Far-red dye probe formulations

Abstract

To provide far-red dye probe formulations.SOLUTION: Disclosed are formulations, including both liquid and lyophilized formulations, comprising a far-red dye probe and a non-linear surfactant or foamban. Also disclosed are related methods for preparing a lyophilized far-red dye probe formulation as well as related kits and diagnostic products. For example, the present invention provides a stabilized far-red dye probe formulation. Here, the formulation includes a far-red dye probe comprising a far-red dye conjugated to a carrier molecule, a non-linear surfactant at a concentration of greater than about 0.05% (v / v), and at least one buffering agent. The formulation is an aqueous solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to Provisional Application No. 62 / 627,040, filed February 6, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on February 5, 2019, is named "DIA.0041-03-PCT__ST25.txt" and is 5 KB in size. [Background technology]

[0003] Probes containing far-red fluorescent dyes are widely used in many bioscience applications, including, for example, in vitro detection assays, conventional and super-resolution localization microscopy, and live-cell imaging. Far-red fluorescent dyes are also particularly convenient for multiplexing due to their limited spectral overlap with other commonly used fluorophores and fluorescent proteins. However, the formulation of far-red dye probes presents significant challenges due to significant loss of fluorescent signal after reconstitution and / or storage in aqueous form. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the present invention provides stabilized far-red dye probe formulations. In some embodiments, the formulation generally comprises a far-red dye probe comprising a far-red dye bound to a carrier molecule, a nonlinear surfactant at a concentration greater than about 0.05% (v / v), and at least one buffer, wherein the formulation is an aqueous solution. In other embodiments, the formulation generally comprises a far-red dye probe comprising a far-red dye bound to a carrier molecule, a formaldehyde at a concentration greater than about 0.05% (v / v), and at least one buffer, wherein the formulation is an aqueous solution. A preferred buffer is Tris, and in some such variations, the Tris buffer is present at a concentration of about 5 mM to about 50 mM. Particularly preferred far-red dyes include far-red cyanine dyes, such as Cyanine 5 or Cyanine 5.5.

[0005] In certain embodiments of the stabilized far-red dye probe formulations containing a nonlinear surfactant, as described above, the nonlinear surfactant is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, polysorbate 40, or polysorbate 60. In other variations, the nonlinear surfactant is digitonin. Suitable nonlinear surfactant concentrations include concentrations of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), or about 0.1% (v / v) to about 10% (v / v). In some embodiments, the nonlinear surfactant concentration is about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v), or about 1% (v / v) to about 2% (v / v).

[0006] In some embodiments of the stabilized far-red dye probe formulations described above, the carrier molecule is a nucleic acid, such as RNA. In other non-mutually exclusive embodiments, the far-red dye probe further comprises a quencher, and in some such variations, the far-red dye probe is a molecular torch, a molecular beacon, or a TaqMan probe. In some nucleic acid probe embodiments, the formulation further comprises a first amplification oligomer, wherein (i) the far-red dye probe comprises a target hybridizing sequence that specifically binds to a first sequence contained within a target region of a target nucleic acid, (ii) the first amplification oligomer comprises a target hybridizing sequence that specifically binds to a second sequence contained within the target region, and (iii) the first amplification oligomer is configured to produce an amplification product containing the target region in an amplification assay that includes the target nucleic acid as a template. In some embodiments further containing a first amplification oligomer as described above, the formulation further contains a second amplification oligomer comprising a target hybridizing sequence that specifically binds to a third sequence contained within the target region, and the first and second amplification oligomers are configured to amplify the target region over multiple cycles of an amplification assay. In some variations of formulations further containing a first amplification oligomer, the first amplification oligomer is a promoter-based amplification oligomer that further comprises a promoter sequence located 5' of the first target hybridizing sequence. Formulations comprising a nucleic acid far-red dye probe and further containing a first amplification oligomer as described above may further comprise one or more additional components suitable for performing an amplification assay, such as, for example, one or more nucleotide triphosphates and / or one or more salts or cofactors.

[0007] In another aspect, the present invention provides a method for preparing a stabilized lyophilized far-red dye probe formulation. The method generally comprises: (a) providing a stabilized far-red dye probe formulation as described above; and (b) lyophilizing the aqueous solution to form the lyophilized far-red dye probe formulation. In another aspect, the present invention provides a stabilized lyophilized far-red dye probe formulation prepared by the aforementioned method.

[0008] In another aspect, the present invention provides stabilized lyophilized far-red dye probe formulations that allow for reconstitution into aqueous formulations as described above.

[0009] In another aspect, the present invention provides a kit comprising (i) a first sealed container containing a lyophilized far-red dye probe formulation as described above, and (ii) a second sealed container containing a diluent. In some embodiments, the diluent comprises a nonlinear surfactant or foam vanadium, and in some such embodiments, the nonlinear surfactant or foam vanadium is present in the diluent at a concentration of greater than about 0.05% (v / v) (e.g., from about 0.06% (v / v) to about 20% (v / v), from about 0.1% (v / v) to about 10% (v / v), or from about 0.5% (v / v) to about 0.5% (v / v) to about 5% (v / v)).

[0010] In yet another aspect, the present invention provides methods for preparing a stabilized aqueous far-red dye probe formulation. In some embodiments, the methods generally comprise: (a) providing a lyophilized far-red dye probe formulation as described above; and (b) dissolving the lyophilized far-red dye probe formulation in a diluent to provide a reconstituted formulation. In some embodiments, the diluent comprises a nonlinear surfactant or foam vanadium. In some such embodiments, the nonlinear surfactant or foam vanadium is present in the diluent at a concentration greater than about 0.05% (v / v) (e.g., from about 0.06% (v / v) to about 20% (v / v), from about 0.1% (v / v) to about 10% (v / v), or from about 0.5% (v / v) to about 0.5% (v / v) to about 5% (v / v)).

[0011] In other embodiments, a method for preparing a stabilized aqueous far-red dye probe formulation generally includes: (a) providing a lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution comprising at least one buffer and a far-red dye probe comprising a far-red dye bound to a carrier molecule; and (b) dissolving the lyophilized far-red dye probe formulation in a diluent to provide a reconstituted formulation, wherein at least one of the lyophilized far-red dye probe formulation and the diluent comprises a nonlinear surfactant or foam vanadium, and the reconstituted formulation comprises a nonlinear surfactant or foam vanadium at a concentration greater than about 0.05% (v / v). In some embodiments, both the lyophilized far-red dye probe formulation and the diluent comprise a nonlinear surfactant or foam vanadium. In some embodiments, the method further includes preparing the lyophilized far-red dye probe formulation by lyophilizing an aqueous solution comprising the far-red dye probe and at least one buffer.

[0012] In another aspect, the present invention provides a kit comprising: (i) a first sealed container containing a lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution comprising at least one buffer and a far-red dye probe comprising a far-red dye bound to a carrier molecule; and (ii) a second sealed container containing a diluent, wherein at least one of the lyophilized far-red dye probe formulation and the diluent comprises a nonlinear surfactant or formaldehyde, and reconstitution of the lyophilized far-red dye probe formulation in the diluent provides a final nonlinear surfactant or formaldehyde concentration of greater than about 0.05% (v / v). In some embodiments, both the lyophilized far-red dye probe formulation and the diluent comprise a nonlinear surfactant or formaldehyde.

