WO2008111981A1

WO2008111981A1 - Compositions, systems, and methods for preservation of macromolecules

Info

Publication number: WO2008111981A1
Application number: PCT/US2007/063982
Authority: WO
Inventors: Tony Baker
Original assignee: Sierra Molecular Corp
Current assignee: Sierra Molecular Corp
Priority date: 2007-03-14
Filing date: 2007-03-14
Publication date: 2008-09-18
Anticipated expiration: 2009-09-14

Abstract

The present disclosure relates to compositions, systems, and methods for preserving and/or stabilizing a macromolecule and/or biomolecule. A macromolecule stabilizing composition may include a chelator, a chelator enhancing component, and a base {e.g., a purine base or a pyrimidine base). A macromolecule stabilizing method may include contacting a macromolecule with a macromolecule stabilizing composition. A macromolecule stabilizing system may include a container suitable for receiving a sample containing a macromolecule and/or biomolecule and a macromolecule stabilizing composition. A macromolecule and/or biomolecule may be preserved and/or stabilized under ambient conditions (e.g., without refrigeration). A macromolecule and/or biomolecule may include a protein and/or a nucleic acid.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR PRESERVATION OF

MACROMOLECULES

FIELD OF THE INVENTION

The present disclosure relates in general to compositions, systems, and methods for the preservation of a macromolecule and/or a biomolecule. For example, compositions, systems, and methods of the disclosure may be used to preserve and/or stabilize a macromolecule and/or a biomolecule in a condition in which it may interact with another molecule in a conformation-specific and/or sequence specific manner.

BACKGROUND

Macromolecules and biomolecules may be unstable under some conditions. A nucleic acid molecule, for example, may be degraded in the presence of a nuclease. Similarly, a protein molecule may be degraded in the presence of a protease. Degradation of macromolecules and biomolecules may increase with time. The efficacy of assays that include detection of a property of such molecules (presence, concentration, sequence, conformation) may be reduced or lost where such degradation occurs. For example, a diagnostic or forensic assay that depends on detection of minute quantities of a biomolecule may be unable to return a reliable result where the biomolecule has been degraded. Sexually-transmitted disease (STD) clinics regularly screen and treat patients for such diseases as gonorrhea and Syphilis. Infectious agents such as gonococci may be detected by analyzing a DNA sample. A genetic transformation test (GTT), such as Gonostat™ (Sierra Diagnostics, Inc., Sonora, Calif), maybe used to detect gonococcal DNA in specimens taken from the urethra of men, and the cervix and anus of women, according to HW Jaffe et al.(J. Inf. Dis. 146:275-279 (1982)). WL Whittington et al. obtained similar results (Abstr. Ann. Meeting Am. Soc. Microbiol, p. 315 (1983)). However, it is not always possible to immediately test a patient for the presence of an infectious agent. For example, clinical laboratories are not readily found in many rural or underdeveloped areas. In such circumstances, it is necessary to transport patient test specimens to a laboratory for analysis, during which time the target of interest may be partially or wholly degraded.

Degradation of a macromolecule and/or biomolecule may be reduced by lowering the temperature of the macromolecule or biomolecule. However, this option may not be available in all situations or it may not be available for a sufficiently long period of time (e.g., from the time of sample collection to the time of analysis). For example, where a sample is collected (e.g., from a patient) in a remote location, it may be difficult or impossible to preserve the target molecule long enough for the sample to be transported to a facility where the sample is analyzed. In addition, cooling may not be uniform across all samples and/or may not be consistent from experiment to experiment.

Degradation of a macromolecule and/or biomolecule may be reduced by heating a composition to a temperature sufficient to inactivate one or more nucleases or proteases. However only a limited number of proteases and nucleases are inactivated by heating. In addition, heating may degrade rather than preserve a target molecule.

