can be visualized by adding the appropriate substrate to generate a colored product indicating that the analyte has been detected.
  • a separate linker may not be needed if there is enough flexibility in the phosphodiester bonds of the two oligonucleotide strands to permit the probe to form a four- way junction with the analyte.
  • probes are called “binary" because the two parts of the probe act synergistically and the detection event occurs only when both are hybridized to the analyte. In the absence of a nucleic acid analyte, the strands are dissociated and the probe does not bind the hemin. Addition of a specific DNA/RNA analyte, some or all of which is complementary to the respective analyte-binding arms on the two halves of the probe, results in hybridization of the analyte - binding arms to the corresponding complementary nucleotides on the analyte. The analyte can and often is longer than the combined length of the two analyte-binding arms on the binary probe.
  • the BOPP is made of a. the first oligonucleotide strand that has at its 5 '-terminus a hemin-binding arm that is flanked by a flexible linker, b. a flexible linker that is flanked by a first oligonucleotide analyte-binding arm and c. a first oligonucleotide analyte-binding arm that is complementary to and selectively hybridizes with a first region of an oligonucleotide analyte.
  • the second strand is antiparallel to the first.
  • the linker may be omitted if there is enough flexibility to permit the probe to form a four- way junction with the analyte.
  • the oligonucleotide hemin-binding arm on the first oligonucleotide strand includes the sequence 3 ' gggatggg 5 SEQ ID NO: 3, and the oligonucleotide hemin-binding arm on the second oligonucleotide strand includes 5' gggttggg 3' SEQ ID NO: 4.
  • the oligonucleotide hemin-binding arm on the first oligonucleotide strand comprises 3 ' ggg 5 SEQ ID NO: 5; and the oligonucleotide hemin-binding arm on the second oligonucleotide strand comprises 5' gggcgggttggg 3' SEQ ID NO: 6, or the oligonucleotide hemin-binding arm on the first oligonucleotide strand comprises 3' gggcgggatggg 5 SEQ ID NO: 7; and the oligonucleotide hemin-binding arm on the second oligonucleotide strand comprises 5 ' ggg 3 ' SEQ ID NO: 5.
  • the analyte -binding arms are customized for each particular analyte.
  • the probe is entirely DNA, but it can be made of RNA or be a chimera.
  • the analyte can be DNA, RNA or a chimera.
  • the hemin binding arms are customized to bind to hemin.
  • the analyte -binding arm of each strand of the probe ranges from 6-20 nucleotides in length, preferably 10, which make total recognizable analyte fragment 12-40 nucleotides long.
  • Analyte-binding arms of about 10 nucleotides are preferred because a combined length of 20 nucleotides will cover any unique sequence in the genome. It is important to note, that the analyte itself can be of any length from 12-40, to many thousand nucleotides.
  • the analyte-binding- and hemin-binding arms are separated by flexible linkers that permit the formation of two full-fledged double helixes when the analyte and hemin are bound to the probe.
  • the flexible linker is Methylene glycole.
  • Flexible linkers are also used if the binary probe is made of RNA since RNA will also form a double helix. Nucleotide linkers can also be used if they permit the formation of a double helix.
  • the binary probes of the present invention are substantially destabilized by a single mismatched base pair, thereby preventing binding to the hemin.
  • the binary probes thus provide an extraordinary level of selectivity.
  • the new probes and analytic methods using them have the following major advantages:
  • DNA probes have an advantage over RNA probes when the analyte is DNA because DNA-DNA duplexes are typically less stable than RNA-DNA duplexes and are therefore more sensitive to SNPs. DNA probes are cheaper also to synthesize and they are more stable to degradation in solution. In those embodiments where the probes are made of RNA oligoribonucleotides, U is substituted for T; otherwise the structures are the same.
  • the analyte binding arms can be DNA, RNA or chimeras; however, the hemin-binding arms are only made of DNA.
  • a stem loop structure forms by adding a nucleotide fragment of from about 3-10 nucleotides in length or up to 40 nucleotides in length (called a structure stabilization arm or SSA) to the free end of the analyte -binding arm on each strand of the probe.
  • the added sequences in the SSA are complementary to all or part of the analyte-binding arm.
  • stem-loop When the complementary sequences in the SSA hybridize to the corresponding sequences in the analyte-binding arm, a stem-loop is formed.
  • the formation of stem-loops represents a conformational constraint that further increases the sensitivity of the binary DNA or RNA probes.
  • SSA can also be added to the free end of the hemin-binding arm if they do not interfere with g-complex formation.
  • each dissociated strand of the probe is stabilized by complementary base pairing to itself via the stem loop in the analyte-binding arms.
  • Additional changes that may increase the selectivity of the probe include shortening the analyte, for example from 20 to 12 nucleotides, or increasing the reaction temperature to 37°C, which is still within physiologic conditions that can eventually permit analyte analysis in live cells in culture or in vitro. Since the oligonucleotide strands of the binary probe are simple nucleotide sequences they can be made to order by various existing companies such as Integrated DNA Technologies (Coralville, IA, USA).
  • Certain embodiments are also directed to a binary oligonucleotide probe hybridization assay to detect RNA or DNA analyte in a sample containing a heterogeneous mixture of nucleic acids including at least one single stranded analyte molecule that has a known nucleotide sequence.
