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WO2012053018A1 - Méthode de détection des produits issus de réactions biologiques - Google Patents

Méthode de détection des produits issus de réactions biologiques Download PDF

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Publication number
WO2012053018A1
WO2012053018A1 PCT/IT2010/000424 IT2010000424W WO2012053018A1 WO 2012053018 A1 WO2012053018 A1 WO 2012053018A1 IT 2010000424 W IT2010000424 W IT 2010000424W WO 2012053018 A1 WO2012053018 A1 WO 2012053018A1
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WO
WIPO (PCT)
Prior art keywords
avidin
hybridization
streptavidin
biotin
derivatives
Prior art date
Application number
PCT/IT2010/000424
Other languages
English (en)
Inventor
Rubina Vecchiato
Original Assignee
Fiore, Marco
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fiore, Marco filed Critical Fiore, Marco
Priority to PCT/IT2010/000424 priority Critical patent/WO2012053018A1/fr
Publication of WO2012053018A1 publication Critical patent/WO2012053018A1/fr

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Classifications

    • 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

  • This invention relates generally to methods for amplifying detectable signals in hybridization assays, particularly nucleic acid hybridization assays.
  • Biomolecules hybridizations are commonly used in biochemical research and diagnostic assays. As an example, a single stranded analyte nucleic acid is hybridized to labeled nucleic acid probe, and resulting nucleic acid duplexes are detected. Hybridization techniques are also commonly used for the detection of proteins. Radioactive and nonradioactive labels are used. Commonly used nonradioactive labeling agent are fluorophores , i.e. functional group which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. Broadly used fluorophores are xanthenes derivatives such as fluorescein or rhodamin and cyanine derivatives, such as Cy3, Cy5.
  • Another commonly used detection method is based on the use of enzymes such as horseradish peroxidase, able to catalyze the conversion of a chemiluminescent substrate into a sensitized reagent in the vicinity of the molecule of interest which, on further oxidation by hydrogen peroxide, produces a triplet (excited) carbonyl which emits light when it decays to the singlet carbonyl.
  • enzymes such as horseradish peroxidase
  • linker are used.
  • said linkers have to be compatible with aqueous solutions, should be able to immobilize functional groups, oligonucleotides, PCR products, peptides, proteins etc., need to be compatible with fluorescent dyes and with enzymes.
  • linker is the avidin-biotin complex or, preferably, streptavidin-biotin .
  • Biotin is a 244 Da water-soluble B-complex vitamin that is composed of an ureido (tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring.
  • a valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring.
  • the valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules. Using these reactive groups, biotin can be easily attached to most proteins and other molecules. Since biotin is a relatively small molecule, it can be conjugated to many proteins without significantly altering their biological activity.
  • Avidin is a 68,000 kDa tetrameric protein (Green
  • the tetrameric protein contains four identical subunits (homotetramer) , each of which can bind to biotin with a high degree of affinity and specificity.
  • the dissociation constant of avidin is measured to be KD*10-15 M, making it one of the strongest known non-covalent bonds.
  • Avidin is produced in the oviducts of birds, reptiles and amphibians and deposited in the whites of their eggs. The natural function of avidin in eggs is not known, although it has been postulated to be made in the oviduct as a bacterial growth-inhibitor, by binding biotin the bacteria need.
  • avidin As a basically charged glycoprotein, avidin exhibits non-specific binding in some applications.
  • Streptavidin is a 52,800 kDa tetrameric protein purified from the bacterium Streptomyces avidinii, where it is thought to serve to inhibit the growth of competing bacteria, in the manner of an antibiotic. It is loosely related to avidin and has an equal biotin affinity and a very similar binding site. Streptavidin has a near-neutral isoelectric point and, for this reason, it exhibits less nonspecific binding than avidin and it is more widely used than avidin in laboratory applications. Dissociation of biotin from streptavidin is reported to be about 30 times faster than dissociation of biotin from avidin.
  • NeutrAvidin an avidin that has been processed to remove the carbohydrate and lower its isoelectric point to reduce background staining.
  • Avidin, streptavidin and NeutrAvidin biotin- binding proteins each bind four biotins per molecule with high affinity and selectivity (Gonzalez M et al. 1997) .
  • a biomolecule can be reacted with several molecules of biotin that, in turn, can each bind a molecule of avidin. This greatly increases the sensitivity of many assay procedures.
  • the biotinylation process comprises the chemical link of biotin to the biomolecule of interest.
  • a biotinylated probe is applied to a sample and then the bound probe is detected with a labeled avidin or streptavidin.
  • avidin or streptavidin are commonly used to localize antigens in cells and tissues and to detect biomolecules in immunoassays and DNA hybridization procedures.
  • immobilized avidins are used to capture and release biotinylated targets.
  • biotinylated nucleic acids are suitable for binding to streptavidin coated surfaces. With the nucleic acid firmly attached to this substrate, various DNA hybridization and immunological assays can be performed. Biotynilated nucleic acids may also be attached to streptavidin coated agarose microspheres, polystyrene or even paramagnetic beads. These complexes are most commonly used for the purification or isolation of nucleic acid binding proteins. Biotinylated biomolecules are suitable to be linked via streptavidin to labeling agents for further detection.