SUMMARY

Therefore, a need has arisen for compositions, systems, and methods for preserving and/or stabilizing a macromolecule and/or a biomolecule. The present disclosure relates to compositions, systems, and methods for preserving and/or stabilizing a macromolecule and/or biomolecule (collectively, "macromolecule"). According to some embodiments, a macromolecule and/or a biomolecule may include a protein and/or a nucleic acid (e.g., DNA and RNA). As will be appreciated by those of ordinary skill in the art, a nucleic acid may include sequences from a plurality of sources. For example, a single nucleic acid may include an artificial sequence (e.g., a primer binding site), a human sequence (e.g., adenomatous polyposis coli (APC), amyloid precursor protein (APP), breast cancer 1 (BRCAl), transmembrane protease serine 2 (TMPRSS2), v-ets erythroblastosis virus E26 oncogene homolog (ERG)), a plant sequence, a microbial sequence (e.g., an antibiotic resistance gene), a viral sequence (e.g., HIV protease), and/or combinations thereof. A single nucleic acid sequence may also include an unusual or artificial fusion of two sequences from a common source (e.g., a TMPRSS2:ERG fusion). A macromolecule may be regarded as preserved as long as the macromolecule, if present, is maintained in a detectable form at least from the time of sample collection to the time of sample analysis, hi some embodiments, the disclosure relates to preservation and/or stabilization of macromolecules in a bodily fluid or excretion (e.g., urine, blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat). In some embodiments, an unexpected improvement in nucleic acid hybridization may be observed in such nucleic acid testing methods (e.g., compared with the same methods practiced in the absence of a preservation composition, system, or method of the disclosure). The present disclosure, according to some embodiments, relates to a composition for preserving and/or stabilizing a macromolecule and/or a biomolecule (a "macromolecule stabilizing composition"). In some embodiments, a macromolecule stabilizing composition may include (a) a chelator selected from the group consisting of ethyl enediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), l,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and salts thereof, (b) at least one chelator enhancing component selected from the group consisting of guanidine, lithium chloride, sodium salicylate, sodium perchlorate, and sodium thiocyanate, and (c) a base selected from the group consisting of a purine base and a pyrimidine base. The concentration of a chelator, a chelator enhancing component, and/or a base may be each selected from any attainable concentration. For example, the concentration of a chelator may be from about 0.00 IM to about 0.1 M, the concentration of a chelator enhancing component may be from about 0.1 M to about 2M; and/or the concentration of a base may be from about 0.1 M to about 5 M. hi some embodiments, a macromolecule stabilizing composition may be formulated as an aqueous solution. In some embodiments, a chelator enhancing component may be selected from the group consisting of sodium perchlorate, sodium thiocyanate, and lithium chloride. A macromolecule stabilizing composition may include, according to some embodiments, at least one enzyme inactivating component selected from the group consisting of manganese chloride, sarkosyl, sodium dodecyl sulfate, and combinations thereof.

A macromolecule stabilizing composition may include a buffer according to some embodiments. For example, a buffer may include a compound selected from the group consisting of potassium acetate, sodium acetate, potassium phosphate, sodium phosphate, tris(hydroxyamino)methane, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 3-(N- morpholino)propane sulfonic acid, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, N-(2- acetamido)2-iminodiacetic acid, 3-[(l,l-dimethyl-2-hydroxyethyl)amino]-2-propanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethylglycine, bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane, 3 -(cyclohexylamino)- 1 - propanesulfonic acid, 3 -(cyclohexylamino)-2 -hydroxy- 1 -propanesulfonic acid, 2-(N- cyclohexylamino)ethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxy- propanesulfonic acid, N-(2-hydroxyethylpiperazine)-N' -(3 -propanesulfonic acid), N-(2- hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid), 2-(N- morpholine)ethanesulfonic acid, triethanolamine buffer, imidazole, glycine, ethanolamine, 3- (N-morplioline)-2-hydroxypropanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), piperazine-N,N' -bis(2-hydroxypropanesulfonic acid), N-tris [(hydroxymethyl)methyl] -3 - aminopropanesulfonic acid, 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-l- propanesulfonic acid, N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, N- [Tris(hydroxymethyl)methyl]glycine, 2-amino-2 -methyl- 1,3 -propanediol, 2-amino-2-methyl- 1-propanol, and combinations thereof.

According to some embodiments, the present disclosure also relates to a method of preserving and/or stabilizing a macromolecule (a "macromolecule stabilizing method"). A macromolecule stabilizing method may include, for example, contacting a macromolecule with a macromolecule stabilizing composition comprising (a) a chelator selected from the group consisting of etbylenediaminetetraacetic acid (EDTA),

[ethyl enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), 1 ,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA), and salts thereof, (b) at least one chelator enhancing component selected from the group consisting of guanidine, lithium chloride, sodium salicylate, sodium perchlorate, and sodium thiocyanate, and (c) a base selected from the group consisting of a purine base and a pyrimidine base.