[0013] In yet another aspect, the present invention provides a diagnostic product comprising a sealed container containing a stabilized far-red dye probe formulation as described above. These and other aspects of the present invention will become apparent upon reference to the following detailed description of the invention.

[0014] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the methods and compositions being described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0015] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0016] As used herein, the term "far-red dye" refers to a fluorescent molecule having an emission maximum of about 630 nm to about 800 nm. In some embodiments, the far-red dye has an emission maximum of about 630 nm to about 750 nm, about 640 nm to about 750 nm, about 630 nm to about 700 nm, about 640 nm to about 700 nm, about 630 nm to about 680 nm, or about 640 nm to about 680 nm. Typically, far-red dyes are excited by a long-wavelength excitation source (e.g., a laser source providing a wavelength of about 625 nm to about 655 nm).

[0017] As used herein, the term "carrier molecule" refers to a biological or non-biological component that can be covalently attached to a far-red dye. Labeled carrier molecules are useful as probes for monitoring or detecting one or more in vitro, in situ, or in vivo biological or biochemical targets, processes, or reactions. Such components can include, for example, nucleosides, nucleotides, oligonucleotides, nucleic acids, amino acids, peptides, proteins, polysaccharides, drugs, hormones, lipids, lipoproteins, lipid assemblies, synthetic polymers, polymeric microparticles, and combinations thereof. In some variations, the carrier molecule includes a moiety or region capable of specific binding interactions with another molecule (e.g., a target-hybridizing sequence of a nucleic acid carrier molecule or a binding site of a protein, such as the antigen-binding site of an antibody).

[0018] As used herein, a "covalent bond" refers to a direct covalent bond or a covalent bond through several atoms corresponding to a linker moiety.

[0019] The term "stabilized" with respect to a lyophilized far-red dye probe formulation containing a surfactant as described herein means that the far-red dye probe formulation, when used in a detection assay, exhibits less than about a 20% decrease in relative fluorescence units (RFU) at day 30 relative to day 0 upon reconstitution from lyophilized to aqueous form (day 0), as well as at day 30 after reconstitution and after storing the reconstituted formulation at 2-8°C for 30 days (day 30). When used to refer to an aqueous far-red dye probe formulation containing a surfactant as described herein, the term "stabilized" means that the aqueous far-red dye probe formulation can either (a) be reconstituted from a stabilized lyophilized formulation as defined above, or (b) be lyophilized to obtain a stabilized lyophilized formulation as defined above. In some variations, the stabilized far-red dye probe formulation exhibits less than about a 15% decrease in RFU, less than about a 12% decrease, or less than about a 10% decrease in RFU.

[0020] As used herein, the term "nonlinear surfactant" refers to a surfactant having a branched chain structure. Nonlinear surfactants may contain, for example, one or more ring structures that may be in the main chain and / or one or more branches. Exemplary nonlinear surfactants include polysorbate 20, polysorbate 40, polysorbate 60, and digitonin. In some variations, nonlinear surfactants are nonionic.

[0021] As used herein, the term "stabilizing surfactant" refers to a non-linear surfactant or foam binder.

[0022] "Nucleic acid" refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs, where the nucleosides are linked together by phosphodiester or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and their analogs. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages (as in "peptide nucleic acids" or PNAs, see PCT Publication No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of a nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy and 2'-halide substitutions (e.g., 2'-F). The nitrogenous bases may be conventional bases (A, G, C, T, U), their analogs (e.g., inosine, 5-methylisocytosine, isoguanine; The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43:617-24), or derivatives of purine or pyrimidine bases (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5 or 6 positions, purine bases with modified or replaced substituents at the 2, 6, and / or 8 positions, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4These include -alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines; U.S. Patent Nos. 5,378,825, 6,949,367, and PCT Publication No. WO93 / 13121. Nucleic acids may contain "abasic" residues, in which the backbone does not contain a nitrogenous base for one or more residues (U.S. Patent No. 5,585,481). Nucleic acids may contain only conventional sugars, bases, and linkages as found in RNA and DNA, or may contain conventional components and substitutions (e.g., conventional bases linked by a 2'-methoxy backbone, or nucleic acids containing a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked into an RNA-mimicking sugar conformation, which enhances hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., Biochemistry 43:13233-41, 2004). Nucleic acids may contain modified bases to alter the function or behavior of the nucleic acid, for example, the addition of 3'-terminal dideoxynucleotides to prevent additional nucleotides from being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art, but nucleic acids may also be purified from natural sources using routine techniques.

[0023] As used herein, a "nucleotide" is a nucleic acid subunit consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of ribose (2'-O-Me).

[0024] As used herein, "target nucleic acid" refers to a nucleic acid containing a target sequence to be detected. The target nucleic acid may be DNA or RNA as described herein, and may be either single-stranded or double-stranded. The target nucleic acid may also contain other sequences other than the target sequence.

[0025] As used herein, the term "target sequence" refers to the specific nucleotide sequence of a target nucleic acid to be detected. The "target sequence" includes a composite sequence to which an oligonucleotide (e.g., a probe oligonucleotide, a priming oligonucleotide, and / or a promoter oligonucleotide) is composited during the detection process (e.g., an amplification-based detection assay such as TMA or PCR). If the target nucleic acid is originally single-stranded, the term "target sequence" will also refer to a sequence complementary to the "target sequence" as present in the target nucleic acid. If the target nucleic acid is originally double-stranded, the term "target sequence" will refer to both the sense (+) strand and the antisense (-) strand. When selecting a target sequence, those skilled in the art will understand that a "unique" sequence should be selected to distinguish closely related target nucleic acids from unrelated target nucleic acids.

[0026] The term "target hybridization sequence" is used herein to refer to a portion of an oligomer configured to hybridize with a target nucleic acid sequence. Preferably, the target hybridization sequence is configured to specifically hybridize with the target nucleic acid sequence. The target hybridization sequence may, but need not, be 100% complementary to the portion of the target sequence to which it is configured to hybridize. The target hybridization sequence may also contain inserted, deleted, and / or substituted nucleotide residues relative to the target sequence. For example, when the target nucleic acid is from multiple strains within a species (e.g., various strains of a bacterial or viral species), the target hybridization sequence may be less than 100% complementary to the target sequence. It is understood that there are other reasons for configuring a target hybridization sequence to have less than 100% complementarity to the target nucleic acid.

[0027] As used herein, the term "region" refers to a portion of nucleic acid, and this portion is smaller than the entire nucleic acid.For example, when the nucleic acid referred to is a promoter-based amplification oligomer, the term "region" can be used to refer to the smaller promoter portion of the entire oligonucleotide.Similarly, and also just for example, when nucleic acid is target nucleic acid, the term "region" can be used to refer to the smaller region of nucleic acid, and this smaller region is targeted by one or more oligonucleotides.

