WO2012053018A1

WO2012053018A1 - Method f or the detection of biological reactions products

Info

Publication number: WO2012053018A1
Application number: PCT/IT2010/000424
Authority: WO
Inventors: Rubina Vecchiato
Original assignee: Fiore, Marco
Priority date: 2010-10-20
Filing date: 2010-10-20
Publication date: 2012-04-26

Abstract

It is claimed a versatile method for amplifying detectable signals in hybridization assays, particularly nucleic acid hybridization assays, based on the (strept) avidin-biotin technology.

Description

"Method for the detection of biological reactions products"

Description

This invention relates generally to methods for amplifying detectable signals in hybridization assays, particularly nucleic acid hybridization assays.

Background

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.

The hybridization techniques share the common need to link biomolecules to one another or onto a solid support. At this purpose, linker are used. To be suitable for different applications, 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.

A widely used "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

1964; DeLange and Huang 1971). 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.

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.

A commercially available alternative to the commonly used avidin and streptavidin is 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.

Given the strength of the avidin-biotin bond, dissociation of the avidin-biotin complex requires extreme conditions that cause protein denaturation.

However, endogenously biotinylated proteins that have carboxylase activity are found in the mitochondria; therefore, sensitive detection of biotinylated targets in cells requires the use of biotin-blocking agents to reduce this background.

The biotinylation process comprises the chemical link of biotin to the biomolecule of interest. In the simplest form, a biotinylated probe is applied to a sample and then the bound probe is detected with a labeled avidin or streptavidin. These techniques are commonly used to localize antigens in cells and tissues and to detect biomolecules in immunoassays and DNA hybridization procedures. In some applications, immobilized avidins are used to capture and release biotinylated targets.

As an example, 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.

The method currently used in hybridization techniques comprising the formation of streptavidin- biotin complexes can be summarized as follows:

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 ;

optionally, 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;

hybridization: the biotinylated biomolecules are hybridized with specific probes;

stringent wash: the unbound biomolecules and the not specifically bounded biomolecules are removed; - signal development: incubation with avidin conjugate and, if needed, substrate;

signal detection: the developed signal is detected via a suitable apparatus.

A large series of kit are available for hybridization, and they all follow the above indicated scheme. In particular, the incubation with avidin conjugate is suggested to be carried on in blocking solution, to prevent aspecific signal.

Here we firstly describe a method, suitable in hybridization techniques, that offers great advantages in term of time and simplicity of execution, without any loose in term of specificity and/or intensity of the revealed signal.

Detailed description

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;

denaturation : if needed, using known techniques available in the state of the art;

hybridization: the biotinylated biomolecules are hybridized with specific probes in the presence of the avidin-conjugate;

stringent wash: the unbound biomolecules and conjugate and the unspecifically bounded biomolecules are removed;

signal detection: 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.

Surprisingly, 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.

It is also of interest to note that it was known from the literature to deeply wash the sample after the hybridization step before adding the conjugate. The here claimed method eliminates this need. Also the need to wash with deionized water after the stringent washes performed after the hybridization step is eliminated in the here claimed method.

The claimed method is suitable when using ( strept ) avidin conjugated to fluorophores or with enzymes as well.

In a different embodiment, 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.

As an example, 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.

When the ( strept ) avidin conjugate is on a solid substrate, beads, microbeads, chips, nylon filters, multi-well plates, plastic culture dishes or any other support suitable for this purpose, 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.

' This versatile method is further described in the examples that follow, examples that are reported in order to better describe the present invention, but that are not intended in any way to limit the applications of the method itself.

Example 1

DNA or RNA hybridization and imaging, comparison between a conventional protocol and the here claimed method by using streptavidin-enzyme conjugated

Two assays have been performed in parallel, one following the conventional protocol, the other one by applying the here claimed method. The assays have- been performed on a solid nitrocellulose support. The steps for the conventional protocol are as follows:

i) target Preparation: RNA or DNA Biotin labeling, 40 min;

ii) pre-hybridization :

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) ;

iv) stringent wash: 15-30 min, by using a conventional buffer described in the literature (Sambrook J and Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Press. 2001);

v) streptavidin-enzyme conjugated labeling: 30 min at room temperature, in a buffer selected according to literature (Sambrook J and Russell · DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Press. 2001) ;

vi) detection: 10-30 min; a substrate is added since an streptavidin-enzyme conjugated was used.

Presently claimed method:

i) target Preparation: RNA or DNA Biotin labeling, _.40 min;

ii) Pre-hybridization :

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;

v) detection: 10-30 min; a substrate is added since a streptavidin-enzyme conjugated has been used. Unexpectedly, the obtained signal is clear, without background, and of a comparable intensity when using both the indicated method. However, 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. Moreover, thanks to the here claimed method, there is the possibility to supply streptavidin already in the hybridization buffer, in a ready to use format, further eliminating manipulation from the operator.

Example 2

DNA or RNA hybridization and imaging, comparison between a conventional protocol and the here claimed method by using streptavidin-fluorophore conjugated

Two assays have been performed in parallel, one following the conventional protocol, the other one by applying the here claimed method. The assays have been performed on a solid nitrocellulose substrate. The steps for the conventional protocol are as follows:

i) target Preparation: RNA or DNA Biotin labeling, 40 min;

ii) pre-hybridization :

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) ;

v) streptavidin-fluorophore conjugated labeling: 30 min at room temperature, in a buffer selected according to literature (Sambrook J and Russell DW . Molecular cloning: a laboratory manual. Cold Spring Harbor Press. 2001);

vi) final washing: 15 min;

vii) detection: 10-30 min.

Presently claimed method:

i) target Preparation: RNA or DNA Biotin labeling, 40 min;

ii) pre-hybridization and labeling:

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;

iii) hybridization: 15-90 min; the hybridization temperature was about 55 °C, at pH 7.5;

iv) stringent wash: 15-30 min, by using a conventional buffer described in the literature (Sambrook J and Russell D . Molecular cloning: a laboratory manual. Cold Spring Harbor Press. 2001);

vii) detection: 10-30 min.

Surprisingly, the obtained signal is clear, without background, and of a comparable intensity when using both the indicated method. However, 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. Moreover, streptavidin is supplied in the denaturing buffer, in a ready to use format, eliminating manipulation from the operator .

Claims

1. A method for amplifying detectable signals in hybridization assays comprising the use of avidin, streptavidin, NeutrAvidin or other avidin derivatives complexed with biotin, comprising the following steps:

i) biotinylation;

ii) optionally, denaturation;

iii) hybridization;

iv) stringent wash;

v) signal detection;

wherein said avidin, streptavidin, NeutrAvidin or other avidin derivatives are added during the denaturation or the hybridization step.

2. A method according to claim 1, where said avidin, streptavidin, NeutrAvidin or other avidin derivatives is conjugated to a fluorophore, said fluorophore being selected among xanthenes derivatives or cyanine derivatives.

3. A method according to claim 1, where said avidin, streptavidin, NeutrAvidin or other avidin derivatives is conjugated to an enzyme, preferably to a peroxidase.

4. A method according to any one of claims 1 to 3, where said hybridization assay is perform to detect a nucleic acid.

5. A method according to any one of claims 1 to 3, where said hybridization assay is performed to detect proteins.

6. A method according to any one of claims 1 to 4, where said hybridization assays is performed to evaluate the presence of viral genome in samples.

7. A method according to any one of claims 1 to 6, performed on a solid substrate, preferably nitrocellulose.

8. A kit to perform an hybridization assay, wherein reagents are provided suitable to perform the method according to claim 1.