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WO2011000977A1 - Procédé miniaturisé appliqué dans des plaques multipuits pour analyser la liaison de ligands à des acides nucléiques à travers l'obtention de courbes de fusion thermique - Google Patents

Procédé miniaturisé appliqué dans des plaques multipuits pour analyser la liaison de ligands à des acides nucléiques à travers l'obtention de courbes de fusion thermique Download PDF

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Publication number
WO2011000977A1
WO2011000977A1 PCT/ES2010/000264 ES2010000264W WO2011000977A1 WO 2011000977 A1 WO2011000977 A1 WO 2011000977A1 ES 2010000264 W ES2010000264 W ES 2010000264W WO 2011000977 A1 WO2011000977 A1 WO 2011000977A1
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WIPO (PCT)
Prior art keywords
nucleic acid
ligand
derivative
temperature
well
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Ceased
Application number
PCT/ES2010/000264
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English (en)
Spanish (es)
Inventor
Alberto DOMINGO GALÁN
Verónica GARCÍA HERNÁNDEZ
Juan José VAQUERO LÓPEZ
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Universidad de Alcala de Henares UAH
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Universidad de Alcala de Henares UAH
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Publication of WO2011000977A1 publication Critical patent/WO2011000977A1/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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

  • the procedure described in this patent has applications in the analysis and characterization of the interaction with DNA of compounds capable of binding to this macromolecule. It is also applicable to other nucleic acids such as RNA, DNA-RNA hybrids, derivatives or analogs of any of these.
  • the miniaturized procedure described allows to study the binding of a specific ligand to oligonucleotides. of different sequences or a collection of ligands, structurally related or not, with oligonucleotides of the same or different sequence.
  • the comparative study of the binding of a particular ligand to different sequences can be applied to the determination of the specificity or sequence preference of said binding.
  • the same type of study also allows to determine differences in local stability of the complexes formed.
  • Covalent oligonucleotide ligands whether this interaction is an end in itself or if it is a means to study other parameters.
  • An example of the latter could be a study of stability or maintenance of the functional capacity of a certain compound with pharmacological application that acts through DNA binding.
  • This method has an applicability to a wide variety of compounds and modes of interaction with nucleic acids, so it can be used to search for new compounds capable of binding to DNA or other nucleic acids. This is of special interest in the search for drugs that have these molecules as targets, or ligands for nucleic acid mapping and localization. STATE OF THE TECHNIQUE
  • calorimetric techniques electrophoretics (gel electrophoresis, capillary), footprinting, spectrophotometric techniques (such as ultraviolet-yisible spectrophotometry, circular dichroism or fluorimetry), crystallography (or diffraction X-ray) and nuclear magnetic resonance (or NMR).
  • the invention consists of a miniaturized process for determining the binding of compounds to nucleic acids in multiwell plates, based on the analysis of thermal fusion curves of the complexes.
  • the performance of the multi-well plate test allows parallel execution for a large number of reaction mixtures, while facilitating the manipulation and automation of the entire process, from liquid handling to data processing.
  • the key to achieving the miniaturization of the assay is to use very sensitive and rapid methods for determining the proportion of associated and dissociated complexes. This is achieved thanks to various mapping strategies with fluorescent groups or molecules.
  • the process can also be carried out using different forms or combinations of fluorescent marking and for the binding of ligands or reagents to different types of nucleic acids or derivatives.
  • the following explanation refers to the binding of a double stranded DNA ligand and using one of the most common and versatile fluorescent marking combinations.
  • One of these oligonucleotides is synthesized with a fluorescent group covalently linked at one of the ends, for example a derivative of the fluorescein at the 5-prime end, and the complement is synthesized with a buffer group ("quencher") of the emission of fluorescence of the above, covalently bound at the opposite end, for example a rhodamine derivative at the 3-prime end.
  • a fluorescent group covalently linked at one of the ends
  • the complement is synthesized with a buffer group (“quencher") of the emission of fluorescence of the above, covalently bound at the opposite end, for example a rhodamine derivative at the 3-prime end.
  • the duplex state of this construction emits very little fluorescence intensity.
  • the separation of the strands by any cause causes an increase in the distance between the fluorescent and buffer groups, so that the effectiveness of the latter in eliminating the emission of the former decreases.
  • the practical result is that an important fluorescence emission occurs in the dissociated state of these derivatized oligonucleotides.
  • the simple determination of the fluorescence emission intensity of the reaction mixture containing these oligonucleotides allows to know the proportion of them that are in associated and dissociated state.
  • dissociation curve also called denaturation or fusion
  • a gradual and programmed temperature change is applied and fluorescence emission readings are obtained simultaneously.
  • each reading of said emission intensity of a given reaction mixture at a certain temperature is directly proportional to the concentration of dissociated strands or inversely proportional to the concentration of associated strands in the form of a duplex at said temperature.
  • fusion curve is really a form of representation of the change with the temperature of the apparent equilibrium constant of the global association-dissociation reaction, according to the Van't Hoff equation.
  • T m melting temperature
