# Patent

## Title
Compositions and methods for RNA hybridization applications

## Abstract

The invention provides methods and compositions for hybridizing at least one molecule to an RNA target. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in RNA hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a U.S. national stage application of International Application No. PCT/IB2009/007725, filed Dec. 2. 2009, and also claims the benefit of U.S. Provisional Patent Application No. 61/155,683, filed Feb. 26, 2009.


FIELD OF THE INVENTION
The present invention relates to aqueous compositions for use in RNA hybridization applications, for example, for use in in situ hybridization (ISH) applications.
In one embodiment, the present invention relates to the field of molecular examination of RNA. In particular, the invention relates to the fields of cytology, histology, and molecular biology. In one aspect, the present invention relates to the energy (e.g., incubation time and heat) required during hybridization between ribonucleic acids, e.g., in in situ hybridization of RNA.
BACKGROUND AND DESCRIPTION
In a basic example of hybridization, nucleic acid probes or primers are designed to bind, or “hybridize,” with a target nucleic acid, for example RNA, in a sample. One type of hybridization application, in situ hybridization (ISH), includes hybridization to a target in a specimen wherein the specimen may be in vivo, in situ, or for example, fixed or adhered to a glass slide (i.e., in vitro). Probes may then be used, for example, to detect genetic abnormalities in a target sequence, providing valuable information about, e.g., prenatal disorders, cancer, and other genetic or infectious diseases.
The efficiency and accuracy of nucleic acid hybridization assays mostly depend on at least one of three major factors: a) denaturation (i.e., separation of, e.g., two nucleic acid strands) conditions, b) renaturation (i.e., re-annealing of, e.g., two nucleic acid strands) conditions, and c) post-hybridization washing conditions.
In order for the probes or primers to bind to the target nucleic acid in the sample, complementary strands of nucleic acid must be separated (i.e., denatured). Although RNA is a single-stranded molecule and, therefore, should not require a strand-separation step in order to bind to the primer/probe, RNA molecules can pair with single-stranded DNA molecules or with other RNA molecules. These associations are stabilized by hydrogen bonding between bases on opposite strands when bases are paired in a particular way (A+T/U or G+C) and by hydrophobic bonding among the stacked bases. In addition, complementary base-pairing and other types of hydrogen bonds can occur between nucleotides in the same RNA molecule, causing parts of the RNA to fold and pair with itself in a double helical configuration. Thus, some RNA hybridization applications may optionally include a denaturation step.
Traditional hybridization experiments, such as ISH assays, use high temperatures (e.g., 95° C. to 100° C.) and/or formamide-containing solutions to denature doubled stranded nucleic acid. Formamide disrupts base pairing by displacing loosely and uniformly bound hydrate molecules and by causing “formamidation” of the Watson-Crick binding sites. Thus, formamide has a destabilizing effect on double stranded nucleic acids and analogs. However, formamide is a toxic, hazardous material, subject to strict regulations for use and waste. In addition, the use of high formamide concentrations appears to cause morphological destruction of cellular, nuclear, and/or chromosomal structure. Heat can also be destructive to the sample because the phosphodiester bonds of the nucleic acids may be broken at high temperatures, leading to a collection of broken single stranded nucleic acids. In addition, heat can lead to complications when small volumes are used, since evaporation of aqueous buffers is difficult to control.
Once any double-stranded nucleic acids have been separated, a “renaturation” or “reannealing” step allows the primers or probes to bind to the target nucleic acid in the sample. This step is also sometimes referred to as the “hybridization” step. The re-annealing step is by far the most time-consuming aspect of traditional hybridization applications. See FIGS. 1 and 2 (presenting examples of traditional hybridization times for DNA templates). In addition, the presence of formamide in a hybridization buffer can significantly prolong the renaturation time, as compared to aqueous denaturation solutions without formamide.
After the probe has annealed to the target nucleic acid in the sample, any unbound and mis-paired probe is removed by a series of post-hybridization washes. The specificity of the interaction between the probe and the target is largely determined by stringency of these post-hybridization washes. Duplexes containing highly complementary sequences are more resistant to high-stringency conditions than duplexes with low complementary. Thus, increased stringency conditions can be used to remove non-specific bonds between the probe and the target nucleic acids. Four variables are typically adjusted to influence the stringency of the post-hybridization washes: (1) temperature (as temperature increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); (2) salt conditions (as salt concentration decreases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); (3) formamide concentration (as the amount of formamide increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); and (4) time (as the wash time increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).
The present invention provides several potential advantages over prior art methods and compositions for RNA hybridization applications. These advantages include faster hybridization times, lower hybridization temperatures, and less toxic hybridization solvents.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide compositions which result in at least one of the following advantages: highly sensitive, technically easy, fast, flexible, and reliable RNA hybridization procedures that are easy to automate, preserve sample morphology, provide safer reagents, reduce background, and reduce evaporation of reagent. Yet another object of the invention is to provide a composition with a low probe concentration. It is another object of the invention to reduce and/or remove the need for blocking of unspecific binding. The compositions and methods of the invention may also permit the use of heterogeneous probes without the need to block, remove, or otherwise disable the binding of, e.g., repetitive sequences in a biological sample or loop formation of RNA.
The compositions and methods of the invention are applicable to any RNA hybridization technique. The compositions and methods of the invention are also applicable to any molecules that hybridize or bind to RNA using base pairing, such as, for example, single-stranded DNA, RNA, PNA, LNA, and synthetic and natural analogs thereof.
In one embodiment, the nucleic acid hybridization method and compositions of the present invention are useful for the in vitro, in vivo, or in situ analysis of cellular RNA. Further, the methods and compositions are useful for detection of RNA from infectious agents as well as changes in levels of expression of RNA.
For example, the method and compositions of the invention may be used for the in vitro, in vivo, or in situ analysis of messenger RNA (mRNA), viral RNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNA and rRNA), transfer messenger RNA (tmRNA), micro RNA (miRNA), piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA (snoRNA), antisense RNA, double-stranded RNA (dsRNA), and heterogeneous nuclear RNA (hnRNA).
The nucleic acid hybridization method and compositions of the present invention are useful for in vitro, in vivo, or in situ analysis of RNA using techniques such as northern blot, flow cytometry, autoradiography, fluorescence microscopy, chemiluminescence, immunohistochemistry, virtual karyotype, gene assay, microarray, gene expression profiling, Gene ID, Tiling array, gel electrophoresis, capillary electrophoresis, and in situ hybridizations such as FISH, SISH, CISH.
The methods and compositions of the invention may be used on in situ, in vitro, and in vivo samples such as bone marrow smears, blood smears, paraffin embedded tissue preparations, enzymatically dissociated tissue samples, bone marrow, amniocytes, cytospin preparations, imprints, etc.
In one embodiment, the invention provides methods and compositions for hybridizing at least one molecule to a target RNA. In one embodiment, the compositions and methods of the invention lower the energy necessary for hybridization applications. The lower energy barrier may reduce the time and/or temperature necessary for hybridization applications. For example, the invention may allow for performing the hybridization step at lower temperatures or may allow for a rapid hybridization step at higher temperatures. In addition, the hybridization step may be performed at room temperature. Thus, the methods and compositions of the invention enable one to tailor the time required for hybridization applications by varying the temperature of the reaction to a much greater degree than is available using prior art methods, overcoming a major time-consuming step in hybridization assays.
The invention may also eliminate the use of, or reduce the dependence on formamide. For example, the methods and compositions of the invention may lower the energy barrier to hybridization without the use of formamide.
One aspect of the invention is a composition or solution for use in RNA hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA. One way to test for whether the amount of polar aprotic solvent is effective to enable hybridization to RNA is to determine whether the polar aprotic solvent, when used in the hybridization methods and compositions described herein, such as example 1, yield a detectable signal.
Non-limiting examples of effective amounts of polar aprotic solvents include, e.g., about 1% to about 95% (v/v). In some embodiments, the concentration of polar aprotic solvent is 5% to 60% (v/v). In other embodiments, the concentration of polar aprotic solvent is 10% to 60% (v/v). In still other embodiments, the concentration of polar aprotic solvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v/v) are also suitable. In some embodiments, the polar aprotic solvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5% (v/v). In other embodiments, the polar aprotic solvent will be present at a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).
According to another aspect of the present invention the aqueous composition comprising a polar aprotic solvent has reduced toxicity. For example, a less-toxic composition than traditional solutions used in hybridization applications may comprise a composition with the proviso that the composition does not contain formamide, or with the proviso that the composition contains less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01% formamide. A less-toxic composition may also comprise a composition with the proviso that the composition does not contain dimethyl sulfoxide (DMSO), or with the proviso that the composition contains less than 10%, 5%, 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01% DMSO.
In one aspect of the invention, suitable polar aprotic solvents for use in the invention may be selected based on their Hansen Solubility Parameters. For example, suitable polar aprotic solvents may have a dispersion solubility parameter between 17.7 to 22.0 MPa1/2, a polar solubility parameter between 13 to 23 MPa1/2, and a hydrogen bonding solubility parameter between 3 to 13 MPa1/2.
According to one aspect of the present invention, suitable polar aprotic solvents for use in the invention are cyclic compounds. A cyclic compound has a cyclic base structure. Examples include the cyclic compounds disclosed herein. In other embodiments, the polar aprotic solvent may be chosen from Formulas 1-4 below:




where X is O and R1 is alkyldiyl.

According to another aspect of the invention, suitable polar aprotic solvents for use in the invention may be chosen from Formula 5 below:




where X is optional and if present, is chosen from O or S;

where Z is optional and if present, is chosen from O or S;

where A and B independently are O or N or S or part of the alkyldiyl or a primary amine;

where R is alkyldiyl; and

where Y is O or S or C.

Examples of suitable polar aprotic solvents according to Formula 5 are provided in Formulas 6-9 below:




According to yet another aspect of the invention the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, or carbonate functionality. Such compounds are distinguished by their relatively high dielectric constants, high dipole moments, and solubility in water.
According to another aspect of the invention the polar aprotic solvent having lactone functionality is γ-butyrolactone (GBL), the polar aprotic solvent having sulfone functionality is sulfolane (SL), the polar aprotic solvent having nitrile functionality is acetonitrile (AN), the polar aprotic solvent having sulfite functionality is glycol sulfite/ethylene sulfite (GS), and the polar aprotic solvent having carbonate functionality is ethylene carbonate (EC), propylene carbonate (PC), or ethylene thiocarbonate (ETC). In yet another aspect of the invention, the compositions and methods of the invention comprise a polar aprotic solvent, with the proviso that the polar aprotic solvent is not acetonitrile (AN) or sulfolane (SL).
In one embodiment, the at least one nucleic acid sequence is a PNA sequence. In another embodiment, the at least one nucleic acid sequence is an LNA sequence.
According to yet another aspect, the invention discloses a hybridization method comprising:

    
    
        providing an RNA sequence,
        providing a second nucleic acid sequence,
        providing an aqueous composition comprising at least one polar aprotic solvent in an amount effective to enable hybridization to RNA, and
        combining the RNA and the second nucleic acid sequence and the aqueous composition for at least a time period sufficient to hybridize the RNA and the second nucleic acid sequence.
    
    


According to yet another aspect, the invention discloses a hybridization method comprising:

    
    
        providing an RNA sequence, and
        applying to said RNA sequence an aqueous composition comprising a second nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA for at least a time period sufficient to hybridize the RNA and the second nucleic acid sequences.
    
    


In one embodiment, the RNA is in a biological sample. In another embodiment, the biological sample is a cytology or histology sample. In one embodiment, the second nucleic acid sequence is a PNA sequence, an LNA sequence, or a DNA sequence.
In one embodiment, the step of hybridizing the RNA sequence to the second nucleic acid sequence occurs in less than 8 hours, such as, for example, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour. According to another aspect the present invention relates to a method wherein the hybridization step takes less than 1 hour. In other embodiments, the hybridization step takes less than 30 minutes. In still other embodiments, the hybridization step takes less than 15 minutes. In other embodiments, the hybridization step takes less than 5 minutes.
In one embodiment, a sufficient amount of energy to hybridize the RNA and the second nucleic acids is provided.
According to yet another aspect of the present invention, the hybridization energy is provided by heating the aqueous composition and nucleic acid sequence. Thus, the step of hybridizing may include the steps of heating and cooling the aqueous composition and nucleic acid sequences.
A further aspect of the invention comprises a method wherein the step of providing a sufficient amount of energy to hybridize the nucleic acids involves a heating step performed by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating, or heat lamps.
According to another aspect of the invention, the method further includes a denaturation step. In one embodiment, the nucleic acid sequences are denatured separately. For example, the specimen may be denatured with a solution without probe and thereafter hybridized with probe. In another embodiment, the nucleic acid sequences are denatured together.
According to a further aspect, the invention relates to the use of a composition comprising between 1 and 95% (v/v) of at least one polar aprotic solvent in an RNA hybridization application.
According to yet another aspect, the invention relates to the use of a composition comprising an aqueous composition as described in this invention for use in an RNA hybridization application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a typical time-course for single locus detection of a DNA target with primary labeled FISH probes on formaldehyde fixed paraffin embedded tissue sections (histological specimens). The bars represent a hybridization assay performed using a traditional solution (top) and a typical time-course for a hybridization assay performed using a composition of the invention (bottom). The first bar on the left in each time-course represents the deparaffination step; the second bar represents the heat-pretreatment step; the third bar represents the digestion step; the fourth bar represents the denaturation and hybridization steps; the fifth bar represents the stringency wash step; and the sixth bar represents the mounting step.
FIG. 2 depicts a typical time-course for single locus detection of a DNA target with primary labeled FISH probes on cytological specimens. The bars represent a hybridization assay performed using a traditional solution (top) and a typical time-course for a hybridization assay performed using a composition of the invention (bottom). The first bar on the left in each time-course represents the fixation step; the second bar represents the denaturation and hybridization steps; the third bar represents the stringency wash step; and the fourth bar represents the mounting step.