  • the assay has the following steps: a) providing a binary oligonucleotide peroxidase probe BOPP described above, wherein the nucleotides in the analyte -binding arms are complementary to the known nucleotide sequence in the analyte, b) adding hemin and a biological sample having the DNA or RNA analyte to the probe to form a mixture, c) maintaining said mixture for a sufficient period of time and under predetermined reaction conditions to allow the analyte to hybridize to the analyte -binding arms on the probe, and for the hemin to bind to the probe, d) adding a peroxidase substrate to the mixture of step c), and e) determining that the analyte is present in the sample if a visible peroxidase substrate reaction product is observed.
  • Certain embodiments of the invention are directed to truncated forms of the binary probe ("the truncated probe").
  • each truncated BOPP probe strand has only the hemin-binding arms and flexible linkers, with optional structure stabilization arms internally complementary to a portion of the respective heme -binding arm. The user can customize the analyte-binding arms to suit the target.
  • Certain embodiments are directed to a diagnostic binary oligonucleotide peroxidase probe hybridization assay kit to detect a known RNA or DNA analyte in a sample containing a heterogeneous mixture of nucleic acids, the kit comprising 1.
  • the kit can further comprise a peroxidase substrate like 3-3'-diaminobenzidine tetrahydrochloride (DAB), 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid) (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), luminol (for chemiluminescence detection) and other standard horseradish peroxidase substrates [0046]
  • DAB 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid)
  • TMB 3,3',5,5'-tetramethylbenzidine
  • luminol for chemiluminescence detection
  • the new binary probe-based technology requires synthesis of only two short DNA, RNA, or chimeric oligonucleotides with specific analyte-binding arms for each different probe.
  • the hemin-binding arms and the linker can be standard. Standard desalting provides sufficient purity for the oligonucleotides of such lengths. All other components of the probe, such as the hemin-binding arms are universal for all assays. If applied for analysis of many different single nucleotide polymorphisms (SNPs), out of several million existing in human genome, the new approach will offer increased accuracy and the ability to work at moderate physiologic conditions. Since DNA-RNA and DNA-DNA hybrids have different structural parameters, the binary constructions should be customized for RNA in order to obtain highly specific and sensitive recognition of RNA targets.
  • SNPs single nucleotide polymorphisms
  • Diagnostic kits can be prepared having the full BOPP with analyte-binding arms (and optional structure stabilization arms) that are already customized to be complementary to and hybridize with high specificity to a known oligonucleotide analyte.
  • the analyte binding arms recognize the target SNP rs242557-that indicates increased risk of developing Alzheimer's disease.
  • Oligonucleotides were custom-made by Integrated DNA Technologies, Inc. (Coralville, IA). Hydrogen peroxide, 3-3'- diaminobenzidine tetrahydrochloride (DAB), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hemin, and HEPES were from Sigma-Aldrich (St. Louis, MO, USA). Electronic spectra were taken on a Spectrophotometer Ultraspec 3300 (Amersham-Biotech, NJ, USA). The data were processed using Microsoft Excel.
  • B. SNP typing assay Binary peroxidase-like DNA enzyme (1 ⁇ M both strands) was incubated in the reaction buffer (50 mM HEPES, pH 7.4, 50 mM MgCl 2 , 20 mM KCl, 120 mM NaCl, 0.03% Triton X-100, 1% DMSO, hemin (125 nM), H 2 O 2 (1 mM), and DAB or ABTS (1 mM)) in the absence or presence of rs242557-A or rs242557-G.
  • the reaction buffer 50 mM HEPES, pH 7.4, 50 mM MgCl 2 , 20 mM KCl, 120 mM NaCl, 0.03% Triton X-100, 1% DMSO, hemin (125 nM), H 2 O 2 (1 mM), and DAB or ABTS (1 mM)
  • Negative control (sample 0) contained no oligonucleotides; Positive control (sample 1) contained 1 ⁇ M proxidase-like DNA enzyme (FIG. IA).
  • the electronic absorption spectra of the samples were recorded after 30 min of incubation at room temperature. The test tubes were photographed using an Olympus FE- 170 digital camera 6 mega pixel.
  • C Native PAGE.
  • the reaction mixtures were analyzed in 12% native PAGE containing the reaction buffer.
  • Each reaction mixture was mixed 1 :10 with the loading buffer (50% Glycerol, 50 mM HEPES, pH 7.4, 50 mM MgCl 2 , 20 mM KCl, 120 mM NaCl, 0.03% Triton X-100, 1% DMSO, 0.01% bromphenol, 0.01% xylencyanol).
  • Two microliters of each sample were loaded on the gel and run 1 h at room temperature (200 V) followed by staining with SYBR Gold (Invitrogen, OR) and photographed using Alphaimager 3400 (Alpha Innotech, CA).
  • ABTS (2, 2 -azino- bis(3-ethylbenzthiazoline-6-sulphonic acid) is a commonly used water soluble substrate of hydrogen peroxidases. Therefore, DAB can be substituted with ABTS in the colorimetric assay for SNP.
  • Triethylene glycol linkers in the sequence of the binary DNA peroxidase probe can be replaced with dithymidine linkers (FIG. 4 A).
  • the intensity of the optical signal was reduced about 6 times (from 0.74 ⁇ 0.06 to 0.12 ⁇ 0.01 OU, compare dark grey bar with white bar in third group, panels B).
  • Such a weak color was hardly recognizable by the naked eye (data not shown).
  • the probes composed of ⁇ tt and ⁇ tt ⁇ generated color intensive enough to be visualized.
  • the highest S/B ratios were found for ⁇ tt ⁇ tt and ⁇ tt combinations, -46 and -30, respectively, due to a very low background reaction.