  • biotinylation a biotin tag is covalently bounded to a molecule or a surface, this normally occurs via primary ammine biotinylation, sulfhydryl biotinylation, carboxyl biotinylation, glycoprotein biotinylation. Photo-reactive biotin compounds that react nonspecifically upon photoactivation are also available ;
  • denaturation this step being particularly needed when dealing with nucleic acid: during denaturation, double-stranded nucleic acid unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases.
  • This process normally is carried out using high temperature, but chemical processes are suitable, too;
  • the developed signal is detected via a suitable apparatus.
  • kits are available for hybridization, and they all follow the above indicated scheme.
  • the incubation with avidin conjugate is suggested to be carried on in blocking solution, to prevent aspecific signal.
  • the here described invention relates generally to methods for amplifying detectable signals in hybridization assays, particularly nucleic acid hybridization assays.
  • the method comprises the following steps:
  • biotinylation a biotin tag is covalently bounded to a molecule or a surface using techniques available in the state of the art;
  • biotinylated biomolecules are hybridized with specific probes in the presence of the avidin-conjugate;
  • the developed signal is detected via a suitable apparatus .
  • the method here claimed is characterized in that the conjugate is added during the hybridization procedure, without the need for a dedicated step.
  • the avidin, or streptavidin or NeutrAvidin or any other avidin derivative when added during the hybridization step, not only is able to bind to the target biotinylated biomolecules, but the formed non-covalent binding is not affected by the following stringent wash. In this manner, the labeled biomolecules can be detected. Moreover, when adding the conjugate during the hybridization step, no undesired not specific conjugate- biomolecules binding has been observed during the detection procedure. No specific blocking solution is needed, but an efficient ( strept ) avidin-biotin binding is obtained by adding ( strept ) avidin in the buffer used for the hybridization step. Contrary to the literature teaching, no aspecific signal is detected when adding the conjugate during the hybridization step and not in a subsequent, dedicated step.
  • the claimed method is suitable when using ( strept ) avidin conjugated to fluorophores or with enzymes as well.
  • the ( strept ) avidin conjugated is added before the hybridization step, during the denaturation process. It has been surprisingly found that the ( strept ) avidin conjugate was able to specifically bind to the biotinylated molecule and the binding was not disturbed by the denaturation conditions, high temperature and/or chemical denaturation as well. This embodiment is preferably suggested for ( strept ) avidine conjugated to fluorophores than for ( strept ) avidine conjugated to enzymes.
  • the here claimed method is indicated when performing assays on a solid substrate, as an example on microbeads, on nylon strip, on chip, and in liquid assays as well.
  • the here claimed method is suitable for the identification of specific DNA and RNA molecules, e.g. viral DNA or tRNA, with complementary biotinylated nucleic acid probes.
  • the here claimed method is suitable also for the isolation of specific DNA and RNA molecules with complementary biotinylated nucleic acid probes, for the isolation of DNA-RNA- binding proteins with biotinylated nucleic acid probes.
  • the method offers also great advantages when performing protein interaction studies, for example in the isolation of receptors by using biotinylated ligands and in the isolation of complexes, organelles, or viruses.
  • RNA fragmentation (RNA targets only) , 40 min;
  • DNA denaturation 5 min 95 °C, or 5 min at room temperature, in the presence of a denaturing solution; iii) hybridization: 15-90 min; the hybridization temperature and the buffer are selected according to the literature (Sambrook J and Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Press. 2001) ;
  • RNA fragmentation (RNA targets only) , 40 min;
  • DNA denaturation 5 min 95°C, or 5 min at room temperature, in the presence of a denaturing solution; iii) hybridization and labeling: 15-90 min; the hybridization temperature was about 55 °C, at pH 7.5, in the presence of streptavidin-enzyme conjugated;
  • RNA fragmentation (RNA targets only) , 40 min; DNA denaturation, 5 min 95 °C, or 5 min at room temperature, in the presence of a denaturing solution; iii) hybridization: 15-90 min; the hybridization temperature and the buffer are selected according to the literature (Sambrook J and Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Press. 2001) ;
  • RNA fragmentation (RNA targets only) , 40 min in the presence of streptavidin-fluorophore conjugated; DNA denaturation, 5 min 95 °C, or 5 min at room temperature, in the presence of a denaturing solution containing the streptavidin-fluorophore conjugated;
  • hybridization temperature was about 55 °C, at pH 7.5;
  • the obtained signal is clear, without background, and of a comparable intensity when using both the indicated method.
  • the here claimed method offers sensible advantage in term of time of execution, amount of buffer needed and, more important, it offers the great advantage of decreasing the manipulation steps by the operator, thus preventing potential contaminations and/or errors.
  • streptavidin is supplied in the denaturing buffer, in a ready to use format, eliminating manipulation from the operator .