[0028] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" generally refer to nucleic acids having fewer than 1,000 nucleotide (nt) residues, including polymers within a range having a lower limit of about 5 nt residues and an upper limit of about 500-900 nt residues. In some embodiments, oligonucleotides fall within a size range having a lower limit of about 12-15 nt and an upper limit of about 50-600 nt, while other embodiments fall within a range having a lower limit of about 15-20 nt and an upper limit of about 22-100 nt. Oligonucleotides may be purified from naturally occurring sources or synthesized using any of a variety of well-known enzymatic or chemical methods. The term "oligonucleotide" does not denote any specific function for the reagent, but rather is used generically to encompass all such reagents described herein. Oligonucleotides may serve a variety of different functions. For example, an oligonucleotide can function as a primer if it is specific for and capable of hybridizing to a complementary strand and can be further extended in the presence of a nucleic acid polymerase; it can function as a primer and provide a promoter if it contains a sequence recognized by an RNA polymerase and allows transcription (e.g., a T7 primer); it can function to detect a target nucleic acid if it is capable of hybridizing to a target nucleic acid or its amplicon and further provides a detectable moiety (e.g., a far-red dye).

[0029] An "amplification oligomer" is an oligomer that has at least its 3' end complementary to a target nucleic acid, hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase during the amplification process. Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (e.g., because it has a 3' blocked end), but participates in or facilitates amplification. For example, the 5' region of an amplification oligonucleotide may contain a promoter sequence that is non-complementary to the target nucleic acid (which may be referred to as a "promoter primer" or "promoter provider"). Those skilled in the art will understand that an amplification oligomer that functions as a primer can be modified to include a 5' promoter sequence and thus function as a promoter primer. Incorporating a 3' blocked end further modifies the promoter primer, which is now capable of hybridizing to the target nucleic acid and providing an upstream promoter sequence that serves to initiate transcription, but does not provide a primer for oligonucleotide extension. Such modified oligos are referred to herein as "promoter provider" oligomers. Amplification oligonucleotides range in size from about 10 to about 70 nt in length (not including any promoter sequence or poly-A tail) and include those containing at least about 10 contiguous bases, or even at least 12 contiguous bases, that are complementary to a region of the target nucleic acid sequence (or its complementary strand). The contiguous bases are at least 80%, or at least 90%, or even fully complementary to the target sequence to which the amplification oligomer binds. Amplification oligomers may optionally contain modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but that are not complementary to or contained in the target nucleic acid or template sequence.

[0030] As used herein, a "promoter-based amplification oligomer" means either a promoter primer or a promoter provider.

[0031] As used herein, a "promoter" is a specific nucleic acid sequence that binds to a nucleic acid and is recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to initiate transcription of RNA at a specific site.

[0032] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, or its complement or fragments. The multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothermal amplification. In some embodiments, isothermal amplification is preferred. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as QB replicase (e.g., U.S. Pat. No. 4,786,600). PCR amplification uses DNA polymerase, primer pairs, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by employing multiple cycles of hybridization, ligation, and denaturation (e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses a restriction endonuclease and a primer containing a recognition site for the endonuclease that nicks one strand of a semi-modified DNA duplex containing the target sequence, thereby causing amplification in a series of primer extension and strand displacement steps (e.g., U.S. Pat. Nos. 5,422,252, 5,547,861, and 5,648,211). Amplification methods include embodiments suitable for amplifying RNA target nucleic acids, such as transcription-mediated amplification (TMA) or NASBA.

[0033] "Transcription-associated amplification," also referred to herein as "transcription-mediated amplification" (TMA), refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template-complementary oligonucleotide containing a promoter sequence, and may optionally include one or more other oligonucleotides. Variations of transcription-associated amplification are well known in the art, as previously disclosed in detail (e.g., U.S. Pat. Nos. 4,868,105, 5,124,246, 5,130,238, 5,399,491, 5,437,990, 5,554,516, and 7,374,885, and PCT Publication Nos. WO 88 / 01302, WO 88 / 10315, and WO 95 / 03430).

[0034] The term "amplicon," used interchangeably with "amplification product," refers to a nucleic acid molecule produced during an amplification procedure that is complementary to or homologous to a sequence contained within a target sequence. These terms can be used to refer to a single-stranded amplification product, a double-stranded amplification product, or one of the strands of a double-stranded amplification product.

[0035] "Detection oligonucleotide" and "detection probe oligomer" are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization, to enable detection of the target sequence or amplified nucleic acid. Detection can be either direct (e.g., a probe directly hybridized to its target sequence) or indirect (e.g., a probe bound to its target via an intermediate molecular structure). A detection probe oligomer can be DNA, RNA, an analog thereof, or a combination thereof. The "target sequence" of a detection probe oligomer generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through standard base pairing. Detection probe oligomers can contain target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication No. 2006 / 0068417).

[0036] The term "TaqMan® probe" refers to a detection oligonucleotide that contains a fluorescent dye, typically at the 5' base, and a non-fluorescent quenching dye (quencher), typically at the 3' base. When illuminated, the excited fluorescent dye transfers energy to a nearby quenching dye molecule rather than fluorescing, resulting in a non-fluorescent substrate. During amplification, the exonuclease activity of the polymerase cleaves the TaqMan probe, separating the fluorophore from the quencher, thereby releasing an unquenched signal from the fluorophore as an indication of amplification.

[0037] As used herein, the structure referred to as a "molecular torch" is designed to contain distinct self-complementary regions ("closing domains") connected by binding regions ("target binding domains") and hybridizing to each other under predetermined hybridization assay conditions. All or part of the nucleotide sequence comprising the target closing domain can also function as the target binding domain. Thus, the target closing sequence can include target binding sequences, non-target binding sequences, and combinations thereof.

[0038] A "polypeptide" or "polypeptide chain" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of about 25 amino acid residues or fewer are commonly referred to as "peptides."

[0039] A "protein" is a macromolecule containing one or more polypeptide chains. Proteins may also contain non-peptide components, such as carbohydrate groups. Carbohydrate and other non-peptide substituents may be added to proteins by the cell in which the protein is produced, and vary depending on the type of cell.

[0040] A "peptide aptamer" is a peptide that specifically binds to a target protein and is embedded as a loop within a protein scaffold. See generally, e.g., Li et al., Curr. Med. Chem. 18:4215-4222, 2011.

[0041] As used herein, the term "antibody" refers to any immunoglobulin protein that specifically binds to an antigen, as well as antigen-binding fragments thereof and recombinant variants thereof. Thus, the term "antibody" includes, for example, polyclonal antibodies, monoclonal antibodies, and antigen-binding antibody fragments containing the paratope of an intact antibody, such as Fab, Fab', F(ab')2, and F(v) fragments. Also included are genetically engineered intact antibodies and fragments, such as chimeric antibodies, humanized antibodies, single-chain Fv fragments, single-chain antibodies, diabodies, minibodies, linear antibodies, and multivalent or multispecific hybrid antibodies. Thus, the term "antibody" is used broadly to include any protein that contains the antigen-binding site of an antibody and is capable of binding to that antigen.

[0042] As used herein, the term "diluent" refers to a solution suitable for modifying or achieving the exemplary or suitable concentrations described herein.

[0043] The term "container" refers to something (e.g., a holder, vessel, container, etc.) in which an object or liquid can be placed or contained, e.g., for storage.

[0044] References herein to numerical ranges (eg, "X to Y" or "X to Y") include the endpoints defining the range, and all values ​​subsumed within the range.