A macromolecule to be preserved and/or stabilized with a macromolecule stabilizing composition and/or method may include, according to some embodiments, a nucleic acid selected from the group consisting of DNA, RNA, mRNA, and cDNA. A nucleic acid may include, for example, prokaryotic and/or eukaryotic DNA. In some embodiments, a macromolecule to be preserved and/or stabilized with a macromolecule stabilizing composition and/or method may be present in a bodily fluid obtained from a human subject. A bodily fluid may include, for example, a material selected from the group consisting of blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat. The present disclosure further relates to a system for preserving and/or stabilizing a macromolecule and/or a biomolecule (a "macromolecule stabilizing system") in some embodiments. A macromolecule stabilizing system may include a sample container configured and arranged to receive and contain a sample comprising the macromolecule and a macromolecule stabilizing composition (e.g., including a chelator, at least one chelator enhancing component, and a base). A system may also include user instructions in some embodiments. The sample container, in some embodiments, may contain the macromolecule stabilizing composition. For example, the sample container may include at least one inner surface and at least one outer surface with a macromolecule stabilizing composition coated onto the latter. A sample container may include at least one vesicle, liposome, and/or micelle in some embodiments. A macromolecule stabilizing composition may be present within the lumen of a vesicle, liposome, and/or micelle.

BRIEF DESCRIPTION OF THE DRAWINGS Some embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings, wherein:

Figure 1 is a bar graph of DNA concentration in preserved urine according to an embodiment of the disclosure;

Figure 2 is a graph of eight day serial data on preserved urine according to an embodiment of the disclosure;

Figure 3 is a graph comparing PCR results in unpreserved and preserved normal urine according to an embodiment of the disclosure;

Figure 4 is a graph of eight day serial data on preserved serum according to an embodiment of the disclosure; Figure 5 is a graph of DNA concentration in preserved serum according to an embodiment of the disclosure;

Figure 6 is a diagram of the system for preserving DNA according to one embodiment of the disclosure;

Figure 7 graphically illustrates a comparison of signal response in PCR assays wherein the DNA has been treated with a preservative of the disclosure, and one which has not; Figure 8 illustrates the efficacy of reagents of the present disclosure to enhance signal response of a branched DNA assay of blood plasma samples subjected to various storage conditions;

Figure 9 illustrates the efficacy of reagents of the present disclosure to enhance signal response of a branched DNA assay of blood serum and plasma samples;

Figure 10 is a graph showing the interference of methemoglobin on PCR absorbance in a PCR amplification assay on hepatitis B sequences MD03/06 in unprotected serum;

Figure 11 is a graph showing the improvement in attenuating the interference of methemoglobin on PCR absorbance in a PCR amplification assay on hepatitis B sequences MD03/06 in serum which has been treated with a preservative of the disclosure;

Figure 12A is a chart showing a representation of results obtained from an example PCR amplification using MD03 and MD06 primers and a hepatitis B template in serum contacted with buffer (no protection), guanidine only, EGTA only, or EGTA+guanidine;

Figure 12B is a chart showing a representation of results obtained from an example PCR amplification using MD03 and MD06 primers and a hepatitis B template in serum contacted with buffer (no protection), EDTA only, sodium perchlorate only, or EDTA+sodium perchlorate;

Figure 12C is a chart showing a representation of results obtained from an example PCR amplification using MD03 and MD06 primers and a hepatitis B template in serum contacted with buffer (no protection), EGTA only, sodium perchlorate only, or EGTA+sodium perchlorate;

Figure 12D is a chart showing a representation of results obtained from an example PCR amplification using MD03 and MD06 primers and a hepatitis B template in serum contacted with buffer (no protection), EDTA only, or EDTA+sodium thiocyanate; Figure 12E is a chart showing a representation of results obtained from an example

PCR amplification using MD03 and MD06 primers and a hepatitis B template in serum contacted with buffer (no protection), EGTA only, or EGTA+sodium thiocyanate;

Figure 12F is a chart showing a representation of results obtained from an example PCR amplification using MD03 and MD06 primers and a hepatitis B template in serum contacted with buffer (no protection) or BAPTA only; Figures 13A-13G are graphs showing the absence of preservative effect on gonococcal DNA in urine stored at room temperature and subsequently subjected to PCR detection offered by the individual addition of certain components which are included in the reagents of the disclosure; Figure 14A is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with cytosine only or sodium thiocyanate+EDTA+cytosine;

Figure 14B is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with guanine only or sodium thiocyanate+EDTA+guanine;

Figure 14C is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with thymine only or sodium thiocyanate+EDTA+thymine;

Figure 14D is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with uracil only or sodium thiocyanate+EDTA+uracil;

Figure 15A is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with IM adenine, IM sodium thiocyanate, IM EDTA, or IM sodium thiocyanate+O.OlM EDTA+1M adenine; Figure 15B is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with IM adenine, IM EDTA, 2M sodium thiocyanate+lM EDTA, or 2M sodium thiocyanate+lM EDTA+1M adenine;