  • the practical application derives from the fact that the binding of a ligand to the DNA produces an increase or decrease in the T m as well as other different alterations in the dissociation curve, so that the data obtained in the presence of different ligand concentrations provide useful information on The binding capacity of said ligand to the nucleic acid and on the complex or product formed in its case.
  • the association-dissociation equilibrium reaction of two complementary oligonucleotides, S 1 • and S 2 can be expressed as S 1 + S 2 ⁇ -> D, where D represents the double stranded DNA. If a ligand L that preferentially binds to double stranded DNA also participates in the reaction, the reaction can be expressed as S + + S 2 + nL ⁇ - + D: L n , where D: L n (DL in the following ) represents the complex formed by the double-stranded DNA and n molecules of ligand L.
  • the equilibrium constant for this reaction is given by the expression:
  • each increase in ligand concentration will cause an increase in the melting temperature. This is because, for a fixed temperature T, the equilibrium constant K must be the same, regardless of the concentration of ligand L. Therefore, in order for the value of K to remain constant, increasing L requires that If and S 2 decrease and, consequently, DL increases.
  • the fusion curve as a representation of percentage of single chain in ordinates (Y axis, vertical) versus at the temperature in abscissa (horizontal axis X 1 ), the increase in ligand concentration motivates a nonlinear displacement "downwards" of all the points that make up the melting curve, which manifests as an apparent displacement "to the right” of the sigmoid curve, that is, towards a higher melting temperature T n , when the concentration of ligand increases.
  • the DNA oligonucleotides with complementary sequence to each other and labeled as indicated above should be available, so that one of them contains a fluorescent group covalently linked in one of the ends, for example a derivative of the fluorescein at the 5-prime end, and the complementary oligonucleotide contains a buffer or quencher group of the fluorescence emission of the former, covalently linked at the opposite end, for example a rhodamine derivative in The 3-bonus end.
  • the double chain structure is obtained by mixing both oligonucleotides in equimolecular proportions. To ensure a correct association, the mixture is brought to 95 ° C and then allowed to cool slowly to room temperature.
  • reaction mixtures are then prepared containing a suitable buffered medium, a fixed concentration (for example 10 "7 M) of labeled oligonucleotides, previously associated, and different concentrations of the ligand under study (for example between 10 " 2 and 10 "9 M)
  • a suitable buffered medium for example 10 "7 M
  • concentrations of the ligand under study for example between 10 " 2 and 10 "9 M
  • These reaction mixtures are arranged in different wells of a suitable multi-well plate
  • the usual total volume of reaction mixture per well is 20 ⁇ l in the case of 96-well plates
  • the reaction mixtures may optionally be subjected to a incubation of controlled duration and temperature to ensure the formation and stabilization of the complexes between the ligand and the DNA.
  • the entire plate is subjected to a gradual temperature increase program (for example between 2O 0 C and 95 ° C, increasing one degree every 45 seconds) taking fluorescence intensity readings from each well after each increase in 1 0 C.
  • a gradual temperature increase program for example between 2O 0 C and 95 ° C, increasing one degree every 45 seconds
  • a quantitative or real-time PCR apparatus suitable for the use of multiwell plates can be used for this process of temperature change and fluorescence emission intensity reading.
  • This device is used here as a programmable and multichannel thermostatted spectrofluorimeter.
  • the apparatus is connected to a computer that stores the fluorescence intensity readings as a function of the temperature for each of the reaction mixtures in the multiwell plate.
  • the data is finally recovered in a comma separated text document format and suitable computer programs are processed and analyzed with help.
  • a typical 96-well plate experiment with 75 temperature steps and reading of four wavelength channels generates 28,800 numerical values.
  • With the option of double temperature increase program (dissociation) followed by temperature decrease (association) represents 57,600 numerical values. Obviously, the manual analysis of this amount of data is impossible, so it must be done with the help of software tools developed specifically for this purpose.
  • the described example uses two oligonucleotides differentially labeled with a fluorescent group and a group that extinguishes the emission of the previous one.
  • the basic idea of the embodiment can be applied to other mapping schemes or to the use of independent fluorescent molecules, not covalently bound to oligonucleotides, such as for example the commercial compound called SYBR® Green, whose emission allows quantifying the chain proportion double and single chain.
  • the differential feature of the procedure described in this patent resides in the use of multiwell plates in conjunction with the use of various mapping strategies with fluorescent groups or molecules. This conjunction enables sufficient miniaturization to allow the application of the method to a large number of samples in a multiwell plate format that facilitates parallel processing, The automation of the calculation and the robotization of the process, including the dispensing of liquids or the load on devices.
  • the process described and claimed in this patent has different industrial applications.
  • the first application consists in its use as a rapid method of determining the binding of compounds to DNA. This method can be applied, in the first place, to identify among a large number of compounds those that bind to DNA, so it can be used to search for new compounds with DNA binding. This is of special interest in the search for drugs that have DNA as a target, or ligands for DNA mapping and localization.
  • Another application of the method is its use for the design of devices intended for its realization, accessories for other devices for the same purpose, or commercialization of programs, separately or associated with devices, intended for data processing and calculations related to the procedure.