[0045] Unless otherwise clear from the context, when a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention provides stabilized formulations of far-red dye probes containing a surfactant selected from a nonlinear surfactant and a formaldehyde. The formulations are based, in part, on the surprising observation that surfactant-containing formulations exhibit reduced loss of fluorescent signal intensity (RFU) of the far-red dye probe when stored in aqueous form over long periods of time compared to formulations that do not contain the stabilizing surfactant. Without wishing to be bound by theory, the inventors believe that in the absence of a stabilizing surfactant, the far-red dye probe in buffer aggregates over time to form organized structures (e.g., micelles) where more nonpolar fluorophore molecules are in close contact and tend to self-quench; in the presence of a stabilizing surfactant (e.g., a nonpolar nonlinear surfactant), the far-red dye probe aggregation is disrupted so that the fluorophore molecules are no longer in close proximity and therefore cannot self-quench. Particularly suitable nonlinear surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, and polysorbate 60) and digitonin.

[0047] In certain embodiments, the stabilized far-red dye probe formulation is an aqueous formulation. Such a formulation may be, for example, a previously lyophilized formulation or one reconstituted from a lyophilized form. In some variations, the formulation is provided as an aqueous solution containing a far-red dye probe comprising a far-red dye conjugated to a carrier molecule, a surfactant selected from nonlinear surfactants and foam buffers at a concentration greater than about 0.05% (v / v), and at least one buffer. In some embodiments, the surfactant is present at a concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.06% (v / v) to about 3% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), or about 0.1% (v / v) to about 0% (v / v). The surfactant is present at a concentration of about 3% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 3% (v / v), about 1% (v / v) to about 20% (v / v), about 1% (v / v) to about 10% (v / v), or about 1% (v / v) to about 3% (v / v). In more specific variations, the surfactant is present at a concentration of about 0.41% (v / v), about 0.62% (v / v), about 1% (v / v), about 1.24% (v / v), about 1.5% (v / v), about 1.6% (v / v), or about 3% (v / v).

[0048] The buffering agent is typically present at a concentration sufficient to maintain a pH suitable for use of the far-red dye probe in biological systems, such as in vitro or in situ assays. In some embodiments, the buffering agent is present at a concentration sufficient to maintain a pH in the range of about 5.5 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 8.0, or about 6.5 to about 7.5. Suitable buffering agents include Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate, citrate, succinate, and histidine. In certain embodiments, the Tris buffer is present at a concentration of about 5 mM to about 50 mM or about 10 mM to about 50 mM. Other suitable concentrations of buffering agent for formulations according to the present invention can be readily determined by one of ordinary skill in the art.

[0049] In certain variations, formulations, such as those suitable for lyophilization, those reconstituted from lyophilized forms, or lyophilized formulations for reconstitution into aqueous formulations as described herein, may contain a cryoprotectant. Exemplary cryoprotectants include glycerol, non-reducing sugars such as sucrose, raffinose, or trehalose, and amino acids such as glycine, arginine, or methionine. The use of cryoprotectants, including the selection of an appropriate concentration to prevent unacceptable amounts of degradation and / or aggregation of carrier molecules during lyophilization, is generally well known in the art. In some variations where the cryoprotectant is glycerol, the cryoprotectant concentration in the aqueous formulation ranges from about 1% (v / v) to about 10% (v / v), from about 2% (v / v) to about 8% (v / v), or from about 2% (v / v) to about 5% (v / v).

[0050] The concentration of the far-red dye probe in the stabilized formulations described herein will vary depending on the particular probe carrier molecule and the desired application, and suitable probe concentrations can be readily determined by one of skill in the art for a particular application. In certain variations, the far-red dye probe (e.g., the far-red dye probe comprising the nucleic acid carrier molecule) is present in the stabilized formulation at a concentration of about 0.01 μM to about 50 mM, about 0.01 μM to about 5 mM, about 0.05 μM to about 500 μM, about 0.05 μM to about 100 μM, about 0.1 μM to about 100 μM, or about 0.1 μM to about 50 μM. In other variations, the far-red dye probe (e.g., the far-red dye probe comprising a nucleic acid carrier molecule) is present in the stabilized formulation at a concentration of about 0.001 mg / mL to about 100 mg / mL, about 0.001 mg / mL to about 50 mg / mL, about 0.01 mg / mL to about 25 mg / mL, or about 0.01 mg / mL to about 10 mg / mL.

[0051] Suitable far-red dyes for use in accordance with the present invention include Cyanine 5 (Cy5), Cyanine 5.5 (Cy5.5), ALEXA FLUOR® 633, ALEXA FLUOR® 635, ALEXA FLUOR® 647, QUASAR® 705, QUASAR® 650, DYLIGHT® 649, DYLIGHT® 650, HILYTE™ 647, ATTO™ 647, and Allophycocyanin (APC). CyLyte Fluor and HILYTE Fluor dyes are available from AnaSpec, Inc., Fremont, California. ALEXA FLUOR dyes are available from Thermo Fisher Scientific, Waltham, Massachusetts. ATTO™ 647 and Allophycocyanin (APC) are available from Millipore-Sigma, St. Louis, Missouri. Cyanine 5 and Cyanine 5.5 are available from Glen Research, Sterling, Virginia. QUASAR 650 and QUASAR 705 are available from LGC Biosearch Technologies, Petaluma, California. Other suppliers include, but are not limited to, Dyomics (Jena, Germany) and Atto-Tec GmbH (Siegen-Weidenau, Germany). In some embodiments, the far-red dye is a far-red cyanine dye such as, for example, Cy5, Cy5.5, ALEXA FLUOR 647, or ATTO 647.

[0052] According to the present invention, various carrier molecules can be used.Suitable carrier molecules can include nucleosides, nucleotides, nucleic acids (e.g., oligonucleotides), amino acids, proteins (e.g., peptides, antibodies), polysaccharides, hormones, drugs, lipids, lipoproteins, lipid aggregates, synthetic polymers, polymeric microparticles, and combinations thereof.Particularly suitable carrier molecules include moieties or regions that can specifically bind with other molecules.

[0053] In certain variations, the probe carrier molecule is a nucleic acid that specifically binds to a target molecule, such as a target nucleic acid. Particularly suitable nucleic acid carrier molecules include oligonucleotides containing a target hybridization sequence that specifically binds to a target sequence contained in a target region of a target nucleic acid. The oligonucleotide carrier can be, for example, a DNA or RNA oligomer, or an oligomer containing a combination of DNA and RNA nucleotides, or an oligomer synthesized with a modified backbone, such as an oligomer containing one or more 2'-methoxy-substituted ribonucleotides. In some embodiments, the probe containing the oligonucleotide carrier molecule contains a quencher in addition to a far-red dye, which is particularly useful in fluorescence resonance energy transfer (FRET) assays; specific variations of such probes include, for example, TaqMan detection probes (Roche Molecular Diagnostics) and "molecular beacons" (see, for example, Tyagi et al., Nature Biotechnol. 16:49-53, 1998; U.S. Patent Nos. 5,118,801 and 5,312,728, each of which is incorporated herein by reference).