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

Abstract

La méthode polyvalente ci-revendiquée permet d'amplifier des signaux détectables dans des dosages d'hybridation, en particulier, des dosages d'hybridation d'acides nucléiques, basés sur la technologie de la (strept)avidine-biotine.
PCT/IT2010/000424 2010-10-20 2010-10-20 Méthode de détection des produits issus de réactions biologiques WO2012053018A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IT2010/000424 WO2012053018A1 (fr) 2010-10-20 2010-10-20 Méthode de détection des produits issus de réactions biologiques

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PCT/IT2010/000424 WO2012053018A1 (fr) 2010-10-20 2010-10-20 Méthode de détection des produits issus de réactions biologiques

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WO2012053018A1 true WO2012053018A1 (fr) 2012-04-26

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175706A1 (en) * 1994-06-22 2003-09-18 Zhang David Y. Nucleic acid amplification methods
WO2007095464A2 (fr) * 2006-02-15 2007-08-23 Indevr, Inc. Amplification de signal d'événements de bioreconnaissance par photopolymérisation en présence d'air

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175706A1 (en) * 1994-06-22 2003-09-18 Zhang David Y. Nucleic acid amplification methods
WO2007095464A2 (fr) * 2006-02-15 2007-08-23 Indevr, Inc. Amplification de signal d'événements de bioreconnaissance par photopolymérisation en présence d'air

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AHMET VAR ET AL: "Impact of hemostatic gene single point mutations in patients with non-diabetic coronary artery disease", MOLECULAR BIOLOGY REPORTS ; AN INTERNATIONAL JOURNAL ON MOLECULAR AND CELLULAR BIOLOGY, KLUWER ACADEMIC PUBLISHERS, DO, vol. 36, no. 8, 3 January 2009 (2009-01-03), pages 2235 - 2243, XP019752177, ISSN: 1573-4978, DOI: DOI:10.1007/S11033-008-9439-5 *
RUSSELL DW: "Molecular cloning: a laboratory manual", 2001, COLD SPRING HARBOR PRESS
SAMBROOK J; RUSSELL DW.: "Molecular cloning: a laboratory manual", 2001, COLD SPRING HARBOR PRESS
SAMBROOK J; RUSSELL DW: "Molecular cloning: a laboratory manual", 2001, COLD SPRING HARBOR PRESS
WANG BAOSHENG ET AL: "Polyploid evolution in Oryza officinalis complex of the genus Oryza", BMC EVOLUTIONARY BIOLOGY, BIOMED CENTRAL LTD., LONDON, GB, vol. 9, no. 1, 14 October 2009 (2009-10-14), pages 250, XP021062063, ISSN: 1471-2148, DOI: DOI:10.1186/1471-2148-9-250 *

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