DETAILED DESCRIPTION
A. Definitions
In the context of the present invention the following terms are to be understood as follows:
“Biological sample” is to be understood as any in vivo, in vitro, or in situ sample of one or more cells or cell fragments. This can, for example, be a unicellular or multicellular organism, tissue section, cytological sample, chromosome spread, purified nucleic acid sequences, artificially made nucleic acid sequences made by, e.g., a biologic based system or by chemical synthesis, microarray, or other form of nucleic acid chip. In one embodiment, a sample is a mammalian sample, such as, e.g., a human, murine, rat, feline, or canine sample.
“Nucleic acid,” “nucleic acid chain,” and “nucleic acid sequence” mean anything that binds or hybridizes using base pairing including, oligomers or polymers having a backbone formed from naturally occurring nucleotides and/or nucleic acid analogs comprising nonstandard nucleobases and/or nonstandard backbones (e.g., a peptide nucleic acid (PNA) or locked nucleic acid (LNA)), or any derivatized form of a nucleic acid.
As used herein, the term “peptide nucleic acid” or “PNA” means a synthetic polymer having a polyamide backbone with pendant nucleobases (naturally occurring and modified), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in, e.g., U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 6,201,103, 6,228,982 and 6,357,163, WO96/04000, all of which are herein incorporated by reference, or any of the references cited therein. The pendant nucleobase, such as, e.g., a purine or pyrimidine base on PNA may be connected to the backbone via a linker such as, e.g., one of the linkers taught in PCT/US02/30573 or any of the references cited therein. In one embodiment, the PNA has an N-(2-aminoethyl)-glycine) backbone. PNAs may be synthesized (and optionally labeled) as taught in PCT/US02/30573 or any of the references cited therein. PNAs hybridize tightly, and with high sequence specificity, with DNA and RNA, because the PNA backbone is uncharged. Thus, short PNA probes may exhibit comparable specificity to longer DNA or RNA probes. PNA probes may also show greater specificity in binding to complementary DNA or RNA.
As used herein, the term “locked nucleic acid” or “LNA” means an oligomer or polymer comprising at least one or more LNA subunits. As used herein, the term “LNA subunit” means a ribonucleotide containing a methylene bridge that connects the 2′-oxygen of the ribose with the 4′-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).
Examples of nucleic acids and nucleic acid analogs also include polymers of nucleotide monomers, including double and single stranded deoxyribonucleotides (DNA), ribonucleotides (RNA), α-anomeric forms thereof, synthetic and natural analogs thereof, and the like. The nucleic acid chain may be composed entirely of deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNA), locked nucleic acids (LNA), synthetic or natural analogs thereof, or mixtures thereof. DNA, RNA, or other nucleic acids as defined herein can be used in the method and compositions of the invention.
“Polar aprotic solvent” refers to an organic solvent having a dipole moment of about 2 debye units or more, a water solubility of at least about 5% (volume) at or near ambient temperature, i.e., about 20° C., and which does not undergo significant hydrogen exchange at approximately neutral pH, i.e., in the range of 5 to 9, or in the range 6 to 8. Polar aprotic solvents include those defined according to the Hansen Solubility Parameters discussed below.
“Alkyldiyl” refers to a saturated or unsaturated, branched, straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene, or alkyne.
“Aqueous solution” is to be understood as a solution containing water, even small amounts of water. For example, a solution containing 1% water is to be understood as an aqueous solution.
“Hybridization application,” “hybridization assay,” “hybridization experiment,” “hybridization procedure,” “hybridization technique,” “hybridization method,” etc. are to be understood as referring to any process that involves hybridization to RNA. Unless otherwise specified, the terms “hybridization” and “hybridization step” are to be understood as referring to the re-annealing step of the RNA hybridization procedure as well as the optional denaturation step (if present).
“Hybridization composition” refers to an aqueous solution of the invention for performing a hybridization procedure, for example, to bind a probe to a nucleic acid sequence. Hybridization compositions may comprise, e.g., at least one polar aprotic solvent, at least one nucleic acid sequence, and a hybridization solution. Hybridization compositions do not comprise enzymes or other components, such as deoxynucleoside triphosphates (dNTPs), for amplifying nucleic acids in a biological sample.
“Hybridization solution” refers to an aqueous solution for use in a hybridization composition of the invention. Hybridization solutions are discussed in detail below and may comprise, e.g., buffering agents, accelerating agents, chelating agents, salts, detergents, and blocking agents.
“Hansen Solubility Parameters” and “HSP” refer to the following cohesion energy (solubility) parameters: (1) the dispersion solubility parameter (δD, “D parameter”), which measures nonpolar interactions derived from atomic forces; (2) the polar solubility parameter (δP, “P parameter”), which measures permanent dipole-permanent dipole interactions; and (3) the hydrogen bonding solubility parameter (δH, “H parameter”), which measures electron exchange. The Hansen Solubility Parameters are further defined below.
“Repetitive Sequences” is to be understood as referring to the rapidly reannealing (approximately 25%) and/or intermediately reannealing (approximately 30%) components of mammalian genomes. The rapidly reannealing components contain small (a few nucleotides long) highly repetitive sequences usually found in tandem (e.g., satellite DNA), while the intermediately reannealing components contain interspersed repetitive DNA. Interspersed repeated sequences are classified as either SINEs (short interspersed repeat sequences) or LINEs (long interspersed repeated sequences), both of which are classified as retrotransposons in primates. SINEs and LINEs include, but are not limited to, Alu-repeats, Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotide repeats and hexa-nucleotide repeats. Alu repeats make up the majority of human SINEs and are characterized by a consensus sequence of approximately 280 to 300 bp that consist of two similar sequences arranged as a head to tail dimer. In addition to SINEs and LINES, repeat sequences also exist in chromosome telomeres at the termini of chromosomes and chromosome centromeres, which contain distinct repeat sequences that exist only in the central region of a chromosome. However, unlike SINEs and LINEs, which are dispersed randomly throughout the entire genome, telomere and centromere repeat sequences are localized within a certain region of the chromosome.
“Non-toxic” and “reduced toxicity” are defined with respect to the toxicity labeling of formamide according to “Directive 1999/45/EC of the European Parliament and of the Council of 31 May 1999 concerning the approximation of the laws, regulations and administrative provisions of the Member States relating to the classification, packaging, and labelling of dangerous preparations” (ecb.jrc.it/legislation/1999L0045EC.pdf) (“Directive”). According to the Directive, toxicity is defined using the following classification order: T+“very toxic”; T “toxic”, C “corrosive”, Xn “harmful”, .Xi “irritant.” Risk Phrases (“R phrases”) describe the risks of the classified toxicity. Formamide is listed as T (toxic) and R61 (may cause harm to the unborn child). All of the following chemicals are classified as less toxic than formamide: acetonitrile (Xn, R11, R20, R21, R22, R36); sulfolane (Xn, R22); γ-butyrolactone (Xn, R22, R32); and ethylene carbonate (Xi, R36, R37, R38). At the time of filing this application, ethylene trithiocarbonate and glycol sulfite are not presently labeled.
“Stringent” and “stringency” in the context of post-hybridization washes are to be understood as referring to conditions for reducing non-complementary base-pairing. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates specific hybridization with little non-complementary base-pairing. The specificity of the interaction between the probe and the target is largely determined by stringency of these post-hybridization washes. Duplexes containing highly complementary sequences are more resistant to high-stringency conditions than duplexes with low complementary. Thus, increased stringency conditions can be used to remove non-specific bonds between the probe and the target nucleic acids. Four variables are typically adjusted to influence the stringency of the post-hybridization washes: (1) temperature (as temperature increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); (2) salt conditions (as salt concentration decreases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); (3) formamide concentration (as the amount of formamide increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences); and (4) time (as the wash time increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences). Other factors such as pH, rate of agitation, and number of washes will also influence the stringency of the wash step.
As used herein, the terms “room temperature” and “RT” refer to about 20° C. to about 25° C., unless otherwise stated.
B. Solvent Selection
Suitable polar aprotic solvents for use in the invention may be selected based on their Hansen Solubility Parameters. Methods for experimentally determining and/or calculating HSP for a solvent are known in the art, and HSP have been reported for over 1200 chemicals.
For example, the D parameter may be calculated with reasonable accuracy based on refractive index, or may be derived from charts by comparison with known solvents of similar size, shape, and composition after establishing a critical temperature and molar volume. The P parameter may be estimated from known dipole moments (see, e.g., McClellan A. L., Tables of Experimental Dipole Moments (W.H. Freeman 1963)) using Equation 1:

δP=37.4(Dipole Moment)/V1/2  Equation 1

where V is the molar volume. There are no equations for calculating the H parameter. Instead, the H parameter is usually determined based on group contributions.

HSP characterizations are conveniently visualized using a spherical representation, with the HSP of an experimentally-determined suitable reference solvent at the center of the sphere. The radius of the sphere (R) indicates the maximum tolerable variation from the HSP of the reference solvent that still allows for a “good” interaction to take place. Good solvents are within the sphere and bad ones are outside. The distance, Ra, between two solvents based on their respective HSP values can be determined using Equation 2:

(Ra)2=4(δD1−δD2)2+(δP1−δP2)2(δH1−δH2)2  Equation 2

where subscript 1 indicates the reference sample, subscript 2 indicates the test chemical, and all values are in MPa1/2. Good solubility requires that Ra be less than the experimentally-determined radius of the solubility sphere Ro. The relative energy difference between two solvents, i.e., RED number, can be calculated by taking the ratio of Ra to Ro, as shown in Equation 3.

RED=Ra/Ro  Equation 3

RED numbers less than 1.0 indicate high affinity; RED numbers equal or close to 1.0 indicate boundary conditions; and progressively higher RED numbers indicate progressively lower affinities.

In some embodiments, the D parameters of the polar aprotic solvents of the invention are between 17.7 to 22.0 MPa1/2. Such relatively high D parameters are generally associated with solvents having cyclic structures and/or structures with sulfur or halogens. Linear compounds are not likely to be among the most suitable polar aprotic solvents for use in the invention, but may be considered if their P and H parameters are within the ranges discussed below. Since the D parameter is multiplied by 4 in Equation 2, the limits are one-half of Ro. In addition, it should be noted that D values of around 21 or higher are often characteristic of a solid.
In some embodiments, the P parameters of the polar aprotic solvents of the invention are between 13 to 23 MPa1/2. Such exceptionally high P parameters are generally associated with solvents having a high dipole moment and presumably also a relatively low molecular volume. For example, for V near 60 cc/mole, the dipole moment should be between 4.5 and 3.1. For V near 90 cc/mole, the dipole moment should be between 5.6 and 3.9.
In some embodiments, the H parameters of the polar aprotic solvents of the invention are between 3 to 13 MPa1/2. Generally, polar aprotic solvents having an alcohol group are not useful in the compositions and methods of the invention, since the H parameters of such solvents would be too high.
The molar volume of the polar aprotic solvent may also be relevant, since it enters into the evaluation of all three Hansen Solubility Parameters. As molar volume gets smaller, liquids tend to evaporate rapidly. As molar volume gets larger, liquids tend to enter the solid region in the range of D and P parameters recited above. Thus, the polar aprotic solvents of the invention are rather close to the liquid/solid boundary in HSP space.
In some embodiments, the polar aprotic solvents of the invention have lactone, sulfone, nitrile, sulfite, and/or carbonate functionality. Such compounds are distinguished by their relatively high dielectric constants, high dipole moments, and solubility in water. An exemplary polar aprotic solvent with lactone functionality is γ-butyrolactone (GBL), an exemplary polar aprotic solvent with sulfone functionality is sulfolane (SL; tetramethylene sulfide-dioxide), an exemplary polar aprotic solvent with nitrile functionality is acetonitrile (AN), an exemplary polar aprotic solvent with sulfite functionality is glycol sulfite/ethylene sulfite (GS), and an exemplary polar aprotic solvents with carbonate functionality are ethylene carbonate (EC), propylene carbonate (PC), or ethylene trithiocarbonate (ETC). The structures of these exemplary solvents are provided below and their Hansen Solubility Parameters, RED numbers, and molar volumes are given in Table 1.
















TABLE 1










Molar







Volume



D
P
H
RED
(cm3/mole)




















Correlation
19.57
19.11
7.71
—
—


(R0 = 3.9)







GBL
19.0
16.6
7.4
0.712
76.5


PC
20.0
18.0
4.1
0.993
85.2


SL
20.3
18.2
10.9
0.929
95.7


EC
19.4
21.7
5.1
0.946
66.0


ETC
n/a
n/a
n/a
n/a
n/a


GS
20.0
15.9
5.1
n/a
75.1





n/a = not available.






Other suitable polar aprotic solvents that may be used in the invention are cyclic compounds such as, e.g., ε-caprolactone. In addition, substituted pyrrolidinones and related structures with nitrogen in a 5- or 6-membered ring, and cyclic structures with two nitrile groups, or one bromine and one nitrile group, may also be suitable for use in the invention. For example, N-methylpyrrolidinone (shown below) may be a suitable polar aprotic solvent for use in the methods and compositions of the invention.




Other suitable polar aprotic solvents may contain a ring urethane group (NHCOO—). However, not all such compounds are suitable, since 1,3-dimethyl-2-imidazolidinone produces no signals when used in the hybridization compositions of the invention. One of skill in the art may screen for compounds useful in the compositions and methods of the invention as described herein. Exemplary chemicals that may be suitable for use in the invention are set forth in Tables 2 and 3 below.












TABLE 2







Solvent
D
P
H





















Acetanilide
20.6
13.3
12.4



N-Acetyl Pyrrolidone
17.8
13.1
8.3



4-Amino Pyridine
20.4
16.1
12.9



Benzamide
21.2
14.7
11.2



Benzimidazole
20.6
14.9
11.0



1,2,3-Benzotriazole
18.7
15.6
12.4



Butadienedioxide
18.3
14.4
6.2



2,3-Butylene Carbonate
18.0
16.8
3.1



Caprolactone (Epsilon)
19.7
15.0
7.4



Chloro Maleic Anhydride
20.4
17.3
11.5



2-Chlorocyclohexanone
18.5
13.0
5.1



Chloronitromethane
17.4
13.5
5.5



Citraconic Anhydride
19.2
17.0
11.2



Crotonlactone
19.0
19.8
9.6



Cyclopropylnitrile
18.6
16.2
5.7



Dimethyl Sulfate
17.7
17.0
9.7



Dimethyl Sulfone
19.0
19.4
12.3



Dimethyl Sulfoxide
18.4
16.4
10.2



1,2-Dinitrobenzene
20.6
22.7
5.4



2,4-Dinitrotoluene
20.0
13.1
4.9



Dipheynyl Sulfone
21.1
14.4
3.4



1,2-Dinitrobenzene
20.6
22.7
5.4



2,4-Dinitrotoluene
20.0
13.1
4.9



Epsilon-Caprolactam
19.4
13.8
3.9



Ethanesulfonylchloride
17.7
14.9
6.8



Furfural
18.6
14.9
5.1



2-Furonitrile
18.4
15.0
8.2



Isoxazole
18.8
13.4
11.2



Maleic Anhydride
20.2
18.1
12.6



Malononitrile
17.7
18.4
6.7



4-Methoxy Benzonitrile
19.4
16.7
5.4



1-Methoxy-2-Nitrobenzene
19.6
16.3
5.5



1-Methyl Imidazole
19.7
15.6
11.2



3-Methyl Isoxazole
19.4
14.8
11.8



N-Methyl Morpholine-N-
19.0
16.1
10.2



Oxide






Methyl Phenyl Sulfone
20.0
16.9
7.8



Methyl Sulfolane
19.4
17.4
5.3



Methyl-4-Toluenesulfonate
19.6
15.3
3.8



3-Nitroaniline
21.2
18.7
10.3



2-Nitrothiophene
19.7
16.2
8.2



9,10-Phenanthrenequinone
20.3
17.1
4.8



Phthalic Anhydride
20.6
20.1
10.1



1,3-Propane Sultone
18.4
16.0
9.0



beta-Propiolactone
19.7
18.2
10.3



2-Pyrrolidone
19.4
17.4
11.3



Saccharin
21.0
13.9
8.8



Succinonitrile
17.9
16.2
7.9



Sulfanilamide
20.0
19.5
10.7



Sulfolane
20.3
18.2
10.9



2,2,6,6-
19.5
14.0
6.3



Tetrachlorocyclohexanone






Thiazole
20.5
18.8
10.8



3,3,3-Trichloro Propene
17.7
15.5
3.4



1,1,2-Trichloro Propene
17.7
15.7
3.4



1,2,3-Trichloro Propene
17.8
15.7
3.4










Table 2 sets forth an exemplary list of potential chemicals for use in the compositions and methods of the invention based on their Hansen Solubility Parameters. Other compounds, may of course, also meet these requirements such as, for example, those set forth in Table 3.









TABLE 3





Chemical (dipole moment)
RED
Melting Point ° C.

