[0054] The oligonucleotide carrier molecule comprising a target hybridization sequence can further comprise a non-target hybridization sequence.Specific embodiments of the oligonucleotide probe comprising a non-target hybridization sequence include probes that form a conformation that is maintained by intramolecular hybridization, such as the conformation that is generally referred to as hairpin.Particularly suitable hairpin probes include "molecular torches" (see, for example, U.S. Patent Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, each of which is incorporated herein by reference) and "molecular beacons" (see, for example, Tyagi et al., supra, U.S. Patent Nos. 5,118,801 and 5,312,728, supra).Methods for using such hairpin probes are well known in the art.In other embodiments, the oligonucleotide carrier molecule is a linear oligomer that does not substantially form a conformation that is maintained by intramolecular bond.

[0055] In other embodiments, the probe carrier molecule is a protein. Particularly suitable protein carrier molecules include antibodies and other proteins that have binding specificity for another molecule, such as peptides (e.g., neuropeptides, peptide hormones), peptide aptamers, antibody-binding proteins, toxins, lectins, growth factors, cytokines, enzymes, and enzyme substrates. Antibody-binding proteins can include, for example, protein A, protein G, soluble Fc receptors, protein L, anti-IgG, anti-IgA, anti-IgM, anti-IgD, anti-IgE, and fragments thereof. In some variations, peptide carrier molecules can function as organelle-localizing peptides by targeting attached far-red dyes for localization within specific cellular substructures via cellular transport mechanisms. In some variations, protein carrier molecules (e.g., antibodies, peptides, peptide aptamers, lectins) specifically bind to cell surface molecules, and cell surface-binding proteins such as antibodies can be used, for example, in various cell imaging and flow cytometry applications, including, for example, microscopy, cell counting, cell sorting, and biomarker detection.

[0056] In yet other embodiments, the carrier molecule comprises a lipid (e.g., a lipid having 6 to 25 carbons), including glycolipids, phospholipids, and sphingolipids. In some variations, the carrier molecule is a lipid aggregate (e.g., a liposome) or a lipoprotein. Some lipophilic substituents are useful for facilitating transport of the conjugated far-red dye into cells or organelles.

[0057] Methods for attaching fluorescent labels to carrier molecules, including biomolecules such as nucleic acids and proteins, to generate labeled probes are generally well known in the art and are readily utilized by those skilled in the art in preparing far-red dye probes in accordance with the present invention.

[0058] Stabilized formulations containing far-red dye probes as described herein may further comprise one or more additional components for conducting assays using the probe. For example, in some embodiments of stabilized far-red dye probe formulations containing oligonucleotide carrier molecules, the formulation further comprises one or more amplification oligomers for producing amplification products that can specifically hybridize with the oligonucleotide in an amplification and detection assay. Thus, in some variations, a far-red dye probe formulation containing an oligonucleotide bound to a far-red dye probe further comprises a first amplification oligomer, where (i) the oligonucleotide comprises a target hybridization sequence that specifically binds to a first sequence contained within a target region of a target nucleic acid, (ii) the first amplification oligomer comprises a target hybridization sequence that specifically binds to a second sequence contained within the target region, and (iii) the first amplification oligomer is configured to produce an amplification product containing the target region in an amplification assay that includes a target nucleic acid as a template. In some such embodiments, the formulation further comprises a second amplification oligomer comprising a target-hybridizing sequence that specifically binds to a third sequence contained within the target region, and the first and second amplification oligomers are configured to amplify the target region over multiple cycles of an amplification assay. In some variations of formulations further containing one or more amplification oligomers, the amplification oligomer(s) are configured to perform transcription-associated amplification of the target region; for example, in some aspects further comprising a first amplification oligomer as described above, the first amplification oligomer is a promoter-based amplification oligomer that further comprises a promoter sequence (e.g., a T7 promoter sequence) located 5' of the first target-hybridizing sequence. In yet other non-mutually exclusive embodiments of formulations further containing one or more amplification oligomers, the amplification oligomer(s) are configured to effect a distinct phase of an amplification procedure comprising two or more distinct phases (also referred to herein as "multiphasic" nucleic acid amplification), such amplification systems being described, for example, in U.S. Pat. No. 9,139,870 to Nelson et al., incorporated herein by reference.Formulations that further contain one or more amplification oligomers as described above may further include one or more additional components suitable for conducting an amplification assay, such as, for example, salts, cofactors, nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase).

[0059] In some embodiments, stabilized far-red dye probe formulations as described herein are concentrated preparations of far-red dye probes (e.g., oligonucleotide far-red dye probes, optionally with one or more additional components for conducting amplification and detection assays), and are often useful as bulk products for use in assays.

[0060] In typical variations, the formulation is stable for an extended period of time. For example, the formulation may be stable for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, or at least about 6 months. In some embodiments, the formulation is stable for at least about 12 months, at least about 18 months, at least about 24 months, or at least about 30 months.

[0061] Stabilized far-red dye probe formulations as described herein can be stored at temperatures of about -80°C to about 40°C, about -20°C to about 25°C, about 0°C to about 25°C, about 0°C to about 15°C, about 0°C to about 10°C, or about 2°C to about 8°C. In various embodiments, the formulations can be stored at about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. Generally, the formulations are stable and retain activity within these ranges. In some variations, the formulations are stable at about -80°C to about 25°C or about 4°C to about 25°C. In more specific variations, the liquid formulations are stable at temperatures of about -80°C to about -20°C, about -80°C to about 4°C, or about -80°C to about 25°C. In other specific variations, the lyophilized formulation is stable at temperatures of about 4° C. to about 25° C. or about 4° C. to about 40° C. Intermediate ranges of temperatures between the above are also contemplated as part of the invention, e.g., about 2° C. to about 18° C. For example, ranges of values ​​using any combination of the above values ​​as upper and / or lower limits are intended to be included.

[0062] In specific embodiments, for long-term storage, aqueous formulations as described herein can be aliquoted into, for example, vials, ampoules, or other containers and lyophilized according to procedures known in the art. The lyophilized product typically appears as a powder or cake. The container is then sealed, and in some such variations, the seal allows for subsequent injection of a diluent into the container through the seal. Methods for preparing such stabilized lyophilized far-red dye probe formulations from aqueous formulations, as well as lyophilized formulations prepared by such methods, are additional aspects of the present invention. In yet another aspect, the present invention provides stabilized lyophilized far-red dye probe formulations that allow for reconstitution into aqueous far-red dye probe formulations as described herein.

[0063] Also encompassed within the present invention are methods for preparing stabilized aqueous far-red dye probe formulations from lyophilized formulations as described herein. Such methods generally involve dissolving the lyophilized far-red dye probe formulation in a suitable diluent to provide a reconstituted formulation. Suitable diluents can be readily selected by those skilled in the art depending, for example, on the intended use of the far-red dye probe, and may include, for example, water or an aqueous solution containing a buffer (e.g., Tris). In some embodiments, the diluent contains a stabilizing surfactant, such as that contained in the stabilized lyophilized formulation. Thus, in some variations, the diluent contains a non-linear surfactant (e.g., a polyoxyethylene sorbitan fatty acid ester or digitonin) or formaldehyde, and in some such embodiments, the stabilizing surfactant is present in the diluent at a concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.06% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 5% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), or about 0.5% (v / v) to about 5% (v / v).