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Abstract

La présente invention concerne une sonde qui modifie la couleur d'une solution en présence d'une seule de deux séquences d'ADN ne différant que d'un seul nucléotide. La sonde est constituée de deux oligodésoxyribonucléotides, formant une enzyme à ADN de type hydrogène peroxydase lorsqu'elle est hybridée aux fragments adjacents de l'analyte complémentaire. La peroxydase active catalyse l'oxydation de substrats incolores pour former des produits colorés. La sonde permet de détecter visuellement une mutation dans l'ADN lié à la maladie d'Alzheimer.
PCT/US2009/065341 2008-11-21 2009-11-20 Enzyme à adn divisé pour le typage visuel du polymorphisme d'un nucléotide unique WO2010059944A1 (fr)

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US13/130,536 US8551768B2 (en) 2008-11-21 2009-11-20 Split DNA enzyme for visual single nucleotide polymorphism typing
BRPI0920985-9A BRPI0920985A2 (pt) 2008-11-21 2009-11-20 Enzima de dna dividida para tipificação visual de polimorfismo de nucleotídeo único
EP09828292A EP2367838A4 (fr) 2008-11-21 2009-11-20 Enzyme à adn divisé pour le typage visuel du polymorphisme d'un nucléotide unique

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8354227B2 (en) 2006-02-03 2013-01-15 Columbia University In The City Of New York Binary deoxyribozyme probes for nucleic acid analysis
CN103014148A (zh) * 2012-10-29 2013-04-03 中国科学院成都生物研究所 一种rna的等温检测方法
US8551768B2 (en) 2008-11-21 2013-10-08 The Trustees Of Columbia University In The City Of New York Split DNA enzyme for visual single nucleotide polymorphism typing
WO2014113585A1 (fr) * 2013-01-16 2014-07-24 Massachusetts Institute Of Technology Peroxydases de dissociation moléculaire et leurs méthodes d'utilisation
US8853134B2 (en) 2009-01-28 2014-10-07 The Trustees Of Columbia University In The City Of New York Microarrays of binary nucleic acid probes for detecting nucleic acid analytes
CN104297306A (zh) * 2014-09-30 2015-01-21 江南大学 基于G-四面体/hemin的光电化学多功能传感器
WO2019012481A3 (fr) * 2017-07-12 2019-02-21 John Katz G-quadruplexes divisés pour la capture et la détection d'acides nucléiques
US20230193359A1 (en) * 2017-07-27 2023-06-22 John Fred KATZ Split G-Quadruplexes for Capture and Detection of Nucleic Acids

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US8551768B2 (en) 2008-11-21 2013-10-08 The Trustees Of Columbia University In The City Of New York Split DNA enzyme for visual single nucleotide polymorphism typing
US8853134B2 (en) 2009-01-28 2014-10-07 The Trustees Of Columbia University In The City Of New York Microarrays of binary nucleic acid probes for detecting nucleic acid analytes
CN103014148A (zh) * 2012-10-29 2013-04-03 中国科学院成都生物研究所 一种rna的等温检测方法
CN103014148B (zh) * 2012-10-29 2014-03-12 中国科学院成都生物研究所 一种rna的等温检测方法
WO2014113585A1 (fr) * 2013-01-16 2014-07-24 Massachusetts Institute Of Technology Peroxydases de dissociation moléculaire et leurs méthodes d'utilisation
US9012170B2 (en) 2013-01-16 2015-04-21 Massachusetts Institute Of Technology Split peroxidases and methods of use
US9969987B2 (en) 2013-01-16 2018-05-15 Massachusetts Institute Of Technology Split peroxidases and methods of use
CN104297306A (zh) * 2014-09-30 2015-01-21 江南大学 基于G-四面体/hemin的光电化学多功能传感器
WO2019012481A3 (fr) * 2017-07-12 2019-02-21 John Katz G-quadruplexes divisés pour la capture et la détection d'acides nucléiques
US20230193359A1 (en) * 2017-07-27 2023-06-22 John Fred KATZ Split G-Quadruplexes for Capture and Detection of Nucleic Acids

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BRPI0920985A2 (pt) 2015-08-18
US8551768B2 (en) 2013-10-08
EP2367838A4 (fr) 2012-10-03
US20120135539A1 (en) 2012-05-31

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