Chloroethylene carbonate (4.02)
0.92
—


2-Oxazolidinone (5.07)
0.48
86-89


2-Imidazole
1.49
90-91


1,5-Dimethyl Tetrazole (5.3)
~1.5
70-72


N-Ethyl Tetrazole (5.46)
~1.5



Trimethylene sulfide-dioxide (4.49)
—
—


Trimethylene sulfite (3.63)
—
—


1,3-Dimethyl-5-Tetrazole (4.02)
—
—


Pyridazine (3.97)
1.16
−8


2-Thiouracil (4.21)
—
—


N-Methyl Imidazole (6.2)
1.28
—


1-Nitroso-2-pyrolidinone
~1.37
—


Ethyl Ethyl Phosphinate (3.51)
—
—


5-cyano-2-Thiouracil (5.19)
—
—


4H-Pyran-4-thione (4.08)
1.35
32-34


4H-Pyran-4-one = gamma pyrone (4.08)
1.49
Boiling Point (BP) 80


2-Nitrofuran (4.41)
1.14
29


Methyl alpha Bromo Tetronate (6.24)
—
—


Tetrahydrothiapyran oxide (4.19)
1.75
60-64


Picolinonitrile (2-cyanopyridine) (5.23)
0.40
26-28 (BP 212-215)


Nitrobenzimidazole (6.0)
0.52
207-209


Isatin (5.76)
—
193-195


N-phenyl sydnone (6.55)
—
—


Glycol sulfate (Ethylene glycol)
—
99° C.