[0064] In a related aspect, the aqueous stabilized far-red dye probe formulations described herein are generally prepared by a method comprising the steps of: (a) providing a lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution comprising at least one buffer and a far-red dye probe comprising a far-red dye bound to a carrier molecule; and (b) dissolving the lyophilized far-red dye probe formulation of (a) in a diluent to provide a reconstituted formulation, wherein at least one of the lyophilized far-red dye probe formulation and the diluent contains a nonlinear surfactant or formaldehyde, and the reconstituted formulation contains the nonlinear surfactant or formaldehyde at a concentration greater than about 0.05% (v / v). In some embodiments, only the lyophilized formulation comprises a stabilizing surfactant, and in some such variations, the stabilizing surfactant is present in the aqueous solution from which the lyophilized formulation is derived at a concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v). In other embodiments, only the diluent comprises a stabilizing surfactant, and in some such variations, the stabilizing surfactant is present in the diluent at a concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v).In still other embodiments, both the lyophilized far-red dye probe formulation and the diluent contain a non-linear surfactant or formaldehyde, and in some such variations, the stabilizing surfactant is present in the lyophilized formulation and diluent at a concentration configured to produce, upon reconstitution of the lyophilized formulation in the diluent, a final surfactant concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v). The above method may further comprise preparing a lyophilized far-red dye probe formulation by lyophilizing an aqueous solution comprising the far-red dye probe and at least one buffer.

[0065] In certain aspects of the present invention, a container containing a stabilized lyophilized far-red dye probe formulation as described herein is provided in a kit with a second container containing a diluent. The diluent may contain a nonlinear surfactant or a formaldehyde, as discussed above with respect to preparing a reconstituted formulation from a lyophilized formulation. The far-red dye probe formulation and diluent may be packaged in a variety of different embodiments, and those skilled in the art will understand that the present invention encompasses many different kit configurations. For example, in embodiments in which the far-red dye probe comprises an oligonucleotide carrier molecule containing a target hybridizing sequence that specifically binds to a nucleic acid target region, the kit may further include a third container containing one or more amplification oligomers for amplifying the target region. In some such variations, where the far-red dye probe formulation includes an amplification oligomer that specifically binds to the target region, a third container may contain a second amplification oligomer configured to specifically bind to the target region and, in an amplification assay, generate an amplification product containing the target region; such kit embodiments may be used in a multiphase amplification system, such as that described, for example, in U.S. Pat. No. 9,139,870 to Nelson et al., incorporated herein by reference. Kits containing oligonucleotide far-red dye probes for use in amplification and detection assays may contain other reagents suitable for performing in vitro amplification, such as, for example, buffers, salt solutions, appropriate triphosphate nucleotides (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase). In certain embodiments, the kit further includes a set of instructions for practicing a method according to the invention; the instructions may be associated with a package insert and / or the packaging of the kit or its components.

[0066] In a related aspect, the present invention provides a kit comprising: (i) a first sealed container containing a lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution comprising at least one buffer and a far-red dye probe comprising a far-red dye bound to a carrier molecule; and (ii) a second sealed container containing a diluent, wherein at least one of the lyophilized far-red dye probe formulation and the diluent comprises a nonlinear surfactant or formaldehyde, and wherein reconstitution of the lyophilized far-red dye probe formulation in the diluent provides a final concentration of the nonlinear surfactant or formaldehyde of greater than about 0.05% (v / v). In some embodiments, only the lyophilized formulation comprises a stabilizing surfactant, and in some such variations, the stabilizing surfactant is present in the aqueous solution from which the lyophilized formulation is derived at a concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v). In other embodiments, only the diluent comprises a stabilizing surfactant, and in some such variations, the stabilizing surfactant is present in the diluent at a concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v).In still other embodiments, both the lyophilized far-red dye probe formulation and the diluent contain a non-linear surfactant or formaldehyde, and in some such variations, the stabilizing surfactant is present in the lyophilized formulation and diluent at a concentration configured to produce, upon reconstitution of the lyophilized formulation in the diluent, a final surfactant concentration of about 0.06% (v / v) to about 20% (v / v), about 0.06% (v / v) to about 10% (v / v), about 0.1% (v / v) to about 20% (v / v), about 0.1% (v / v) to about 10% (v / v), about 0.5% (v / v) to about 20% (v / v), about 0.5% (v / v) to about 10% (v / v), about 1% (v / v) to about 20% (v / v), or about 1% (v / v) to about 10% (v / v). As previously mentioned, the far-red dye probe formulation and diluent may be packaged in a variety of different embodiments, and one of skill in the art will appreciate that the present invention encompasses many different kit configurations.

[0067] In yet another aspect, the present invention provides a diagnostic product comprising a sealed container containing the stabilized far-red dye probe formulation described above. In some variations, the stabilized far-red dye formulation is a lyophilized formulation as described herein.

[0068] The present invention is further illustrated by the following non-limiting examples. [Example]

[0069] Unless otherwise stated, reagents commonly used in the RT-TMA-based assays described herein include the following: Target Capture Reagent (TCR) formulation: 250 mM HEPES, 1.88 M LiCl, 310 mM LiOH, 100 mM EDTA, pH 6.4, and 250 μg / mL covalently bound (dT) 14Paramagnetic particles (0.7-1.05 micron particles, Sera-Mag™ MG-CM) with oligomer (SEQ ID NO: 20). Wash solution formulation: 10 mM HEPES, 150 mM NaCl, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v / v) ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate, pH 7.5. Amplification and promoter reagent formulation: 11.61 mM Tris base, 14.94 mM Tris-HCl, 28.5 mM MgCl. 2、 23.30 mM KCl, 3.3% glycerol, 0.02% PRO CLIN 300, 0.05 mM zinc acetate dihydrate, 0.76 mM each of dATP, dCTP, dGTP, and dTTP, 6.50 mM each of ATP, CTP, and GTP, 7.50 mM UTP, plus primers. Enzyme reagent formulation: 57.46 mM HEPES, 49.58 mM N-acetyl-L-cysteine, 0.98 mM EDTA free acid, 0.039 mM EDTA disodium dihydrate, 0.10 v / v TRITON® X-100, 49.61 mM KCl, 0.20 v / v glycerol, 0.03 w / v trehalose dihydrate, MMLV reverse transcriptase (RT), and T7 RNA polymerase.

[0070] Amplification and detection reactions were performed using a Stratagene Mx3000 in a biphasic real-time TMA format. Briefly, samples were incubated with 100 μl of TCR containing target capture oligomers (SEQ ID NOS: 1, 7, 11, and 16, each at 15 pmol / rxn) and T7 primers (SEQ ID NOS: 3, 4, 9, 13, and 18, each at 5 pmol / rxn) for 30 min at 62°C, then cooled to room temperature for 20 min to allow hybridization complexes (magnetic beads-dT 14The hybridization complex was washed and eluted in amplification reagent containing non-T7 primers (SEQ ID NOS: 2, 8, 12, and 17, each at 15 pmol / rxn). The sample was incubated at 43°C during the addition of enzyme reagent (25 μl) followed by promoter reagent (25 μl). The promoter reagent contained T7 primers (SEQ ID NOS: 3, 4, 9, 13, and 18, each at 15 pmol / rxn) and torch oligos (SEQ ID NOS: 5, 6, 10, 14, 15, and 19, each at 15 pmol / rxn). Fluorescence emission, reflecting torch binding to the target amplicon and resulting in dye separation from the quencher, was measured in real time every 30 seconds for 1 hour on a Stratagene instrument. Fluorescence curve profiles were analyzed for target amplification. See, e.g., U.S. Pat. No. 9,139,870 B2. The target nucleic acid for each condition was Lactobacillus Lysates from Bacillus crispatus, Gardnerella vaginalis, Eggerthella lenta, or in vitro transcripts containing at least sequences for hybridization with target capture oligomers, T7 primers, non-T7 primers, and torch for the target to perform the capture, amplification, and detection reactions. (Bacterial strains for preparing lysates were purchased from ATCC, Manassas, VA, catalog numbers ATCC33820, ATCC14018, and ATCC25559.)