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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)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (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)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé miniaturisé appliqué dans des plaques multipuits pour analyser la liaison de ligands à des acides nucléiques au moyen de courbes de fusion thermique. Afin de parvenir à la miniaturisation, on utilise des stratégies de marquage fluorescent qui permettent de déterminer la proportion de complexes associés et dissociés en phase homogène. Les mélanges réactionnels à analyser sont disposés dans différents puits d'une plaque multipuits. Ensuite, on soumet la plaque à une augmentation de température graduelle programmée dans le temps, on effectue des lectures d'intensité de fluorescence de chaque puits après chaque étape d'augmentation de température. Un programme de diminution graduelle de température peut également être exécuté, des lectures d'intensité de fluorescence étant également effectuées à chaque modification. La modification programmée de température et la lecture d'intensité d'émission de fluorescence peuvent être effectuées dans un appareil de PCR en temps réel ou d'autres équipements qui permettent d'effectuer ces opérations sur des plaques multipuits.
PCT/ES2010/000264 2009-06-30 2010-06-16 Procédé miniaturisé appliqué dans des plaques multipuits pour analyser la liaison de ligands à des acides nucléiques à travers l'obtention de courbes de fusion thermique Ceased WO2011000977A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200901508A ES2351570B1 (es) 2009-06-30 2009-06-30 Procedimiento miniaturizado en placas multipocillo para analizar la unión de ligandos a ácidos nucleicos mediante la obtención de curvas de fusión térmica.
ESP200901508 2009-06-30

Publications (1)

Publication Number Publication Date
WO2011000977A1 true WO2011000977A1 (fr) 2011-01-06

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PCT/ES2010/000264 Ceased WO2011000977A1 (fr) 2009-06-30 2010-06-16 Procédé miniaturisé appliqué dans des plaques multipuits pour analyser la liaison de ligands à des acides nucléiques à travers l'obtention de courbes de fusion thermique

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ES (1) ES2351570B1 (fr)
WO (1) WO2011000977A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2182708C2 (ru) * 2000-04-17 2002-05-20 Институт молекулярной биологии им. В.А. Энгельгардта РАН Способ множественного параллельного скрининга специфичности связывания биологически активных соединений с нуклеиновыми кислотами с использованием биочипа (варианты)
WO2007018734A2 (fr) * 2005-06-14 2007-02-15 Bio-Rad Laboratories, Inc. Fusion q pour la detection de polymorphisme

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2182708C2 (ru) * 2000-04-17 2002-05-20 Институт молекулярной биологии им. В.А. Энгельгардта РАН Способ множественного параллельного скрининга специфичности связывания биологически активных соединений с нуклеиновыми кислотами с использованием биочипа (варианты)
WO2007018734A2 (fr) * 2005-06-14 2007-02-15 Bio-Rad Laboratories, Inc. Fusion q pour la detection de polymorphisme

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DARBY, R.A.J. ET AL.: "High Throughput Measurement of Duplex, Triplex and Quadruplex Melting Curves Using Molecular Beacons and a LightCycler", NUCLEIC ACIDS RES., vol. 30, no. 9, 2002, pages 8PP, XP055196746, DOI: doi:10.1093/nar/30.9.e39 *
DATABASE WPI Week 200263, Derwent World Patents Index; AN 2002-588369 *
DROBYSHEV, A.L. ET AL.: "Massive Parallthe Analysis of DNA-Hoechst 33258 Binding Specificity with a Generic Oligodeoxyribonucleotide Microchip", NUCLEIC ACIDS RES., vol. 27, no. 20, 1999, pages 4100 - 4105, XP002267643, DOI: doi:10.1093/nar/27.20.4100 *

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ES2351570A1 (es) 2011-02-08
ES2351570B1 (es) 2011-11-30

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