Note: not soluble at 40%











Some of the chemicals listed in Tables 2 and 3 have been used in hybridization and/or PCR applications in the prior art (e.g., dimethyl sulfoxide (DMSO) has been used in hybridization and PCR applications, and sulfolane (SL), acetonitrile (AN), 2-pyrrolidone, ε-caprolactam, and ethylene glycol have been used in PCR applications). Thus, in some embodiments, the polar aprotic solvent is not DMSO, sulfolane, acetonitrile, 2-pyrrolidone, ε-caprolactam, or ethylene glycol. However, most polar aprotic solvents have not been used in prior art hybridization applications. Moreover, even when such compounds were used, the prior art did not recognize that they may be advantageously used to decrease hybridization times and/or temperatures in RNA hybridizations, as disclosed in this application.
In addition, not all of the chemicals listed in Tables 2 and 3 are suitable for use in the compositions and methods of the invention. For example, although DMSO is listed in Table 2 because its Hansen Solubility Parameters (HSPs) fall within the ranges recited above, DMSO likely does not function to decrease hybridization times and/or temperatures in the compositions and methods of the invention. However, it is well within the skill of the ordinary artisan to screen for suitable compounds using the guidance provided herein including testing a compound in one of the examples provided. For example, in some embodiments, suitable polar aprotic solvents will have HSPs within the ranges recited above and a structure shown in Formulas 1-9 above.
C. Compositions, Buffers, and Solutions
(1) Hybridization Solutions
Traditional hybridization solutions for use in RNA applications are known in the art. Such solutions may comprise, for example, buffering agents, accelerating agents, chelating agents, salts, detergents, and blocking agents.
For example, the buffering agents may include SSC, HEPES, SSPE, PIPES, TMAC, TRIS, SET, citric acid, a phosphate buffer, such as, e.g., potassium phosphate or sodium pyrrophosphate, etc. The buffering agents may be present at concentrations from 0.01× to 50×, such as, for example, 0.01×, 0.1×, 0.5×, 1×, 2×, 5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, or 50×. Typically, the buffering agents are present at concentrations from 0.1× to 10×.
The accelerating agents may include polymers such as FICOLL, PVP, heparin, dextran sulfate, proteins such as BSA, glycols such as ethylene glycol, glycerol, 1,3 propanediol, propylene glycol, or diethylene glycol, combinations thereof such as Denhardt's solution and BLOTTO, and organic solvents such as formamide, dimethylformamide, DMSO, etc. The accelerating agent may be present at concentrations from 1% to 80% or 0.1× to 10×, such as, for example, 0.1% (or 0.1×), 0.2% (or 0.2×), 0.5% (or 0.5×), 1% (or 1×), 2% (or 2×), 5% (or 5×), 10% (or 10×), 15% (or 15×), 20% (or 20×), 25% (or 25×), 30% (or 30×), 40% (or 40×), 50% (or 50×), 60% (or 60×), 70% (or 70×), or 80% (or 80×). Typically, formamide is present at concentrations from 25% to 75%, such as 25%, 30%, 40%, 50%, 60%, 70%, or 75%, while DMSO, dextran sulfate, and glycol are present at concentrations from 5% to 10%, such as 5%, 6%, 7%, 8%, 9%, or 10%.
The chelating agents may include EDTA, EGTA, etc. The chelating agents may be present at concentrations from 0.1 mM to 10 mM, such as 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. Typically, the chelating agents are present at concentrations from 0.5 mM to 5 mM, such as 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM.
The salts may include sodium chloride, sodium phosphate, magnesium phosphate, etc. The salts may be present at concentrations from 1 mM to 750 mM, such as 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, or 750 mM. Typically, the salts are present at concentrations from 10 mM to 500 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM.
The detergents may include Tween, SDS, Triton, CHAPS, deoxycholic acid, etc. The detergent may be present at concentrations from 0.001% to 10%, such as, for example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Typically, the detergents are present at concentrations from 0.01% to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.
The nucleic acid blocking agents may include, yeast tRNA, homopolymer DNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA, COT1 DNA, etc. The blocking nucleic acids may be present at concentrations of 0.05 mg/mL to 100 mg/mL.
A great variation exists in the literature regarding traditional hybridization solutions. For example, a traditional hybridization solution may comprise 5× or 6×SSC, 0.01 M EDTA, 5×Denhardt's solution, 0.5% SDS, and 100 mg/mL sheared, denatured salmon sperm DNA. Another traditional hybridization solution may comprise 50 mM HEPES, 0.5 M NaCl, and 0.2 mM EDTA. Yet another traditional hybridization solution may comprise 2×SSC, 10% dextran sulfate, 50% formamide, and e.g., 0.3 mg/mL salmon sperm DNA or 0.1 mg/mL COT1 DNA. A typical hybridization solution for FISH on biological specimens for RNA detection may comprise, e.g., 2×SSC, 10% dextran sulfate, 2 mM vanadyl-ribonucleoside complex, 50% formamide, 0.02% RNAse-free BSA, and 1 mg/mL E. coli tRNA. Other typical hybridization solutions for DNA detection may comprise 40% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, Alu-PNA (blocking PNA) or COT-1 DNA, and in some cases 0.1 μg/μL total human DNA (THD).
The compositions of the invention may comprise a hybridization solution comprising any of the components of traditional hybridization solutions recited above in combination with at least one polar aprotic solvent. The traditional components may be present at the same concentrations as used in traditional hybridization solutions, or may be present at higher or lower concentrations, or may be omitted completely.
For example, if the compositions of the invention comprise salts such as NaCl and/or phosphate buffer, the salts may be present at concentrations of 0-1200 mM NaCl and/or 0-200 mM phosphate buffer. In some embodiments, the concentrations of salts may be, for example, 300 mM NaCl and 5 mM phosphate buffer, or 600 mM NaCl and 10 mM phosphate buffer.
If the compositions of the invention comprise accelerating agents such as dextran sulfate, glycol, or DMSO, the dextran sulfate may be present at concentrations of from 5% to 40%, the glycol may be present at concentrations of from 0.1% to 10%, and the DMSO may be from 0.1% to 10%. In some embodiments, the concentration of dextran sulfate may be 10% or 20% and the concentration of ethylene glycol, 1,3 propanediol, or glycerol may be 1% to 10%. In some embodiments, the concentration of DMSO may be 1%. In some embodiments, the aqueous composition does not comprise DMSO as an accelerating agent. In some embodiments, the aqueous composition does not comprise formamide as an accelerating agent, or comprises formamide with the proviso that the composition contains less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01%.
If the compositions of the invention comprise citric acid, the concentrations may range from 1 mM to 50 mM and the pH may range from 5.0 to 8.0. In some embodiments the concentration of citric acid may be 10 mM and the pH may be 6.2.
The compositions of the invention may comprise agents that reduce non-specific binding to, for example, the cell membrane, such as salmon sperm DNA or small amounts of total human DNA or, for example, they may comprise blocking agents to block binding of, e.g., repeat sequences in the target, such as larger amounts of total human DNA or repeat-enriched DNA, or specific blocking agents such as PNA or LNA fragments and sequences. These agents may be present at concentrations of from 0.01-100 μg/μL or 0.01-100 μM. For example, in some embodiments, these agents will be 0.1 μg/μL total human DNA, or 0.1 μg/μL non-human DNA, such as herring sperm, salmon sperm, or calf thymus DNA, or 5 μM blocking PNA.
One aspect of the invention is a composition or solution for use in RNA hybridization applications. Compositions for use in the invention include an aqueous composition comprising a nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA. One way to test for whether the amount of polar aprotic solvent is effective to enable hybridization to RNA is to determine whether the polar aprotic solvent, when used in the hybridization methods and compositions described herein, such as example 1, yield a detectable signal.
Non-limiting examples of effective amounts of polar aprotic solvents include, e.g., about 1% to about 95% (v/v). In some embodiments, the concentration of polar aprotic solvent is 5% to 60% (v/v). In other embodiments, the concentration of polar aprotic solvent is 10% to 60% (v/v). In still other embodiments, the concentration of polar aprotic solvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v/v) are also suitable. In some embodiments, the polar aprotic solvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5% (v/v). In other embodiments, the polar aprotic solvent will be present at a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).
In some embodiments, the at least one nucleic acid sequence comprises one or more nucleic acid probes. The probes may be directly or indirectly labeled with detectable compounds such as enzymes, chromophores, fluorochromes, and haptens. DNA probes may be present at concentrations of 0.1 to 100 μg/μL. For example, in some embodiments, DNA probes may be present at concentrations of 1 to 10 ng/μL. PNA probes may be present at concentrations of 0.5 to 5000 nM. For example, in some embodiments, PNA probes may be present at concentrations of 5 to 1000 nM.
In one embodiment, a composition of the invention comprises a mixture of 40% polar aprotic solvent (v/v) (e.g., ethylene carbonate, “EC”), 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, and 1-10 ng/μL probe. Another exemplary composition of the present invention comprises a mixture of 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 μg/μl total human DNA. Yet another exemplary composition comprises 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid pH 6.2, and 0.1 μg/μL non-human DNA (e.g., herring sperm, salmon sperm, or calf thymus) OR 0.5% formamide OR 1% glycol (e.g., ethylene glycol, 1,3 propanediol, or glycerol). Another exemplary composition comprises 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer, pH 6.0.
(2) Polar Aprotic Solvent(s)
Different polar aprotic solvents may impart different properties on the compositions of the invention. For example, the choice of polar aprotic solvent may contribute to the stability of the composition, since certain polar aprotic solvents may degrade over time. For example, the polar aprotic solvent ethylene carbonate breaks down into ethylene glycol, which is a relatively stable molecule, and carbon dioxide, which can interact with water to form carbonic acid, altering the acidity of the compositions of the invention. Without being bound by theory, it is believed that the change in pH upon breakdown of ethylene carbonate and DNA damage from long storage makes the compositions of the invention less effective for hybridization. However, stability can be improved by reducing the pH of the composition, by adding citric acid as a buffer at pH 6.2 instead of the traditional phosphate buffer, which is typically used at about pH 7.4, and/or by adding ethylene glycol at concentrations, e.g., between 0.1% to 10%, or between 0.5% to 5%, such as, for example, 1%, 2%, 3%, etc. For example, with 10 mM citrate buffer, the compositions of the invention are stable at 2-8° C. for approximately 8 months. Stability can also be improved if the compositions are stored at low temperatures (e.g., −20° C.).
In addition, certain polar aprotic solvents may cause the compositions of the invention to separate into multi-phase systems under certain conditions. The conditions under which multi-phase systems are obtained may be different for different polar aprotic solvents. Generally, however, as the concentration of polar aprotic solvent increases, the number of phases increases. For example, compositions comprising low concentrations ethylene carbonate (i.e., less than 20%) may exist as one phase, while compositions comprising higher concentrations of ethylene carbonate may separate into two, or even three phases. For instance, compositions comprising 15% ethylene carbonate exist as a single phase at room temperature, while compositions comprising 40% ethylene carbonate consist of a viscous lower phase (approximately 25% of the total volume) and a less viscous upper phase (approximately 75% of the total volume) at room temperature.
On the other hand, some polar aprotic solvents may exist in two phases at room temperature even at low concentrations. For example, sulfolane, γ-butyrolactone, ethylene trithiocarbonate, glycol sulfite, and propylene carbonate exist as two phases at concentrations of 10, 15, 20, or 25% (20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer) at room temperature.
It may also be possible to alter the number of phases by adjusting the temperature of the compositions of the invention. Generally, as temperature increases, the number of phases decreases. For example, at 2-8° C., compositions comprising 40% ethylene carbonate may separate into a three-phase system.
It may also be possible to alter the number of phases by adjusting the concentration of dextran sulfate and/or salt in the composition. Generally speaking, lowering the dextran sulfate concentration (traditional concentration is 10%) and/or salt concentration may reduce the number of phases. However, depending on the particular polar aprotic solvent and its concentration in the composition, single phases may be produced even with higher concentrations of salt and dextran sulfate. For example, a composition comprising low amounts of EC (e.g., 15%, 10%, or 5%) can work well by increasing the dextran sulfate and salt concentrations, while still keeping a one phase system. In a particular embodiment, compositions comprising a HER2 gene DNA probe, a CEN7 PNA probe, 15% EC, 20% dextran sulfate, 600 mM NaCl, and 10 mM phosphate buffer are frozen at −20° C. In other embodiments, the compositions are liquid at −20° C.
Some polar aprotic solvents may produce stronger signals in one phase or another. For example, 40% glycol sulfite produces strong signals in the lower phase and no signals in the upper phase. Similarly, certain types of probes may produce stronger signals in one phase or another. For example, PNA probes tend to show stronger signals in the lower phase than the upper phase.
Accordingly, the multiphase systems of the invention may be used to conveniently examine different aspects of a sample. For example, a two-phase system could be used to separate samples labeled with PNA probes from samples labeled with DNA probes. Other uses include isolation of a specific phase exhibiting, e.g., certain advantages such that the isolated phase can be used as a single phase system. The probe and/or sample may be added prior to, or after isolation of a particular phase.
Hybridization applications may be performed with a one-phase composition of the invention, with individual phases of the multiphase compositions of the invention, or with mixtures of any one or more of the phases in a multiphase composition of the invention. For example, in a one phase system, a volume of the sample may be extracted for use in the hybridization. In a multiphase system, one may extract a volume of sample from the phase of interest (e.g., the upper, lower, or middle phase) to use in the hybridization. Alternatively, the phases in a multiphase system may be mixed prior to extracting a volume of the mixed sample for use in the hybridization. However, the multiphase system may yield strong and uneven local background staining depending on the composition. While, the addition of low amounts of formamide will reduce background in a one phase system, it has little effect on a multiphase system with high concentrations (e.g., 40%) of a polar aprotic solvent. In addition, as the concentration of formamide increases, higher concentrations of probe and/or longer hybridization times are required to maintain strong signal intensity.
(3) Optimization for Particular Applications
The compositions of the invention can be varied in order to optimize results for a particular application. For example, the concentration of polar aprotic solvent, salt, accelerating agent, blocking agent, and/or hydrogen ions (i.e. pH) may be varied in order to improve results for a particular application.
For example, the concentration of polar aprotic solvent may be varied in order to improve signal intensity and background staining. Generally, as the concentration of polar aprotic solvent increases, signal intensity increases and background staining decreases. For example, compositions comprising 15% EC tend to show stronger signals and less background than compositions comprising 5% EC. However, signal intensity may be improved for compositions having low concentrations of polar aprotic solvent (e.g., 0% to 20%) if the concentrations of salt and/or dextran sulfate are increased. For example, strong signals may be observed with 5% to 10% EC when the salt concentration is raised approximately 8 to 16 times traditional salt concentrations (i.e., approximately 1200 mM NaCl, 20 mM phosphate buffer). Likewise, as lower concentrations of polar aprotic solvent are used, higher concentrations of dextran sulfate are generally required to maintain good signal and background intensity.
Accordingly, the concentrations of salt and dextran sulfate may also be varied in order to improve signal intensity and background staining. Generally, as the concentrations of salt and dextran sulfate increase, the signal intensity increases and background decreases. For example, salt concentrations that are approximately two to four times traditional concentrations (i.e., 300 mM NaCl 5 mM phosphate buffer) produce strong signals and low background. Surprisingly, however, hybridization occurs using the compositions of the invention even in the complete absence of salt. Signal intensities can be improved under no-salt conditions by increasing the concentrations of accelerating agent and/or polar aprotic solvent.
Likewise, signal intensity increases as dextran sulfate concentration increases from 0% to 20%. However, good signals may even be observed at dextran sulfate concentrations of 0%. Signal intensity may be improved under low dextran sulfate conditions by increasing the polar aprotic solvent and/or salt concentrations.
In addition, the types probes used in the compositions of the invention may be varied to improve results. For example, in some aspects of the invention, combinations of DNA/DNA probes may show less background than combinations of DNA/PNA probes in the compositions of the invention or vice versa. On the other hand, PNA probes tend to show stronger signals than DNA probes under low salt and/or low polar aprotic solvent concentrations. In fact, PNA probes also show signals when no polar aprotic solvent is present, whereas DNA probes show weak or no signals without polar aprotic solvent.
D. Applications, Methods, and Uses
(1) Analytical Samples
The methods and compositions of the invention may be used fully or partly in all types of RNA hybridization applications in the fields of cytology, histology, or molecular biology. According to one embodiment, the RNA sequence in the methods of the invention is present in a biological sample. Examples of such samples include, e.g., tissue samples, cell preparations, cell fragment preparations, and isolated or enriched cell component preparations. The sample may originate from various tissues such as, e.g., breast, lung, colorectal, prostate, lung, head & neck, stomach, pancreas, esophagus, liver, and bladder, or other relevant tissues and neoplasia thereof, any cell suspension, blood sample, fine needle aspiration, ascites fluid, sputum, peritoneum wash, lung wash, urine, feces, cell scrape, cell smear, cytospin or cytoprep cells.
The sample may be isolated and processed using standard protocols. Cell fragment preparations may, e.g., be obtained by cell homogenizing, freeze-thaw treatment or cell lysing. The isolated sample may be treated in many different ways depending of the purpose of obtaining the sample and depending on the routine at the site. Often the sample is treated with various reagents to preserve the tissue for later sample analysis, alternatively the sample may be analyzed directly. Examples of widely used methods for preserving samples are formalin-fixed followed by paraffin-embedding and cryo-preservation.
For metaphase spreads, cell cultures are generally treated with colcemid, or another suitable spindle pole disrupting agent, to stop the cell cycle in metaphase. The cells are then fixed and spotted onto microscope slides, treated with formaldehyde, washed, and dehydrated in ethanol. Probes are then added and the samples are analyzed by any of the techniques discussed below.
Cytology involves the examination of individual cells and/or nucleic acid spreads from a biological sample. Cytological examination of a sample begins with obtaining a specimen of cells, which can typically be done by scraping, swabbing or brushing an area, as in the case of cervical specimens, or by collecting body fluids, such as those obtained from the chest cavity, bladder, or spinal column, or by fine needle aspiration or fine needle biopsy, as in the case of internal tumors. In a conventional manual cytological preparation, the sample is transferred to a liquid suspending material and the cells in the fluid are then transferred directly or by centrifugation-based processing steps onto a glass microscope slide for viewing. In a typical automated cytological preparation, a filter assembly is placed in the liquid suspension and the filter assembly both disperses the cells and captures the cells on the filter. The filter is then removed and placed in contact with a microscope slide. The cells are then fixed on the microscope slide before analysis by any of the techniques discussed below.
In a traditional DNA hybridization experiment using a cytological sample, slides containing the specimen are immersed in a formaldehyde buffer, washed, and then dehydrated in ethanol. The probes are then added and the specimen is covered with a coverslip. The slide is optionally incubated at a temperature sufficient to denature any double-stranded nucleic acid in the specimen (e.g., 5 minutes at 82° C.) and then incubated at a temperature sufficient to allow hybridization (e.g., overnight at 45° C.). After hybridization, the coverslips are removed and the specimens are subjected to a high-stringency wash (e.g., 10 minutes at 65° C.) followed by a series of low-stringency washes (e.g., 2×3 minutes at room temperature). The samples are then dehydrated and mounted for analysis.
In a traditional RNA hybridization experiment using cytological samples, cells are equilibrated in 40% formamide, 1×SSC, and 10 mM sodium phosphate for 5 min, incubated at 37° C. overnight in hybridization reactions containing 20 ng of oligonucleotide probe (e.g mix of labeled 50 bp oligos), 1×SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20 mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. coli tRNA (10 mg/ml), and 10 mM sodium phosphate. Then washed twice with 4×SSC/40% formamide and again twice with 2×SSC/40% formamide, both at 37° C., and then with 2×SSC three times at room temperature. Digoxigenin-labeled probes can then e.g. be detected by using a monoclonal antibody to digoxigenin conjugated to Cy3. Biotin-labeled probes can then e.g. be detected by using streptavidin-Cy5. Detection can be by fluorescence or CISH.
Histology involves the examination of cells in thin slices of tissue. To prepare a tissue sample for histological examination, pieces of the tissue are fixed in a suitable fixative, typically an aldehyde such as formaldehyde or glutaraldehyde, and then embedded in melted paraffin wax. The wax block containing the tissue sample is then cut on a microtome to yield thin slices of paraffin containing the tissue, typically from 2 to 10 microns thick. The specimen slice is then applied to a microscope slide, air dried, and heated to cause the specimen to adhere to the glass slide. Residual paraffin is then dissolved with a suitable solvent, typically xylene, toluene, or others. These so-called deparaffinizing solvents are then removed with a washing-dehydrating type reagent prior to analysis of the sample by any of the techniques discussed below. Alternatively, slices may be prepared from frozen specimens, fixed briefly in 10% formalin or other suitable fixative, and then infused with dehydrating reagent prior to analysis of the sample.
In a traditional DNA hybridization experiment using a histological sample, formalin-fixed paraffin embedded tissue specimens are cut into sections of 2-6 μm and collected on slides. The paraffin is melted (e.g., 30-60 minutes at 60° C.) and then removed (deparaffinated) by washing with xylene (or a xylene substitute), e.g., 2×5 minutes. The samples are rehydrated, washed, and then pre-treated (e.g., 10 minutes at 95-100° C.). The slides are washed and then treated with pepsin or another suitable permeabilizer, e.g., 3-15 minutes at 37° C. The slides are washed (e.g., 2×3 minutes), dehydrated, and probe is applied. The specimens are covered with a coverslip and the slide is optionally incubated at a temperature sufficient to denature any double-stranded nucleic acid in the specimen (e.g. 5 minutes at 82° C.), followed by incubation at a temperature sufficient to allow hybridization (e.g., overnight at 45° C.). After hybridization, the coverslips are removed and the specimens are subjected to a high-stringency wash (e.g., 10 minutes at 65° C.) followed by a series of low-stringency washes (e.g., 2×3 minutes at room temperature). The samples are then dehydrated and mounted for analysis.
In a traditional RNA hybridization experiment using a histological sample, slides with FFPE tissue sections are deparaffinized in xylene for 2×5 min, immerged in 99% ethanol 2×3 min, in 96% ethanol 2×3 min, and then in pure water for 3 min. Slides are placed in a humidity chamber, Proteinase K is added, and slides are incubated at RT for 5 min-15 min. Slides are immersed in pure water for 2×3 min, immersed in 96% ethanol for 10 sec, and air-dried for 5 min. Probes are added to the tissue section and covered with coverslip. The slides are incubated at 55° C. in humidity chamber for 90 min. After incubation, the slides are immersed in a Stringent Wash solution at 55° C. for 25 min, and then immersed in TBS for 10 sec. The slides are incubated in a humidity chamber with antibody for 30 min. The slides are immersed in TBS for 2×3 min, then in pure water for 2×1 min, and then placed in a humidity chamber. The slides are then incubated with substrate for 60 min, and immersed in tap water for 5 min.
In a traditional northern blot procedure, the RNA target sample is denatured for 10 minutes at 65° C. in RNA loading buffer and immediately placed on ice. The gels are loaded and electrophoresed with 1×MOPS buffer (10×MOPS contains 200 mM morpholinopropansulfonic acid, 50 mM sodium acetate, 10 mM EDTA, pH 7.0) at 25 V overnight. The gel is then pre-equilibrated in 20×SSC for 10 min and the RNA is transferred to a nylon membrane using sterile 20×SSC as transfer buffer. The nucleic acids are then fixed on the membrane using, for example, UV-cross linking at 120 mJ or baking for 30 min at 120° C. The membrane is then washed in water and air dried. The membrane is placed in a sealable plastic bag and prehybridized without probe for 30 min at 68° C. The probe is denatured for 5 min at 100° C. and immediately placed on ice. Hybridization buffer (prewarmed to 68° C.) is added and the probe is hybridized at 68° C. overnight. The membrane is then removed from the bag and washed twice for 5 min each with shaking in a low stringency wash buffer (e.g., 2×SSC, 0.1% SDS) at room temperature. The membrane is then washed twice for 15 min each in prewarmed high stringency wash buffer (e.g., 0.1×SSC, 0.1% SDS) at 68° C. The membrane may then be stored or immediately developed for detection.