[0071] Example 1 Several experiments were performed, demonstrating a 25-70% decrease in Cy5.5 RFU signal observed after 30 days of incubation of Cy5.5 dye-containing solutions. The incubated Cy5.5 dye-containing solutions were used in real-time (RT) TMA assays, generally as described above. In this example, the Cy5.5 dye was attached to a torch oligonucleotide to detect an endogenous control target nucleic acid. While FAM, HEX, and ROX torches only decreased by approximately 10% after 30 days of incubation, some representative data are shown in Table 1 below, demonstrating a 35% decrease in the endogenous control Cy5.5 torch. Additional studies were performed with various far-red dyes as well as alternative buffer formulations, and similar signal decreases were observed over time. Stored far-red dye-containing reagents that exhibit a significant decrease in RFU signal become unusable, as unreliable signals would provide invalid assay results. Therefore, it is not recommended to store far-red dye-containing solutions for later use in assays utilizing RFU signals from these dyes; instead, it is recommended to discard any unused portions. [Table 1]

[0072] The Cy5.5 dye-containing solutions were incubated for 30 days as described above. Before using the stored solutions in real-time amplification and detection reactions, a portion of the Cy5.5 dye-containing solution was heated to 80°C for 10 minutes, while another portion was not. Each Cy5.5 dye-containing condition was then used in amplification and detection reactions, and the results are shown in Table 2. These results show that, compared to the unheated control, the 80°C / 10 minute heating step completely restored the loss of Cy5.5 RFU signal. This indicates that the loss of far-red dye signal in the incubated solutions is not due to dye degradation, but rather to micelle formation, which brings the Cy5.5 fluorophores into close proximity and quenches each other. [Table 2]

[0073] To resolve micelle formation over time, the 30-day storage measurements were repeated for far-red dye-containing solutions that also contained a non-ionic surfactant. In this experiment, TRITON® X-100 or TWEEN® 20 (Millipore Sigma, St. Louis, MO, catalog numbers 93443 and P1379) were used as the non-ionic surfactant. The far-red dye-containing solutions were promoter reagents containing Cy5.5-labeled molecular torch and various concentrations of either TRITON® X-100 or TWEEN® 20, as described above. The solutions were stored for 38 or 40 days and then used in real-time isothermal amplification and detection reactions. The results are shown in Tables 3 and 4.

[0074] Addition of TRITON® X-100 to the promoter reagent reduced Cy5.5 RFU signal by more than 70% from 0 to 40 days of storage compared to the control condition without TRITON® X-100, which reduced it by 23% (see Table 3). [Table 3]

[0075] The addition of TWEEN® 20 to the promoter reagent resulted in minimal reduction in Cy5.5 RFU signal from days 0 to 38 of storage, compared to the control condition without TWEEN® 20, which decreased by 29% from days 0 to 38. As shown in Table 4, 1% to 20% TWEEN® 20 resulted in only a 4.6% to 10.4% reduction in Cy5.5 RFU signal from days 0 to 38, compared to the control condition (i.e., no TWEEN® 20 added), which decreased by 29% from days 0 to 38. [Table 4]

[0076] Concentrations of 1% or less TWEEN® 20 were added to several promoter reagents and tested in amplification and detection reactions as described above. Promoter reagents for use in amplification and detection of target nucleic acids were formulated to contain Cy5.5-labeled torch oligonucleotides and also contain 1% TWEEN® 20, 0.03% TWEEN® 20, or 0.001% TWEEN® 20. The various promoter reagent conditions were then used in real-time isothermal amplification and detection reactions on day 0 and after 42 days of storage (day 42). The results are shown in Table 5. The decrease in Cy5.5 RFU signal from day 0 to day 42 was more pronounced in conditions in which the promoter reagent contained lower concentrations of TWEEN® 20. [Table 5]

[0077] In further experiments, other surfactants were added to the far-red dye-containing solution. Promoter reagents were prepared for use in amplification and detection reactions. Promoter reagents containing Cy5.5 Torch were prepared in bulk solution. The bulk solution was then separated into several conditions, each containing one of the following surfactants (1% v / v): TWEEN® 40 (Millipore Sigma, Cat. No. P1504), TWEEN® 60 (Millipore Sigma, Cat. No. P1629), Synperonic (Millipore Sigma, Cat. No. 7579), Formban (Munzing, Cat. No. MS-575), and digitonin (Millipore Sigma, Cat. No. D141). Each condition was then used in real-time isothermal amplification and detection reactions on day 0, after 17 days of storage (day 17), and after 38 days of storage (day 38). As shown in Table 6, TWEEN® 40, TWEEN® 60, Formban, and digitonin resulted in minimal degradation of Cy5.5 RFU signal from 0 to 38 days of storage. Synperonic did not reduce the degradation of Cy5.5 RFU signal. Like TRITON® X-100, Synperonic has a linear structure, which may reduce the efficiency of these surfactants in interfering with micelle formation of far-red dye molecules. TWEEN® as well as digitonin have branched structures, which make them more promiscuous and therefore less conducive to micelle formation. [Table 6]

[0078] The structures of additional digitonin and synperonic detergents are shown below. Digitonin: [ka] Synperonic F108: [ka]

[0079] Several solutions containing a far-red dye and a surfactant were prepared and lyophilized. The lyophilized compositions were then reconstituted with a diluent also containing a surfactant. The reconstituted solutions were then used in real-time amplification and detection assays. In one configuration, the promoter reagent described above was prepared containing Cy5.5-labeled torch and TWEEN®-20, as shown in Table 7. The physical properties of the lyophilized pellets were evaluated, and pellets with acceptable properties were then reconstituted with a diluent containing 1% TWEEN®-20 to prepare solutions containing 1.41% TWEEN®-20 to 2.24% TWEEN®-20 in the reconstituted promoter reagent. The reconstituted promoter reagent was then used in real-time amplification and detection assays on days 0 and 30, yielding robust Cy5.5 RFU signals without significant degradation between days 0 and 30. Poor physical properties observed after the lyophilization cycle included that pre-lyophilization solutions containing excess surfactant did not freeze dry completely (remained partially liquid), and pre-lyophilization solutions containing little surfactant exhibited blue spots (indicating micelle formation of the far-red dye component). Thus, depending on the formulation of the far-red dye-containing solution, the surfactant concentration in the pre-lyophilization solution only needs to be minimal to prevent micelle formation during the short incubation period before lyophilization. The remaining surfactant required to provide longer-term incubation protection of the far-red dye component is then provided using a surfactant-containing reconstitution solution (diluent). [Table 7] [Table 8-1] [Table 8-2]

[0080] Illustrative Embodiments Embodiment 1 1. A stabilized far-red dye probe formulation comprising: a far-red dye probe comprising a far-red dye bound to a carrier molecule; a nonlinear surfactant at a concentration greater than about 0.05% (v / v); at least one buffer; A formulation, wherein the formulation is an aqueous solution.