Additional examples of traditional hybridization techniques can be found, for example, in Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, (1989) at sections 1.90-1.104, 2.108-2.117, 4.40-4.41, 7.37-7.57, 8.46-10.38, 11.7-11.8, 11.12-11.19, 11.38, and 11.45-11.57; and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1998) at sections 2.9.1-2.9.6, 2.10.4-2.10.5, 2.10.11-2.10.16, 4.6.5-4.6.9, 4.7.2-4.7.3, 4.9.7-4.9.15, 5.9.18, 6.2-6.5, 6.3, 6.4, 6.3.3-6.4.9, 5.9.12-5.9.13, 7.0.9, 8.1.3, 14.3.1-14.3.4, 14.9, 15.0.3-15.0.4, 15.1.1-15.1.8, and 20.1.24-20.1.25.
(2) Hybridization Techniques
The compositions and methods of the present invention can be used fully or partly in all types of RNA hybridization techniques known in the art for cytological and histological samples. Such techniques include, for example, in situ hybridization (ISH), fluorescent in situ hybridization (FISH; including multi-color FISH, Fiber-FISH, etc.), chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH), and arrays. The compositions of the invention will improve the efficiency of traditional RNA hybridization applications, e.g., by reducing the denaturation and hybridization temperatures and/or the time.
Molecular probes that are suitable for use in the hybridizations of the invention are described, e.g., in U.S. Patent Publication No. 2005/0266459, which is incorporated herein by reference. In general, probes may be prepared by chemical synthesis, PCR, or by amplifying a specific DNA sequence by cloning, inserting the DNA into a vector, and amplifying the vector an insert in appropriate host cells. Commonly used vectors include bacterial plasmids, cosmids, bacterial artificial chromosomes (BACs), PI diverted artificial chromosomes (PACs), or yeast artificial chromosomes (YACs). The amplified DNA is then extracted and purified for use as a probe. Methods for preparing and/or synthesizing probes are known in the art, e.g., as disclosed in PCT/US02/30573.
The nucleic acid probe may be a double or single stranded nucleic acid fragment or sequence, such as a DNA, RNA, or analogs such as PNA or LNA. The probes may be labeled to make identification of the probe-target hybrid possible by use, for example, of a fluorescence or bright field microscope/scanner. In some embodiments, the probe may be labeled using radioactive labels such as 31P, 33P, or 32S, non-radioactive labels such as digoxigenin and biotin, or fluorescent labels
In general, the type of probe determines the type of feature one may detect in a hybridization assay. For example, large insert probes may be used to target unique single-copy sequences. With these large probes, the hybridization efficiency is inversely proportional to the probe size. Smaller probes can be used to detect aberrations such as deletions, amplifications, inversions, duplications, and aneuploidy. For example, differently-colored locus-specific probes can be used to detect translocations via split-signal in situ hybridization.
In general, the ability to discriminate between closely related sequences is inversely proportional to the length of the hybridization probe because the difference in thermal stability decreases between wild type and mutant complexes as probe length increases. Probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique organism or clinical condition of interest. On the other hand, sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences.
In one embodiment, at least one set of the hybridization probes may comprise one or more PNA probes, as defined above and as described in U.S. Pat. No. 7,105,294, which is incorporated herein by reference. Methods for synthesizing PNA probes are described in PCT/US02/30573. Alternatively, or in addition, at least one set of the hybridization probes in any of the techniques discussed above may comprise one or more locked nucleic acid (LNA) probes, as described in WO 99/14226, which is incorporated herein by reference. Due to the additional bridging bond between the 2′ and 4′ carbons, the LNA backbone is pre-organized for hybridization. LNA/RNA interactions are stronger than the corresponding DNA/RNA interactions, as indicated by a higher melting temperature. Thus, the compositions and methods of the invention, which decrease the energy required for hybridization, are particularly useful for hybridizations with LNA probes.
In one embodiment, the probes may comprise a detectable label (a molecule that provides an analytically identifiable signal that allows the detection of the probe-target hybrid), as described in U.S. Patent Publication No. 2005/0266459, which is incorporated herein by reference. The detectable label may be directly attached to a probe, or indirectly attached to a probe, e.g., by using a linker. Any labeling method known to those in the art, including enzymatic and chemical processes, can be used for labeling probes used in the methods and compositions of the invention. In other embodiments, the probes are not labeled.
In general, in situ hybridization techniques such as FISH, CISH, and SISH for DNA detection, employ large, mainly unspecified, nucleic acid probes that hybridize with varying stringency. Using large probes renders the in situ hybridization technique very sensitive. However, the successful use of large probes in traditional hybridization assays depends on blocking the undesired background staining derived from, e.g., repetitive sequences that are present throughout the genome. Traditional methods for decreasing nonspecific probe binding include saturating the binding sites on proteins and tissue by incubating tissue with prehybridization solutions containing ficoll, bovine serum albumin (BSA), polyvinyl pyrrolidone, and nucleic acids. Such blocking steps are time-consuming and expensive. As discussed below, the methods and compositions of the invention advantageously reduce and/or eliminate the need for such blocking steps and blocking reagents. However, in one embodiment, repetitive sequences may be suppressed according to the methods known in the art, e.g., as disclosed in PCT/US02/30573.
Bound probes may be detected in cytological and histological samples either directly or indirectly with fluorochromes (e.g., FISH), organic chromogens (e.g., CISH), silver particles (e.g., SISH), or other metallic particles (e.g., gold-facilitated fluorescence in situ hybridization, GOLDFISH). Thus, depending on the method of detection, populations of cells obtained from a sample to be tested may be visualized via fluorescence microscopy or conventional brightfield light microscopy.
Hybridization assays on cytological and histological samples are important tools for determining the number, size, and/or location of specific RNA sequences.
FISH is typically used when multiple color imaging is required and/or when the protocol calls for quantification of signals. The technique generally entails preparing a cytological sample, labeling probes, optionally denaturing the target and the probe, hybridizing the probe to the target sequence, and detecting the signal. Typically, the hybridization reaction fluorescently stains the targeted sequences so that their location, size, or number can be determined using fluorescence microscopy, flow cytometry, or other suitable instrumentation. RNA sequences ranging from megabases down to several kilobases can be studied using FISH. With enhanced fluorescence microscope techniques, such as, for example, deconvolution, even a single mRNA molecule can be detected. FISH may also be used on metaphase spreads and interphase nuclei.
One of the most important applications for FISH has been in detecting single-copy sequences, in particular disease related sequences in humans and other eukaryotic model species, and the detection of infections agents. FISH may be used to detect, e.g., chromosomal aneuploidy in prenatal diagnoses, hematological cancers, and solid tumors; gene abnormalities such as oncogene amplifications, gene deletions, or gene fusions; translocations, duplications, insertions, or inversions; contiguous gene syndromes such as microdeletion syndrome; the genetic effects of various therapies; viral nucleic acids in somatic cells and viral integration sites in chromosomes; etc. FISH techniques include multiplex FISH (m-FISH), spectral karyotyping (SKY), combined binary ration labeling (COBRA), color-changing karyotyping, cross-species color banding, high resolution multicolor banding, telomeric multiplex FISH (TM-FISH), split-signal FISH (ssFISH), and fusion-signal FISH.
CISH and SISH may be used for many of the same applications as FISH, and have the additional advantage of allowing for analysis of the underlying tissue morphology, for example in histopathology applications. If FISH is performed, the hybridization mixture may contain sets of distinct and balanced pairs of probes, as described in U.S. Pat. No. 6,730,474, which is incorporated herein by reference. For CISH, the hybridization mixture may contain at least one set of probes configured for detection with one or more conventional organic chromogens, and for SISH, the hybridization mixture may contain at least one set of probes configured for detection with silver particles, as described in Powell R D et al., “Metallographic in situ hybridization,” Hum. Pathol., 38:1145-59 (2007).
The compositions of the invention may also be used fully or partly in all types of molecular biology techniques involving hybridization, including blotting and probing (e.g., northern blotting, etc.), and arrays.
(3) Hybridization Conditions
The method of the present invention involves the use of polar aprotic solvents in RNA hybridization applications. The compositions of the present invention are particularly useful in said method.
Hybridization methods using the compositions of the invention may involve applying the compositions to a sample comprising a target RNA sequence. The polar aprotic solvent will interact with the probe and the target and facilitate the annealing of the probe to the target. The polar aprotic solvent will also facilitate the denaturation of any double stranded sequences, if necessary, to secure access for the probe to hybridize with the target sequence. The polar aprotic solvents specified in the present invention speed up the hybridization process considerably and reduce the harshness and toxicity of the hybridization conditions compared to traditional formamide-containing buffers.
Hybridizations using the compositions of the invention may be performed using the same assay methodology as for RNA hybridizations performed with traditional compositions. For example, the heat pre-treatment, digestion, denaturation, hybridization, washing, and mounting steps may use the same conditions in terms of volumes, temperatures, reagents and incubation times as for traditional compositions. However, the compositions of the invention allow for shorter hybridization times.
A great variation exists in the traditional RNA hybridization protocols known in the art. The compositions of the invention may be used in any of the traditional hybridization protocols known in the art.
Alternatively, assays using the compositions of the invention can be changed and optimized from traditional methodologies, for example, by decreasing the hybridization time, increasing or decreasing the hybridization temperatures, and/or increasing or decreasing the hybridization volumes.
For example, in some embodiments, the compositions of the invention will produce strong signals when the hybridization temperature is from 20 to 60° C. In other embodiments, the compositions of the invention will produce strong signals when the hybridization temperature is from 20 to 30° C., 30 to 40° C., 40 to 50° C., or 50 to 60° C. In other embodiments, the compositions of the invention will produce strong signals when the hybridization temperature is 37, 40, 45, or 50° C.
In other embodiments, the compositions of the invention will produce strong signals when the hybridization time is from 0 minutes to 24 hours. In other embodiments, the compositions of the invention will produce strong signals when the hybridization time is from 0 minute to 8 hours. In other embodiments, the compositions of the invention will produce strong signals when the hybridization time is 0 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 180 minutes, or 240 minutes.
Accordingly, hybridizations using the compositions of the invention may be performed in less than 8 hours. In other embodiments, the hybridization step is performed in less than 6 hours. In still other embodiments, the hybridization step is performed within 4 hours. In other embodiments, the hybridization step is performed within 3 hours. In yet other embodiments, the hybridization step is performed within 2 hours. In other embodiments, the hybridization step is performed within 1 hour. In still other embodiments, the hybridization step is performed within 30 minutes. In other embodiments, the hybridization step can take place within 15 minutes. The hybridization step can even take place within 10 minutes or in less than 5 minutes. FIGS. 1 and 2 illustrate a typical time-course for DNA hybridization applications performed on histological and cytological samples, respectively, using the compositions of the invention compared to hybridization applications using a traditional compositions.
If the hybridization applications of the invention include an optional denaturation step, the denaturation temperature may be from 60 to 100° C. In other embodiments, the compositions of the invention will produce strong signals when the denaturation temperature is from 60 to 70° C., 70 to 80° C., 80 to 90° C. or 90 to 100° C. In other embodiments, the compositions of the invention will produce strong signals when the denaturation temperature is 72, 82, or 92° C. The denaturation time may be from 0 to 10 minutes. In other embodiments, the compositions of the invention will produce strong signals when the denaturation time is from 0 to 5 minutes. In other embodiments, the compositions of the invention will produce strong signals when the denaturation time is 0, 1, 2, 3, 4, or 5 minutes. It will be understood by those skilled in the art that in most cases, RNA detection does not require a denaturation step.
The concentration of probe may also be varied in order to produce strong signals and/or reduce background. For example, as hybridization time decreases, the amount of probe may be increased in order to improve signal intensity. On the other hand, as hybridization time decreases, the amount of probe may be decreased in order to improve background staining.
The compositions of the invention also eliminate the need for a blocking step during hybridization applications by improving signal and background intensity by blocking the binding of, e.g., repetitive sequences to the target DNA. Thus, there is no need to use total human DNA, blocking-PNA, COT-1 DNA, or DNA from any other source as a blocking agent. However, background levels can be further reduced by adding agents that reduce non-specific binding, such as to the cell membrane, such as small amounts of total human DNA or non-human-origin DNA (e.g., salmon sperm DNA) to a hybridization reaction using the compositions of the invention.
The aqueous compositions of the invention furthermore provide for the possibility to considerably reduce the concentration of nucleic acid sequences included in the composition. Generally, the concentration of probes may be reduced from 2 to 8-fold compared to traditional concentrations. For example, if HER2 DNA probes and CEN17 PNA probes are used in the compositions of the invention, their concentrations may be reduced by 1⁄4 and 1⁄2, respectively, compared to their concentrations in traditional hybridization compositions. This feature, along with the absence of any requirement for blocking DNA, such as blocking-PNA or COT1, allows for an increased probe volume in automated instrument systems compared to the traditional 10 μL volume used in traditional compositions systems, which reduces loss due to evaporation, as discussed in more detail below.
Reducing probe concentration also reduces background. However, reducing the probe concentration is inversely related to the hybridization time, i.e., the lower the concentration, the higher hybridization time required. Nevertheless, even when extremely low concentrations of probe are used with the aqueous compositions of the invention, the hybridization time is still shorter than with traditional compositions.
The compositions of the invention often allow for better signal-to-noise ratios than traditional hybridization compositions. For example, with certain probes, a one hour hybridization with the compositions of the invention will produce similar background and stronger signals than an overnight hybridization in a traditional compositions. Background is not seen when no probe is added.
Traditional assay methods may also be changed and optimized when using the compositions of the invention depending on whether the system is manual, semi-automated, or automated. For example, a semi-automated or a fully automated system will benefit from the short hybridization times obtained with the compositions of the invention. The short hybridization time may reduce the difficulties encountered when traditional compositions are used in such systems. For example, one problem with semi-automated and fully automated systems is that significant evaporation of the sample can occur during hybridization, since such systems require small sample volumes (e.g., 10-150 μL), elevated temperatures, and extended hybridization times (e.g., 14 hours). Thus, proportions of the components in traditional hybridization compositions are fairly invariable. However, since the compositions of the invention allow for faster hybridizations, evaporation is reduced, allowing for increased flexibility in the proportions of the components in hybridization compositions used in semi- and automated systems.
For example, two automated instruments have been used to perform hybridizations to DNA targets using the compositions of the invention. Compositions comprising 40% ethylene carbonate (v/v) have been used in the apparatus disclosed in PCT application DK2008/000430, and compositions comprising 15% ethylene carbonate (v/v) have been used in the HYBRIMASTER HS-300 (Aloka CO. LTD, Japan). When the compositions of the invention are used in the HYBRIMASTER HS-300, the instrument can perform rapid FISH hybridization with water in place of the traditional toxic formamide mix, thus improving safety and reducing evaporation. If water wetted strips are attached to the lid of the inner part of the Aloka instrument's reaction unit (hybridization chamber), e.g., as described in U.S. patent application Ser. No. 11/031,514, which is incorporated herein by reference, evaporation is reduced even further.
Another problem with automated imaging analysis is the number of images needed, the huge amount of storage place required, and the time required to take the images. The compositions of the invention address this problem by producing very strong signals compared to traditional compositions. Because of the very strong signals produced by the compositions of the invention, the imaging can be done at lower magnification than required for traditional compositions and can still be detected and analyzed, e.g., by algorithms. Since the focal plane becomes wider with lower magnification, the compositions of the invention reduce or eliminate the requirement to take serial sections of a sample. As a result, the overall imaging is much faster, since the compositions of the invention require fewer or no serial sections and each image covers much greater area. In addition, the overall time for analysis is faster, since the total image files are much smaller.
Thus, the compositions and methods of the invention solve many of the problems associated with traditional hybridization compositions and methods.
The disclosure may be understood more clearly with the aid of the non-limiting examples that follow, which constitute preferred embodiments of the compositions according to the disclosure. Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope are approximations, the numerical values set forth in the specific example are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements. The examples that follow illustrate the present invention and should not in any way be considered as limiting the invention.
EXAMPLES
Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with these embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.
The reagents used in the following examples are from Dako's Histology FISH Accessory Kit (K5599) and Cytology FISH Accessory Kit (K5499) (Dako Denmark A/S, Glostrup Denmark). The kits contain all the key reagents, except for probe, required to complete a FISH procedure for formalin-fixed, paraffin-embedded tissue section specimens. All samples were prepared according to the manufacturer's description. The Dako Hybridizer (S2451, Dako) was used for the digestion, denaturation, and hybridization steps.
Evaluation of FISH slides was performed within a week after hybridization using a Leica DM6000B fluorescence microscope, equipped with DAN, FITC, Texas Red single filters and FITC/Texas Red double filter under 10×, 20×, 40×, and 100× oil objective.
Evaluation of CISH slides was performed using an Olympus BX51 light microscope, under 4×, 10×, 20×, 40×, and 60× objective.
In the Examples that follow, “dextran sulfate” refers to the sodium salt of dextran sulfate (D8906, Sigma) having a molecular weight Mw>500,000. All concentrations of polar aprotic solvents are provided as v/v percentages. Phosphate buffer refers to a phosphate buffered solution containing NaH2PO4, 2H2O (sodium phosphate dibasic dihydrate) and Na2HPO4, H2O (sodium phosphate monobasic monohydrate). Citrate buffer refers to a citrate buffered solution containing sodium citrate (Na3C6H5O7, 2H2O; 1.06448, Merck) and citric acid monohydrate (C6H8O7, H2O; 1.00244, Merck).
General histology FISH/CISH Procedure for Examples 1-20
The slides with cut formalin-fixed paraffin embedded (FFPE) multiple tissue array sections from humans (tonsils, mammacarcinoma, kidney and colon) were baked at 60° C. for 30-60 min, deparaffinated in xylene baths, rehydrated in ethanol baths and then transferred to Wash Buffer. The samples were then pre-treated in Pre-Treatment Solution at a minimum of 95° C. for 10 min and washed 2×3 min. The samples were then digested with Pepsin RTU at 37° C. for 3 min, washed 2×3 min, dehydrated in a series of ethanol evaporations, and air-dried. The samples were then incubated with 10 μL FISH probe as described under the individual experiments. The samples were then washed by Stringency Wash at 65° C. 10 min, then washed 2×3 min, then dehydrated in a series of ethanol evaporations, and air-dried. Finally, the slides were mounted with 15 μL Antifade Mounting Medium. When the staining was completed, observers trained to assess signal intensity, morphology, and background of the stained slides performed the scoring.
General Cytology FISH Procedure for Examples 21-22
Slides with metaphases preparation were fixed in 3.7% formaldehyde for 2 min, washed 2×5 min, dehydrated in a series of ethanol evaporations, and air-dried. The samples were then incubated with 10 μL FISH probe as described under the individual experiments. The samples were then washed by Stringency Wash at 65° C. 10 min, then washed 2×3 min, then dehydrated in a series of ethanol evaporations, and air-dried. Finally, the slides were mounted with 15 μL Antifade Mounting Medium. When the staining was completed, observers trained to assess signal intensity and background of the stained slides performed the scoring as described in the scoring for guidelines for tissue sections.
Histology CISH Procedure for Example 23
The reagents used in Example 23 are from Dako's PNA ISH Detection Kit for fluorescein-labeled PNA probes (K5201) (Dako Denmark A/S, Glostrup Denmark). The kit contains all reagents necessary for performing an in situ hybridization, except for the specific probe. All samples were prepared according to the manufacturer's description. The Dako Hybridizer (S2451, Dako) was used as indicated.
Slides with tissue sections were immersed in xylene for 2×5 min, then in 99% ethanol for 2×3 min, then in 96% ethanol for 2×3 min, and then in pure water for 3 min. The slides were then placed in a humidity chamber and 200 μL Proteinase K was added. The samples that were designated to receive PNA probes were incubated at RT for 5 min, and the samples that were designated to receive LNA probes were incubated at RT for 15 min. The slides were then immersed in pure water for 2×3 min, immersed in 96% ethanol for 10 sec, and air-dried for 5 min. Fluorescein-conjugated PNA or LNA probe (85 μL) was added to the tissue section and the slides were covered with coverslip. The samples with PNA probe were incubated at 55° C., and the samples with LNA probe were incubated at 42° C. (Hybridizer, S2451). After incubation, the coverslips were removed, the slides were immersed in a Stringent Wash solution at 55° C. (for PNA probes) or 30° C. (for LNA probes) for 25 min, and then immersed in TBS for 10 sec. The slides were transferred to a humidity chamber and five to six drops of the Anti-FITC/AP antibody was added. The slides were incubated for 30 min., the antibody was tapped off, the slides were immersed in TBS for 2×3 min, then in pure water for 2×1 min, and then placed in a humidity chamber. Five to six drops of the BCIP/NBT/levamisole chromogenic substrate was added to the sample, the slides were incubated for 60 min, the substrate was tapped off, and the slides were immersed in tap water for 5 min. The slides were then counterstained with Dako Nuclear Fast Red (S1963) for 1 min and mounted in Tissue-Mount (Sakura, The Netherlands).
Histology CISH Procedure for Example 24 and 25
The reagents used in Example 24 are from Dako's PNA ISH Detection Kit for fluorescein-labeled PNA probes (K5201) (Dako Denmark A/S, Glostrup Denmark). The kit contains all reagents necessary for performing an in situ hybridization, except for the specific probe. All samples were prepared according to the manufacturer's description. The Dako Hybridizer (S2451, Dako) was used as indicated.
Slides with tissue sections were immersed in xylene for 2×5 min, then in 99% ethanol for 2×3 min, then in 96% ethanol for 2×3 min, and then in RNase-free water for 3 min. The slides were then placed in a humidity chamber and 200 μL Proteinase K was added, and the slides were incubated at RT for 5 min. The slides were then immersed in RNase-free water for 2×3 min, immersed in 96% ethanol for 10 sec, and air-dried for 5 min. Fluorescein-conjugated PNA or LNA probe (50 μL) was added to the tissue section and the slides were covered with a coverslip and sealed with Coverslip Sealant (K5999, Dako). The samples were incubated at 35° C. or 45° C. in a Hybridizer (S2451, Dako) for 90 min. After incubation, the coverslips were removed, the slides were immersed in a Stringent Wash solution at 35° C. or 45° C. for 25 min, and then immersed in TBS. The slides were transferred to a Dako Autostainer (S3400, Dako), and the staining was performed using two drop zones of 2×200 μL with the following staining protocol: Rinse TBS, Anti-FITC/AP antibody incubation for 30 min, 2× rinse in TBS, 2× rinse in pure water, BCIP/NBT substrate for 30 min, rinse in TBS, BCIP/NBT substrate for 30 min, rinse in pure water, Nuclear Fast Red (S1963) 1 min, and 6× rinse in pure water. The slides were then removed from the Dako Autostainer and mounted in Tissue-Mount (Sakura, The Netherlands).
Scoring Guidelines of Tissue Sections
The signal/staining intensities were evaluated on a 0-3 scale with 0 meaning no signal and 3 equating to a strong signal. The cell/tissue structures are evaluated on a 0-3 scale with 0 meaning no structure and no nuclei boundaries and 3 equating to intact structure and clear nuclei boundaries. Between 0 and 3 there are additional grades 0.5 apart from which the observer can assess signal intensity, tissue structure, and background.
The signal/staining intensity is scored after a graded system on a 0-3 scale.

    
    
        0 No signal/staining is seen.
        1 The signal/staining intensity is weak.
        2 The signal/staining intensity is moderate.
        3 The signal/staining intensity is strong.
    
    


The scoring system allows the use of 1⁄2 grades.
The tissue and nuclear structure is scored after a graded system on a 0-3 scale.

    
    
        0 The tissue structures and nuclear borders are completely destroyed.
        1 The tissue structures and/or nuclear borders are poor. This grade includes situations where some areas have empty nuclei.
        2 Tissue structures and/or nuclear borders are seen, but the nuclear borders are unclear. This grade includes situations where a few nuclei are empty.
        3 Tissue structures and nuclear borders are intact and clear.
    
    


The scoring system allows the use of 1⁄2 grades.
The background is scored after a graded system on a 0-3 scale.

    
    
        0 Little to no background is seen.
        1 Some background.
        2 Moderate background.
        3 High Background.
    
    


The scoring system allows the use of 1⁄2 grades.
Example 1
This example compares the signal intensity and cell morphology from samples treated with the compositions of the invention or traditional hybridization solutions as a function of denaturation temperature.
FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide (15515-026, Invitrogen), 5 μM blocking PNAs (see Kirsten Yang Nielsen et al., PNA Suppression Method Combined with Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10 (Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006)), 10 ng/μL Texas Red labeled CCND1 gene DNA probe (RP11-1143E20, size 192 kb).
FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate (03519, Fluka), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe (RP11-1143E20, size 192 kb).
Phases of different viscosity, if present, were mixed before use. The FISH probes were denatured as indicated for 5 min and hybridized at 45° C. for 60 minutes.
Results:













Signal












(I)
(II)
Cell morphology











Denaturation temperature
Formamide
EC
Formamide
EC





72° C.
0
2
Good
Good


82° C.
1⁄2
3
Good
Good


92° C.
1⁄2
3
Not good
Not good





Signals scored as “3” were clearly visible in a 20x objective.






Example 2
This example compares the signal intensity and background staining from samples treated with the compositions of the invention or traditional hybridization solutions as a function of hybridization time.
FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 14 hours, 4 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 0 minutes.
Results:
















Signal














(I)
(II)
Background staining













Hybridization time
Formamide
EC
Formamide
EC







14 hours
3
3
+1⁄2
+2



 4 hours
1
3
+1⁄2
+1



 2 hours
1⁄2
3
+0
+1



60 min.
1⁄2
3
+0
+1



30 min.
0
21⁄2
+0
+1



15 min.
0
2
+0
+1



 0 min.
0
1
+0
+1⁄2







Signals scored as “3” were clearly visible in a 20x objective.






Example 3
This example compares the signal intensity from samples treated with the compositions of the invention having different polar aprotic solvents or traditional hybridization solutions.
FISH Probe composition I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide, 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe composition II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate (EC), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe composition III: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Propylene carbonate (PC) (540013, Aldrich), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe composition IV: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Sulfolane (SL) (T22209, Aldrich), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe composition V: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Aceto nitrile (AN) (C02CIIX, Lab-Scan), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe composition VI: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% γ-butyrolactone (GBL) (B103608, Aldrich), 5 μM blocking PNAs, 7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes.
Results:










Signal














(I)
(II)
(III)
(IV)
(V)
(VI)



Formamide
EC
PC
SL
AN
GBL







1⁄2
3
3
3
2
3







Signals scored as “3” were clearly visible in a 20x objective.






Example 4
This example compares the signal intensity from samples treated with the compositions of the invention having different concentrations of polar aprotic solvent.
FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 10-60% Ethylene carbonate (as indicated), 5 μM blocking PNAs, 7.5 ng/μL Texas Red labeled IGK-constant DNA gene probe ((CTD-3050E15, RP11-1083E8; size 227 kb) and 7.5 ng/μL FITC labeled IGK-variable gene DNA probe (CTD-2575M21, RP11-122B6, RP11-316G9; size 350 and 429 kb).
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes.
Results:













Ethylene carbonate (EC)













10%
20%
30%
40%
60%

















Signal
Texas Red
11⁄2
2
3
3
2


intensity
FITC
1
11⁄2
2
21⁄2
2





Signals scored as “3” were clearly visible in a 20x objective.






Example 5
This example compares the signal intensity and background intensity from samples treated with the compositions with and without PNA blocking.
FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes.
Results:















Ethylene carbonate (EC)












PNA- blocking
Non- PNA blocking















Signal intensity
3
3



Background intensity
1⁄2+
1⁄2+







Signals scored as “3” were clearly visible in a 20x objective.






Example 6
This example compares the signal intensity from samples treated with the compositions of the invention as a function of probe concentration and hybridization time.
FISH Probe Compositions: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, and 10, 7.5, 5 or 2.5 ng/μL Texas Red labeled CCND1 gene DNA probe (as indicated).
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 3 hours, 2 hours and 1 hours.
Results:















Signal Intensity













Hybridization
(I)
(II)
(III)
(IV)



time
10 ng/μL
7.5 ng/μL
5 ng/μL
2.5 ng/μL







3 hours
3
3
3
3



2 hours
3
3
3
1



1 hours
3
3
3
1⁄2







Signals scored as “3” were clearly visible in a 20x objective.






Example 7
This example compares the signal intensity from samples treated with the compositions of the invention as a function of salt, phosphate, and buffer concentrations.
FISH Probe Compositions: 10% dextran sulfate, ([NaCl], [phosphate buffer], [TRIS buffer] as indicated in Results), 40% Ethylene carbonate, 7.5 ng/μL Texas Red labeled CCND1 gene DNA probe.
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes.
Results:















[NaCl]













300 mM
100 mM
0 mM
















Signal intensity
2
1
1⁄2



phosphate [0 mM]






Signal intensity
3
21⁄2
1⁄2



phosphate [5 mM]






Signal intensity
—
—
3



phosphate [35 mM]






Signal intensity
—
—
2



TRIS [40 mM]







Signals scored as “3” were clearly visible in a 20x objective.






Example 8
This example compares the signal intensity from samples treated with the compositions of the invention as a function of dextran sulfate concentration.
FISH Probe Compositions: 0, 1, 2, 5, or 10% dextran sulfate (as indicated), 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 5 ng/μL Texas Red labeled SIL-TAL1 gene DNA probe (RP1-278O13; size 67 kb) and 6 ng/μL FITC SIL-TAL1 (ICRFc112-112C1794, RP11-184J23, RP11-8J9, CTD-2007B18, 133B9; size 560 kb).
Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes. No blocking.
Results:












Signal Intensity









% Dextran Sulfate
Texas Red Probe
FITC Probe





 0%
1
1


 1%
1
1


 2%
11⁄2
11⁄2


 5%
2
21⁄2


10%
2
21⁄2





NOTE:


this experiment did not produce results scored as “3” because the SIL-TAL1 Texas Red labeled probe is only 67 kb and was from a non-optimized preparation.






Example 9
This example compares the signal intensity from samples treated with the compositions of the invention as a function of dextran sulfate, salt, phosphate, and polar aprotic solvent concentrations.
FISH Probe Composition Ia: 34% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition Ib: 34% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition Ic: 34% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 0% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IIa: 32% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IIb: 32% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IIc: 32% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 5% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IIIc: 30% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IIIb: 30% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IIIc: 30% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 10% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IVa: 28% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IVb: 28% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Composition IVc: 28% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 15% ethylene carbonate, 10 ng/μL Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
FISH Probe Reference V: Standard sales vial of HER2 PharmDx probe mix (K5331, Dako) containing blocking PNA. Overnight hybridization for 20 hours.
All compositions were present as a single phase. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes with no blocking, except for FISH Probe Reference V, which had PNA blocking and was hybridized for 20 hours.
Results:














Signal Strength











DNA Probes
PNA Probes















Composition Ia
0
 1⁄2



Composition Ib
0
 1⁄2



Composition Ic
1⁄2
21⁄2



Composition IIa
1⁄2
3



Composition IIb
1
2



Composition IIc
1⁄2
3



Composition IIIa
1
21⁄2



Composition IIIb
11⁄2 
21⁄2



Composition IIIc
2
3



Composition IVa
21⁄2-3
3



Composition IVb
3
3



Composition IVc
3
3



Reference V
2
21⁄2







NOTE:



Composition IVa gave strong DNA signals with no salt. This is not possible with standard FISH compositions, where DNA binding is salt dependent.






Example 10
This example compares the signal intensity from samples treated with the compositions of the invention as a function of polar aprotic solvent and dextran sulfate concentration under high salt (4× normal) conditions.
FISH Probe Composition I: 0% ethylene carbonate, 29% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition was a single phase.
FISH Probe Composition II: 5% ethylene carbonate, 27% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition was a single phase.
FISH Probe Composition III: 10% ethylene carbonate, 25% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition was a single phase.
FISH Probe Composition IV (not tested): 20% ethylene carbonate, 21% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/μL Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition had two phases.
Results:














Signal Strength











DNA Probes
PNA Probes















Composition I
1⁄2
3



Composition II
2
21⁄2



Composition III
3
3



Composition IV
—
—







Note:



Composition II gave good DNA signals with only 5% EC and strong DNA signals with 10% EC.






Example 11
This example compares the signal intensity and background from samples treated with different phases of the compositions of the invention.
FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 8 ng/μL Texas Red labeled HER2 gene DNA probe and 600 nM FITC-labeled CEN-17 PNA probe. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes. No blocking.
Results:














Signal Intensity












DNA Probe
PNA Probe
Background














Upper Phase
3
11⁄2
+2


Lower Phase
3
21⁄2
+1


Mix of Upper and
21⁄2
3
+1⁄2


Lower Phases





NOTE:


the upper phase had more background than the lower phase in these experiments.






Example 12
This example is similar to the previous example, but uses a different DNA probe and GBL instead of EC.
FISH Probe Composition: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% GBL, 10 ng/μL Texas Red labeled CCND1 gene DNA probe and 600 nM FITC-labeled CEN-17 PNA probe.
The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes. No blocking.
Results:
















Signal Strength












DNA Probe
PNA Probe
Background
















Top Phase
3
0-1⁄2
+11⁄2



Bottom Phase
2
1⁄2
+3



Mixed Phases
21⁄2
1⁄2
+21⁄2










Example 13
This example examines the number of phases in the compositions of the invention as a function of polar aprotic solvent and dextran sulfate concentration.
FISH Probe Compositions: 10 or 20% dextran sulfate; 300 mM NaCl; 5 mM phosphate buffer; 0, 5, 10, 15, 20, 25, 30% EC; 10 ng/μL probe.
Results:













Number of Phases
Number of Phases


% EC
10% Dextran
20% Dextran

















0
1
1


5
1
1


10
1
1


15
1
1


20
2
2


25
2
2


30
2
2





NOTE:


15% EC, 20% dextran sulfate produces the nicest high signal intensities of the above one phase solution. Two phases 20% EC has even higher signal intensities than 15%. (Data not shown).






Example 14
This example compares the signal intensity and background from samples treated with different compositions of the invention as a function of probe concentration and hybridization time.
FISH Probe Composition I: 10 ng/μL HER2 TxRed labeled DNA probe (standard concentration) and standard concentration of CEN7 FITC labeled PNA probe (50 nM); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.
FISH Probe Composition II: 5 ng/μL HER2 TxRed labeled DNA probe (1⁄2 of standard concentration) and standard concentration (50 nM) of FITC labeled CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.
FISH Probe Composition III: 2.5 ng/μL HER2 TxRed labeled DNA probe (1⁄4 of standard concentration) and 1⁄2 of the standard concentration (25 nM) of CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.
Compositions I-III existed as a single phase. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 3 hours, 2 hours and 1 hours.
Results:











Hybrid-
Signal Intensity










ization
I
II
III
















time
DNA
PNA
B.G.
DNA
PNA
B.G.
DNA
PNA
B.G.



















3 hours
3
3
+3
3
3
+2.5
3
3
+1.5


2 hours
2.5
2.5
+3
3
3
+3
3
3
+1.5


1 hours
2.5
2.5
+3
3
3
+1.5
2.5
3
+1





Signals scored as “3” were clearly visible in a 20x objective.


B.G.: Back ground.






Example 15
This example compares the signal intensity and background from samples treated with the compositions of the invention as a function of blocking agent.
FISH Probe Compositions: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer; 2.5 ng/μL HER2 TxRed labeled DNA probe (1⁄4 of standard concentration) and 1⁄2 of the standard concentration (300 nM) FITC labeled CEN17 PNA probe. Samples were blocked with: (a) nothing; (b) 0.1 μg/μL COT1 (15279-011, Invitrogen); (c) 0.3 μg/μL COT1; or (d) 0.1 μg/μL total human DNA before hybridization using the compositions of the invention.
All samples were present as a single phase. The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes.
Results:














Signal Intensity











Blocking Agent
Background
DNA
PNA





Nothing
+1-1.5
3
2.5


0.1 μg/μL COT1
+1
3
2.5


0.3 μg/μL COT1
+1.5
3
2.5


0.1 μg/μL total human DNA
+1⁄2
3
2.5





NOTE:


Background levels without blocking are significantly lower than what is normally observed by standard FISH with no blocking. In contrast, if a standard FISH composition does not contain a blocking agent, signals normally cannot be read.