[0081] Embodiment 2 2. The formulation of embodiment 1, wherein the far-red dye is a far-red cyanine dye.

[0082] Embodiment 3 The formulation of embodiment 2, wherein the far-red cyanine dye is selected from the group consisting of Cyanine 5 and Cyanine 5.5.

[0083] Embodiment 4 The formulation of any of embodiments 1-3, wherein the non-linear surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters and digitonin.

[0084] Embodiment 5 The formulation of embodiment 4, wherein the non-linear surfactant is a polyoxyethylene sorbitan fatty acid ester.

[0085] Embodiment 6 The formulation of embodiment 5, wherein the polyoxyethylene sorbitan fatty acid ester is selected from the group consisting of polysorbate 20, polysorbate 40, and polysorbate 60.

[0086] Embodiment 7 7. The formulation of any one of embodiments 1-6, wherein the nonlinear surfactant concentration is from about 0.06% (v / v) to about 20% (v / v), from about 0.06% (v / v) to about 10% (v / v), from about 0.1% (v / v) to about 20% (v / v), from about 0.1% (v / v) to about 10% (v / v), or from about 0.1% (v / v) to 3% (v / v).

[0087] Embodiment 8 7. The formulation of any one of embodiments 1-6, wherein the nonlinear surfactant concentration is from about 0.5% (v / v) to about 20% (v / v), from about 0.5% (v / v) to about 10% (v / v), from about 1% (v / v) to about 20% (v / v), from about 1% (v / v) to about 10% (v / v), or from about 1% (v / v) to about 3% (v / v).

[0088] Embodiment 9 The formulation of any one of embodiments 1-8, wherein at least one buffering agent is Tris.

[0089] Embodiment 10 The formulation of embodiment 9, wherein the Tris buffer is present at a concentration of about 5 mM to about 50 mM.

[0090] Embodiment 11 The formulation of any of embodiments 1-10, wherein the carrier molecule is a nucleic acid.

[0091] Embodiment 12 The formulation of embodiment 11, wherein the nucleic acid carrier molecule is RNA.

[0092] Embodiment 13 The formulation of any of embodiments 1-10, wherein the far-red dye probe further comprises a quencher.

[0093] Embodiment 14 The formulation of embodiment 13, wherein the far-red dye probe is selected from the group consisting of a molecular torch, a molecular beacon, and a TaqMan probe.

[0094] Embodiment 15 15. The formulation of any of embodiments 10-14, further comprising a first amplification oligomer, wherein the far-red dye probe comprises a target hybridizing sequence that specifically binds to a first sequence contained within a target region of a target nucleic acid, and the first amplification oligomer comprises a target hybridizing sequence that specifically binds to a second sequence contained within the target region, and wherein the first amplification oligomer is configured to produce an amplification product containing the target region in an amplification assay comprising the target nucleic acid as a template.

[0095] Embodiment 16 16. The formulation of embodiment 15, further comprising a second amplification oligomer, the second amplification oligomer comprising a target-hybridizing sequence that specifically binds to a third sequence contained within said target region, and wherein the first and second amplification oligomers are configured to amplify said target region over multiple cycles of an amplification assay.

[0096] Embodiment 17 The formulation of embodiment 15 or 16, wherein the first amplification oligomer is a promoter-based amplification oligomer further comprising a promoter sequence located 5' to the first target hybridizing sequence.

[0097] Embodiment 18 18. The formulation of any of embodiments 15 to 17, further comprising one or more nucleotide triphosphates suitable for performing said amplification assay.

[0098] Embodiment 19 19. The formulation of any of embodiments 15-18, further comprising one or more salts or cofactors suitable for performing said amplification assay.

[0099] Embodiment 20 1. A stabilized far-red dye probe formulation comprising: a far-red dye probe comprising a far-red dye bound to a carrier molecule; Foamban at a concentration greater than about 0.05% (v / v); at least one buffer; A formulation, wherein the formulation is an aqueous solution.

[0100] Embodiment 21 1. A method for preparing a stabilized lyophilized far-red dye probe formulation, comprising: providing a formulation according to any one of embodiments 1 to 20; and lyophilizing the aqueous solution to form a lyophilized far-red dye probe formulation.

[0101] Embodiment 22 22. A stabilized lyophilized far-red dye probe formulation prepared by the method of embodiment 21.

[0102] Embodiment 23 A stabilized lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution according to any one of embodiments 1 to 20.

[0103] Embodiment 24 1. A method for preparing a stabilized aqueous far-red dye probe formulation, comprising: (a) providing a lyophilized far-red dye probe formulation according to embodiment 22 or 23; (b) dissolving the lyophilized far-red dye probe formulation in a diluent to provide a reconstituted formulation.

[0104] Embodiment 25 a first sealed container containing the lyophilized far-red dye probe formulation of embodiment 22 or 23; a second sealed container containing a diluent.

[0105] Embodiment 26 26. The kit of embodiment 25, wherein the diluent comprises a non-linear surfactant.

[0106] Embodiment 27 1. A method for preparing a stabilized aqueous far-red dye probe formulation, comprising: (a) providing a lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution comprising at least one buffer and a far-red dye probe comprising a far-red dye bound to a carrier molecule; (b) dissolving the lyophilized far-red dye probe formulation in a diluent to provide a reconstituted formulation; A method wherein at least one of the lyophilized far-red dye probe formulation and the diluent comprises a nonlinear surfactant, and the reconstituted formulation comprises a nonlinear surfactant at a concentration greater than about 0.05% (v / v).

[0107] Embodiment 28 28. The method of embodiment 27, wherein both the lyophilized far-red dye probe formulation and the diluent comprise a non-linear surfactant.

[0108] Embodiment 29 29. The method of embodiment 27 or 28, further comprising preparing a lyophilized far-red dye probe formulation by lyophilizing an aqueous solution comprising the far-red dye probe and at least one buffer.

[0109] Embodiment 30 a first sealed container containing a lyophilized far-red dye probe formulation that allows for reconstitution into an aqueous solution comprising at least one buffer and a far-red dye probe comprising a far-red dye bound to a carrier molecule; a second sealed container containing a diluent, The kit, wherein at least one of the lyophilized far-red dye probe formulation and the diluent comprises a nonlinear surfactant, and reconstitution of the lyophilized far-red dye probe formulation in the diluent provides a final nonlinear surfactant concentration of greater than about 0.05% (v / v).

[0110] Embodiment 31 31. The kit of embodiment 30, wherein both the lyophilized far-red dye formulation and the diluent comprise a non-linear surfactant.

[0111] Embodiment 32 A diagnostic product comprising a sealed container containing the stabilized far-red dye probe formulation of any of embodiments 1 to 20, 22, and 23.

[0112] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

[Claim 1] The invention described in this specification.

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