Example 16
This experiment compares different ways of removing background staining using the compositions of the invention.
All compositions contained 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 2.5 ng/μL HER2 DNA probes (1⁄4 of standard concentration), 300 nM CEN17 PNA probe (1⁄2 of standard concentration), and one of the following background-reducing agents:
A) 5 μM blocking-PNA (see Kirsten Yang Nielsen et al., PNA Suppression Method Combined with Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10 (Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006))

B) 0.1 μg/μL COT-1 DNA

C) 0.1 μg/μL total human DNA (THD) (sonicated unlabelled THD)

D) 0.1 μg/μL sheared salmon sperm DNA (AM9680, Ambion)

E) 0.1 μg/μL calf thymus DNA (D8661, Sigma)

F) 0.1 μg/μL herring sperm DNA (D7290, Sigma)

G) 0.5% formamide

H) 2% formamide

I) 1% ethylene glycol (1.09621, Merck)

J) 1% glycerol (1.04095, Merck)

K) 1% 1,3-Propanediol (533734, Aldrich)

L) 1% H20 (control)

All samples were present as a single phase. The probes were incubated at 82° C. for 5 minutes and then at 45° C. on FFPE tissue sections for 60 and 120 minutes.
Results:













Signal Intensity











Background blocking
Hybridization/min
Background
DNA
PNA














Blocking-PNA
60
+1
3
2.5


Blocking-PNA
120
+1-11⁄2
3
2.5


COT-1
60
+1⁄2
3
2.5


COT-1
120
+0-1⁄2 
3
2.5


THD
60
+0
3
3


THD
120
+1⁄2
3
2.5


Salmon DNA sperm
60
+0
3
3


Salmon DNA sperm
120
+0
3
3


Calf Thymus DNA
60
+0
2.5
3


Calf Thymus DNA
120
+1⁄2
3
2.5


Hearing sperm DNA
60
+0
3
3


Hearing sperm DNA
120
+1⁄2
2.5
3


0.5% formamide
60
+0
2.5
3


0.5% formamide
120
+0
3
3


2% formamide
60
+1⁄2
2.5
3


2% formamide
120
+1⁄2
3
3


1% Ethylene Glycol
60
+1⁄2
2.5
3


1% Ethylene Glycol
120
+11⁄2 
3
2.5


1% Glycerol
60
+1⁄2
0.5
3


1% Glycerol
120
+1
3
2.5


1% 1,3-Propanediol
60
+0
3
2.5


1% 1,3-Propanediol
120
+1
3
2.5


Nothing
60
+1
2.5
2.5


Nothing
120
+11⁄2 
3
2.5





NOTE:


all background reducing reagents, except for blocking-PNA, showed an effect in background reduction. Thus, specific blocking against repetitive DNA sequences is not required.






Example 17
This experiment compares the signal intensity from the upper and lower phases using two different polar aprotic solvents.
FISH Probe Composition I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene trithiocarbonate (ET) (E27750, Aldrich), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
FISH Probe Composition II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% glycol sulfite (GS) (G7208, Aldrich), 5 μM blocking PNAs, 10 ng/μL Texas Red labeled CCND1 gene DNA probe.
The FISH probes were incubated at 82° C. for 5 min and then at 45° C. for 60 minutes.
Results:














Signal Intensity











I (ET)
II (GS)















Upper Phase
11⁄2
0



Lower Phase
0
3



Mix of Upper and Lower Phases
21⁄2
3










Example 18
This experiment examines the ability of various polar aprotic solvents to form a one-phase system.
All compositions contained: 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and either 10, 15, 20, or 25% of one of the following polar aprotic solvents:
Sulfolane
γ-Butyrolactone
Ethylene trithiocarbonate
Glycol sulfite
Propylene carbonate
Results: all of the polar aprotic solvents at all of the concentrations examined produced at least a two-phase system in the compositions used. However, this does not exclude that these compounds can produce a one-phase system under other composition conditions.
Example 19
This experiment examines the use of the compositions of the invention in chromogenic in situ hybridization (CISH) analysis on multi FFPE tissue sections.
FISH Probe Composition I: 4.5 ng/μL TCRAD FITC labelled gene DNA probe (1⁄4 of standard concentration) (RP11-654A2, RP11-246A2, CTP-2355L21, RP11-158G6, RP11-780M2, RP11-481C14; size 1018 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
FISH Probe Composition II: 4.5 ng/μL TCRAD FITC labelled gene DNA probe (1⁄4 of standard concentration) (size 1018 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon DNA sperm.
FISH Probe Composition III: 300 nM of each individual FITC labelled PNA CEN17 probe (1⁄2 of standard concentration); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
All samples were analyzed using the Dako DuoCISH protocol (SK108) and compositions for split probes with the exception that the stringency wash was conducted for 20 minutes instead of 10 minutes, and without using the DuoCISH red chromogen step.
Results:














Signal Strength












Composition
FITC DNA
FITC PNA







I
3
—



II
3
—



III
—
3







Note:



The signal intensities were very strong. Due to the high levels of background, it was not possible to discriminate if addition of salmon sperm DNA in Composition II reduced the background. Signals were clearly visible using a 10x objective in e.g. tonsils, which in general had less background. If tissues possessed high background, the signals were clearly visible using a 20x objective.






Example 20
This example compares the signal intensity and background from FFPE tissue sections treated with the compositions of the invention with two DNA probes.
FISH Probe Composition I: 9 ng/μL IGH FITC labelled gene DNA probe (RP11-151B17, RP11-112H5, RP11-101G24, RP11-12F16, RP11-47P23, CTP-3087C18; size 612 kb); 6.4 ng/μL MYC Tx Red labeled DNA probe (CTD-2106F24, CTD-2151C21, CTD-2267H22; size 418 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
FISH Probe Composition II: 9 ng/μL IGH FITC labelled gene DNA probe; 6.4 ng MYC TxRed labeled DNA probe; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.














Signal Strength











Salmon DNA
FITC probe
Texas Red probe
Background





−
21⁄2
21⁄2
+2.5


+
3
3
+1.5





NOTE:


the high background was probably due to the fact that standard probe concentrations were used.






Example 21
This experiment examines the use of the compositions of the invention on cytological samples.
FISH Probe Composition: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer; 5 ng/μL HER2 TxRed labeled DNA probe (1⁄2 of standard concentration) and 1⁄2 of the standard concentration of CEN7 (25 nM).
The FISH probes were incubated on metaphase chromosome spreads at 82° C. for 5 minutes, then at 45° C. for 30 minutes, all without blocking.
Results:













Signal Strength










DNA Probe
PNA Probe
Background





3
3
+1





No chromosome banding (R-banding pattern) was observed with the compositions of the invention, in contrast with traditional ISH solutions, which typically show R-banding. A low homogenously red background staining of the interphase nuclei and metaphase chromosomes was observed.






Example 22
This example compares the signal intensity and background from DNA probes on cytology samples, metaphase spreads, with and without blocking.
FISH Probe Composition I: 6 ng/μL TCRAD Texas Red labelled gene DNA probe (standard concentration) (CTP-31666K20, CTP-2373N7; size 301 kb) and 4.5 ng/μL FITC labelled gene DNA probe (1⁄4 of standard concentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
FISH Probe Composition II: 6 ng/μL TCRAD Texas Red labelled gene DNA probe (standard concentration) (size 301 kb) and 4.5 ng/μL FITC labelled gene DNA probe (1⁄4 of standard concentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.
The FISH probes were incubated on metaphase spreads at 82° C. for 5 min, then at 45° C. for 60 min.
Results:














Signal Intensity













Blocking Agent
Background
Tx Red
FITC







Nothing
+0
3
3



0.1 μg/μL Salmon DNA
+0
3
3







Again, no chromosome banding (R-banding pattern) was observed with the compositions of the invention. In addition, no background staining of the interphase nuclei or the metaphase chromosomes were observed.






Example 23
This example compares the staining intensity from mRNA in samples treated with the compositions of the invention or formamide hybridization solutions as a function of hybridization time and temperature.
FISH Probe Composition I: 20 nM fluorescein-labelled kappa PNA probe (Y5202, Dako); 30% formamide; 10% dextran sulfate; 10 mM NaCl; 50 mM Tris, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidon, 0.2% Ficoll, 5 mM Na2EDTA
FISH Probe Composition II: 20 nM fluorescein-labelled kappa PNA probe (Y5202, Dako), 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
FISH Probe Composition III: 20 nM fluorescein-labelled kappa LNA probe, 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
The stringent wash was performed at 55° C. for the PNA probe (FISH Probe Compositions I and II) and at 30° C. for the LNA probe (FISH Probe Composition III) for 25 min.
Results:















Hybridization
Staining Intensity













Probe
time/temp
Tonsil
Colon







PNA I
 5 min/55° C.
3
2



PNA I
15 min/55° C.
3
—



PNA I
30 min/55° C.
—
2



PNA I
60 min/55° C.
—
21⁄2



PNA I
90 min/55° C.
—
21⁄2



PNA II
 5 min/55° C.
3
21⁄2-3



PNA II
15 min/55° C.
 2-3
  2-21⁄2



PNA II
30 min/55° C.
3
—



PNA II
60 min/55° C.
  2-21⁄2
  2-21⁄2



PNA II
90 min/55° C.
  2-21⁄2
  2-21⁄2



LNA III
 5 min/42° C.
11⁄2-2
11⁄2



LNA III
15 min/42° C.
21⁄2
21⁄2



LNA III
30 min/42° C.
21⁄2-3
21⁄2-3



LNA III
60 min/42° C.
21⁄2-3
21⁄2-3



LNA III
90 min/42° C.
3
3







*— indicates that the tissue was over-digested and could not be read properly.






Negative controls having a Fluorescein-conjugated random PNA probe in FISH Probe Composition I and negative controls having no probe in FISH Probe Compositions II and III gave no signals (data not shown).
A positive control having a fluorescein-conjugated PNA probe directed against glyceraldehyde 3-phosphate dehydrogenase in FISH Probe Composition I provided strong signals (data not shown).
No staining were observed for tissue types other than colon and tonsil on the small multi tissue section (data not shown).
These results show that RNA can be detected using the new buffer with both LNA and PNA based probes. These results also show that signals for PNA in FISH Probe Compositions II and III were stronger than for PNA in FISH Probe Composition I at 5 min. Thus, the buffers of the invention can be used in place of traditional formamide buffers for affecting the stringency conditions of both natural and artificial bases such as e.g. DNA, RNA, PNA and LNA.
Example 24
This example compares staining intensity of PNA and LNA probes hybridized to mRNA in biological samples exposed to the same digestion incubation time.
FISH Probe Composition I: Fluorescein-conjugated random PNA probes, 30% formamide; 10% dextran sulfate; 10 mM NaCl; 50 mM Tris, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidon, 0.2% Ficoll, 5 mM Na2EDTA.
FISH Probe Composition II: 20 nM fluorescein-labelled kappa PNA probe (Flu-Flu-CTGCTGAGGCTGTAG, Panagen, Korea), 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
FISH Probe Composition III: 20 nM fluorescein-labelled kappa LNA probe (Flu-CTGCTGAGGCTGTAG-Flu, Exicon, Denmark), 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
The digestion time for all samples was 5 min.
Results:















Hybridization
Staining Intensity













Probe
time/temp
Tonsil
Colon







Control I
90 min/35° C.
0
0



EC PNA II
90 min/45° C.
3
3



EC LNA III
90 min/35° C.
3
11⁄2







No staining was observed for kidney and mamacarcinoma tissues. The samples showed low background, a nice morphology, and were not overdigested.






These results further illustrate that the buffers of the invention can be used for RNA hybridizations using both PNA and LNA probes.
Example 25
This example compares staining intensity of PNA and LNA probes hybridized to mRNA in biological samples exposed to the same digestion incubation time and hybridization temperature.
FISH Probe Composition I: Fluorescein-conjugated random PNA probes, 30% formamide (FM); 10% dextran sulfate; 10 mM NaCl; 50 mM Tris, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidon, 0.2% Ficoll, 5 mM Na2EDTA.
FISH Probe Composition II: 15% GBL, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.
FISH Probe Composition III: 15% SL, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.
FISH Probe Composition IV: 20 nM fluorescein-labelled kappa PNA probe (Flu-Flu-CTGCTGAGGCTGTAG, Panagen, Korea), 15% GBL, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.
FISH Probe Composition V: 40 nM fluorescein-labelled kappa LNA probe (Flu-CTGCTGAGGCTGTAG-Flu, Exicon, Denmark), 15% GBL, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.
FISH Probe Composition VI: 20 nM fluorescein-labelled kappa PNA probe (Flu-Flu-CTGCTGAGGCTGTAG, Panagen, Korea), 15% SL, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.
FISH Probe Composition VII: 40 nM fluorescein-labelled kappa LNA probe (Flu-CTGCTGAGGCTGTAG-Flu, Exicon, Denmark), 15% SL, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2.
The digestion time for all samples was 5 min. The samples were hybridized at 45° C. for 90 min and stringent washed at 45° C. for 25 min.
Results:













Staining Intensity












Probe
Background
Tonsil
Colon
Mamma
Kidney





FM PNA I
+0
2
1⁄2
0
0


GBL control II
+0
0
0
0
0


SL Control III
+0
0
0
0
0


GBL PNA IV
+0
21⁄2
11⁄2 
0
0


GBL LNA V
+0
2
1⁄2
0
0


SL PNA VI
+0
2-21⁄2
1⁄2
0
0


SL LNA VII
+0
3
11⁄2-2
0
0





No staining was observed for kidney and mamacarcinoma tissues. The samples showed no background, nice morphology, and were not over-digested.






The results showed that signals for PNA in FISH Probe Compositions IV and VI were stronger than for PNA in FISH Probe Composition I (i.e., traditional formamide buffer).
Further Embodiments
Embodiment 1
A hybridization composition for RNA hybridization applications comprising at least one nucleic acid sequence, at least one polar aprotic solvent in an amount effective to enable hybridization to RNA, and a hybridization solution, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).
Embodiment 2
The hybridization composition according to embodiment 1, wherein the concentration of polar aprotic solvent is about 1% to 95% (v/v)
Embodiment 3
The hybridization composition according to embodiment 1 or 2, wherein the concentration of polar aprotic solvent is 5% to 10% (v/v).
Embodiment 4
The hybridization composition according to embodiment 1 or 2, wherein the concentration of polar aprotic solvent is 10% to 20% (v/v).
Embodiment 5
The hybridization composition according to embodiment 1 or 2, wherein the concentration of polar aprotic solvent is 20% to 30% (v/v).
Embodiment 6
The hybridization composition according to any one of embodiments 1 to 5, wherein the polar aprotic solvent is non-toxic.
Embodiment 7
The hybridization composition according to any one of embodiments 1 to 6, with the proviso that the composition does not contain formamide.
Embodiment 8
The hybridization composition according to embodiment 6, with the proviso that the composition contains less than 10% formamide.
Embodiment 9
The hybridization composition according to embodiment 8, with the proviso that the composition contains less than 2% formamide.
Embodiment 10
The hybridization composition according to embodiment 9, with the proviso that the composition contains less than 1% formamide.
Embodiment 11
The hybridization composition according to any of embodiments 1 to 10, wherein the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, and/or carbonate functionality.
Embodiment 12
The hybridization composition according to any one of embodiments 1 to 11, wherein the polar aprotic solvent has a dispersion solubility parameter between 17.7 to 22.0 MPa1/2, a polar solubility parameter between 13 to 23 MPa1/2, and a hydrogen bonding solubility parameter between 3 to 13 MPa1/2.
Embodiment 13
The hybridization composition according to any one of embodiments 1 to 12, wherein the polar aprotic solvent has a cyclic base structure.
Embodiment 14
The hybridization composition according to any one of embodiments 1 to 13, wherein the polar aprotic solvent is selected from the group consisting of:




where X is O and R1 is alkyldiyl, and





where X is optional and if present, is chosen from O or S,

where Z is optional and if present, is chosen from O or S,

where A and B independently are O or N or S or part of the alkyldiyl or a primary amine,

where R is alkyldiyl, and

where Y is O or S or C.

Embodiment 15
The hybridization composition according to any one of embodiments 1 to 14, wherein the polar aprotic solvent is selected from the group consisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide, benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone, epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate, N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile, 1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methyl morpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone, methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline, nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone, 2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenyl sydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone, propylene carbonate, 4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone, saccharin, succinonitrile, sulfanilamide, sulfolane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide, tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil, 3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloro propene, trimethylene sulfide-dioxide, and trimethylene sulfite.
Embodiment 16
The hybridization composition according to any one of embodiments 1 to 14, wherein the polar aprotic solvent is selected from the group consisting of:




Embodiment 17
The hybridization composition according to any one of embodiments 1 to 14, wherein the polar aprotic solvent is:




Embodiment 18
The hybridization composition according to any one of embodiments 1 to 17, further comprising at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, and blocking agents.
Embodiment 19
The hybridization composition according to embodiment 18, wherein the accelerating agent is dextran sulfate and the salts are NaCl and/or phosphate buffer.
Embodiment 20
The hybridization composition according to embodiment 19, wherein the dextran sulfate is present at a concentration of 5% to 40%, the NaCl is present at a concentration of 0 mM to 1200 mM, and/or the phosphate buffer is present at a concentration of 0 mM to 50 mM.
Embodiment 21
The hybridization composition according to embodiment 20, wherein the dextran sulfate is present at a concentration of 10% to 30%, the NaCl is present at a concentration of 300 mM to 600 mM, and/or the phosphate buffer is present at a concentration of 5 mM to 20 mM.
Embodiment 22
The hybridization composition according to embodiment 18, wherein the accelerating agent is selected from the group consisting of: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer.
Embodiment 23
The hybridization composition according to embodiment 22, wherein the formamide is present at a concentration of 0.1-5%, the DMSO is present at a concentration of 0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol are present at a concentration of 0.1% to 10%, and the citric acid buffer is present at a concentration of 1 mM to 50 mM.
Embodiment 24
The hybridization composition according to embodiment 18, wherein the blocking agent is selected from the group consisting of: total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA.
Embodiment 25
The hybridization composition according to embodiment 24, wherein the total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA are present at a concentration of 0.01 to 10 μg/μL.
Embodiment 26
The hybridization composition according to any one of embodiments 1-25, comprising 40% of at least one polar aprotic solvent, 10% dextran sulfate, 300 mM NaCl, and 5 mM phosphate buffer.
Embodiment 27
The hybridization composition according to any one of embodiments 1-25, comprising 15% of at least one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 μg/μl total human DNA.
Embodiment 28
The hybridization composition according to any one of embodiments 1-25, comprising 15% of at least one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2, and 0.1 μg/μL herring sperm DNA, or salmon sperm DNA, or calf thymus DNA, or 0.5% formamide, or 1% ethylene glycol, or 1% 1,3 propanediol.
Embodiment 29
The hybridization composition according to any one of embodiments 1-28, comprising more than one phase at room temperature.
Embodiment 30
The hybridization composition according to embodiment 29, comprising two phases at room temperature.
Embodiment 31
The hybridization composition according to embodiment 29, comprising three phases at room temperature.
Embodiment 32
The hybridization composition according to any one of embodiments 1-31, wherein the RNA is messenger RNA (mRNA), viral RNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNA and rRNA), transfer messenger RNA (tmRNA), piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA (snoRNA), antisense RNA, double-stranded RNA (dsRNA), or heterogeneous nuclear RNA (hnRNA).
Embodiment 33
The hybridization composition according to any one of embodiments 1-32, wherein the at least one nucleic acid sequence is a PNA sequence, an LNA sequence, or a DNA sequence.
Embodiment 34
A hybridization method comprising:

    
    
        providing an RNA sequence,
        providing a second nucleic acid sequence,
        providing an aqueous composition comprising at least one polar aprotic solvent in an amount effective to enable hybridization to RNA, and
        combining the RNA and the second nucleic acid sequence and the aqueous composition for at least a time period sufficient to hybridize the RNA and the second nucleic acid sequence,
        wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).
    
    


Embodiment 35
A hybridization method comprising:

    
    
        providing an RNA sequence, and
        applying to said RNA sequence an aqueous composition comprising a second nucleic acid sequence and at least one polar aprotic solvent in an amount effective to enable hybridization to RNA for at least a time period sufficient to hybridize the RNA and the second nucleic acid sequences,
        wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).
    
    


Embodiment 36
A hybridization method comprising:

    
    
        providing an RNA sequence, and
        applying a hybridization composition according to any of embodiments 1-31 to said RNA sequence for at least a time period sufficient to hybridize the RNA and the second nucleic acid sequences.
    
    


Embodiment 37
The method according to embodiments 34 or 35, wherein the polar aprotic solvent is defined according to any of embodiments 2-6 or 11-17.
Embodiment 38
The method according to any of embodiments 34-37, wherein a sufficient amount of energy to hybridize the RNA and the second nucleic acids is provided.
Embodiment 39
The method according to embodiment 38, wherein the energy is provided by heating the hybridization composition and nucleic acid sequence.
Embodiment 40
The method according to embodiment 39, wherein the heating step is performed by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating or heat lamps.
Embodiment 41
The method according to any one of embodiments 34-40, further comprising a denaturation step.
Embodiment 42
The method according to embodiment 41, wherein the denaturation and hybridization steps occur separately.
Embodiment 43
The method according to any one of embodiments 34-42, wherein the step of hybridizing includes the steps of heating and cooling the hybridization composition and nucleic acid sequences.
Embodiment 44
The method according to any one of embodiments 34-43, wherein the hybridization step takes less than 8 hours.
Embodiment 45
The method according to embodiment 44, wherein the hybridization step takes less than 1 hour.
Embodiment 46
The method according to embodiment 45, wherein the hybridization step takes less than 30 minutes.
Embodiment 47
The method according to embodiment 46, wherein the hybridization step takes less than 15 minutes.
Embodiment 48
The method according to embodiment 47, wherein the hybridization step takes less than 5 minutes.
Embodiment 49
The method according to any one of embodiments 34-48, wherein the second nucleic acid is a PNA sequence, an LNA sequence, or a DNA sequence.
Embodiment 50
The method according to any one of embodiments 34-49, wherein the RNA is messenger RNA (mRNA), viral RNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNA and rRNA), transfer messenger RNA (tmRNA), micro RNA (miRNA), piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA (snoRNA), antisense RNA, double-stranded RNA (dsRNA), and heterogeneous nuclear RNA (hnRNA).
Embodiment 51
The method according to any one of embodiments 34-50, wherein the RNA is in a biological sample.
Embodiment 52
The method according to embodiment 51, wherein the biological sample is a cytology or histology sample.
Embodiment 53
The method according to any one of embodiments 34-52, wherein the hybridization composition comprises one phase at room temperature.
Embodiment 54
The method according to any one of embodiments 34-52, wherein the hybridization composition comprises multiple phases at room temperature.
Embodiment 55
The method according to embodiment 54, wherein the hybridization composition comprises two phases at room temperature.
Embodiment 56
The method according to embodiment 54 or 55, wherein the phases of the hybridization composition are mixed.
Embodiment 57
The method according to any one of embodiments 34-56, further comprising a blocking step.
Embodiment 58
Use of a hybridization composition comprising between 1 and 95% (v/v) of at least one polar aprotic solvent in an RNA hybridization application.
Embodiment 59
Use of a composition according to embodiment 58, wherein the hybridization composition is according to any one of embodiments 1 to 33.

## Claims

1. A composition comprising at least one polar aprotic solvent in an amount effective to enable hybridization of a nucleic acid sequence to an RNA sequence within a cell in a sample having a preserved cell morphology, and at least 10% dextran sulfate, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

2. The composition according to claim 1, wherein the concentration of polar aprotic solvent is about 1% to 90% (v/v).

3. The composition according to claim 2, wherein the concentration of polar aprotic solvent is 5% to 10% (v/v).

4. The composition according to claim 2, wherein the concentration of polar aprotic solvent is 10% to 20% (v/v).

5. The composition according to claim 2, wherein the concentration of polar aprotic solvent is 20% to 30% (v/v).

6. The composition according to claim 1, wherein the polar aprotic solvent is non-toxic.

7. The composition according to claim 1, with the proviso that the composition does not contain formamide.

8. The composition according to claim 6, with the proviso that the composition contains less than 10% formamide.

9. The composition according to claim 8, with the proviso that the composition contains less than 2% formamide.

10. The composition according to claim 9, with the proviso that the composition contains less than 1% formamide.

11. The composition according to claim 1, wherein the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, and/or carbonate functionality.

12. The composition according to claim 1, wherein the polar aprotic solvent has a dispersion solubility parameter between 17.7 to 22.0 MPa1/2, a polar solubility parameter between 13 to 23 MPa1/2, and a hydrogen bonding solubility parameter between 3 to 13 MPa1/2.

13. The composition according to claim 1, wherein the polar aprotic solvent has a cyclic base structure.

14. The composition according to claim 1, wherein the polar aprotic solvent is selected from the group consisting of:



where X is O and R1 is alkyldiyl, and




where X is optional and if present, is chosen from O or S,
where Z is optional and if present, is chosen from O or S,
where A and B independently are O or N or S or part of the alkyldiyl or a primary amine,
where R is alkyldiyl, and
where Y is O or S or C, and wherein if Y is C, then either X or Z is not present or both X and Z are not present.

15. The composition according to claim 1, wherein the polar aprotic solvent is selected from the group consisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide, benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone, epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate, N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile, 1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methyl morpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone, methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline, nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrrolidinone, 2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenyl sydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone, propylene carbonate, 4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone, saccharin, succinonitrile, sulfanilamide, sulfolane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide, tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil, 3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloro propene, trimethylene sulfide-dioxide, and trimethylene sulfite.

16. The composition according to claim 1, wherein the polar aprotic solvent is selected from the group consisting of:

17. The composition according to claim 1, wherein the polar aprotic solvent is:

18. The composition according to claim 1, further comprising at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, and blocking agents.

19. The composition according to claim 18, wherein the salt is NaCl and/or the buffering agent is phosphate buffer.

20. The composition according to claim 19, wherein the dextran sulfate is present at a concentration of 10% to 40%, the NaCl is present at a concentration of 0 mM to 1200 mM, and/or the phosphate buffer is present at a concentration of 0 mM to 50 mM.

21. The composition according to claim 20, wherein the dextran sulfate is present at a concentration of 10% to 30%, the NaCl is present at a concentration of 300 mM to 600 mM, and/or the phosphate buffer is present at a concentration of 5 mM to 20 mM.

22. The composition according to claim 18, wherein the accelerating agent is selected from the group consisting of: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer.

23. The composition according to claim 22, wherein the formamide is present at a concentration of 0.1-5%, the DMSO is present at a concentration of 0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol are present at a concentration of 0.1% to 10%, and the citric acid buffer is present at a concentration of 1 mM to 50 mM.

24. The composition according to claim 18, wherein the blocking agent is selected from the group consisting of: total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA.

25. The composition according to claim 24, wherein the total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA are present at a concentration of 0.01 to 10 μg/μL.

26. The composition according to claim 1, comprising 40% of at least one polar aprotic solvent, 10% dextran sulfate, 300 mM NaCl, and 5 mM phosphate buffer.

27. The composition according to claim 1, comprising 15% of at least one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 μg/μl total human DNA.

28. The composition according to claim 1, comprising 15% of at least one polar aprotic solvent, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2, and 0.1 μg/μL herring sperm DNA, or salmon sperm DNA, or calf thymus DNA, or 0.5% formamide, or 1% ethylene glycol, or 1% 1,3 propanediol.

29. The composition according to claim 1, comprising more than one phase at room temperature.

30. The composition according to claim 29, comprising two phases at room temperature.

31. The composition according to claim 29, comprising three phases at room temperature.

32. The composition according to claim 1, wherein the RNA within the sample having a preserved cell morphology is messenger RNA (mRNA), viral RNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), transfer messenger RNA (tmRNA), piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA (snoRNA), antisense RNA, double-stranded RNA (dsRNA), or heterogeneous nuclear RNA (hnRNA).

33. A hybridization method comprising:
combining a sample having a preserved cell morphology comprising an RNA sequence, a nucleic acid sequence, and an aqueous composition for at least a time period sufficient to hybridize the RNA sequence within the cell with the nucleic acid sequence,

wherein the aqueous composition comprises at least one polar aprotic solvent in an amount effective to enable hybridization of the nucleic acid sequence to an RNA sequence within a cell and the sample morphology is preserved, and at least 10% dextran sulfate, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

34. A hybridization method comprising:
combining a nucleic acid sequence with the composition according to claim 1;
applying the combination of the nucleic acid sequence and the composition according to claim 1 to a sample having a preserved cell morphology Comprising an RNA sequence for at least a time period sufficient to hybridize the RNA with the nucleic acid sequences within the cell and the sample morphology is preserved.

35. The method according to claim 34, wherein a sufficient amount of energy to hybridize the RNA within the cell and the nucleic acid sequences is added.

36. The method according to claim 35, wherein the energy is provided by heating the composition, the cell comprising the RNA, and the nucleic acid sequence.

37. The method according to claim 36, wherein the heating step is performed by the use of one or more microwaves, hot baths, hot plates, heat wires, peltier elements, induction heating or heat lamps.

38. The method according to claim 34, wherein the nucleic acid is double stranded and further comprising a denaturation step.

39. The method according to claim 38, wherein the denaturation and hybridization steps occur separately.

40. The method according to claim 34, wherein the step of hybridizing includes the steps of heating and cooling the composition, the cell comprising the RNA, and the nucleic acid sequence.

41. The method according to claim 34, wherein the hybridization step takes less than 8 hours.

42. The method according to claim 41, wherein the hybridization step takes less than 1 hour.

43. The method according to claim 42, wherein the hybridization step takes less than 30 minutes.

44. The method according to claim 43, wherein the hybridization step takes less than 15 minutes.

45. The method according to claim 44, wherein the hybridization step takes less than 5 minutes.

46. The method according to claim 34, wherein the cell comprising the RNA sequence is in a biological sample.

47. The method according to claim 46, wherein the biological sample is a cytology or histology sample.

48. The method according to claim 34, wherein the composition comprises one phase at room temperature.

49. The method according to claim 34, wherein the composition comprises multiple phases at room temperature.

50. The method according to claim 49, wherein the composition comprises two phases at room temperature.

51. The method according to claim 49, wherein the phases of the composition are mixed.

52. The method according to claim 34, further comprising a blocking step.

53. The composition according to claim 1, further comprising at least one nucleic acid sequence.

54. The composition according to claim 53, wherein the at least one nucleic acid sequence is a PNA sequence, an LNA sequence, or a DNA sequence.