Figure 15C is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with IM adenine, IM guanidine, IM guanidine+O.OlM EDTA, 2M sodium thiocyanate+lM EGTA, or IM GuanidineΗCl+lM EGTA+2M adenine;

Figure 15D is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with IM adenine, IM guanidine, IM guanidine+O.OlM EDTA, IM lithium chloride+lM BAPTA, or 2M guanidine thiocyanate+lM BAPTA+2M adenine; Figure 15E is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with IM sodium perchlorate, 1 M sodium thiocyanate+2M EDTA, or 1 M sodium perchlorate+lM EDTA;

Figure 16A is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with IM guanidine-HCl or IM guanidine-HCl+0.01M BAPTA+4M adenine; and

Figure 16A is a chart showing the results of an example PCR amplification using a gonococcal DNA template in fresh urine contacted with 0.0 IM EDTA, 2M sodium thiocyanate, IM sodium thiocyanate+O.lM EDTA+1M adenine, or 2M sodium thiocyanate+0.1 M EGTA+2M adenine.

DESCRIPTION

The present disclosure relates to compositions, systems, and methods for delaying degradation of a macromolecule and/or biomolecule ("macromolecule"). Degradation may be regarded as any change in molecular structure that renders undetectable a molecule of interest or a collection of molecules of interest. For example, degradation of a protein may include any modification of the primary, secondary, tertiary or quaternary structure (e.g., reduction of disulfide bonds, hydrolysis of peptide bonds, or any other cleavage of a covalent, ionic, hydrophobic, hydrogen, or Van der Waals bond). Degradation of a nucleic acid may include any modification of the hybridization state (e.g., single, double, or triple stranded), helical structure (e.g., A, B, or Z), supercoiling, or sequence (e.g., pyrimidine dimerization, deamination, oxidation, depurination, or any other cleavage of a covalent, ionic, hydrophobic, or hydrogen bond). This delay in degradation may be regarded as preserving the macromolecule in a desired form for a long or indefinite period of time. This delay may also be regarded as preserving or stabilizing the macromolecule in a desired form for a defined period (e.g., from the time of sample collection to the time of assay).

Compositions, systems, and methods according to some embodiments of the disclosure may reduce or eliminate degradation of a macromolecule in a biological fluid and/or excretion. For example, a composition, system, and/or method of the disclosure may, in some embodiments, eliminate enzymatic destruction of a nucleic acid of interest in a bodily fluid (e.g., urine). Nucleic acids that may be preserved and/or stabilized include, for example natural and/or synthetic forms of DNA, RNA, RNA/DNA hybrids, and variants thereof. DNA that may be preserved and/or stabilized may include, for example, human DNA, mammalian DNA, bacterial DNA, fungal DNA, and viral DNA. Bacterial DNA that may be preserved and/or stabilized may include, for example, gonococcal DNA, Haemophilus influenzae DNA, and Bacillus subtilis DNA.

A macromolecule (and/or biomolecule) to be preserved and/or stabilized may be comprised in a bodily fluid and/or excretion, a tissue {e.g., biopsy tissue), and/or an object {e.g., bone). For example, a macromolecule may be comprised in a food particle, a soil sample, a forensic sample {e.g., an article of clothing, a hair, a finger print), a fabric, a bacterial matrix, a slime, an environmental specimen, and/or a biowarfare specimen. A macromolecule (and/or biomolecule) to be preserved and/or stabilized may be comprised in a whole cell and/or purified {e.g., fully or partially purified) from a whole cell.

Compositions, systems, and methods may preserve and/or stabilize a macromolecule {e.g., at room temperature) for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about a week, at least about 2 weeks, at least about 3 weeks, and/or at least about 4 weeks. Compositions, systems, and methods may preserve and/or stabilize a macromolecule {e.g., at room temperature) for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about a week, up to about 2 weeks, up to about 3 weeks, and/or up to about 4 weeks. Compositions, systems, and methods, in some embodiments, may preserve and/or stabilize a macromolecule for any of the foregoing periods without refrigeration. For example, preservation and/or stabilization may be achieved where the ambient temperature and/or temperature of the composition does not exceed about 70° C, about 60° C, about 55° C, about 50° C, about 45° C, and/or about 40° C. Preservation and/or stabilization may be achieved where the ambient temperature and/or temperature of the composition is from about 0° C to about 10° C, from about 10° C to about 20° C, from about 15° C to about 25° C, from about 20° C to about 30° C, from about 15° C to about 35° C, and/or from about 30° C to about 40° C. The choice of temperature range, in some embodiments, may be chosen based on the expected and/or desired storage conditions for a specific sample. For example, compositions, systems, and methods may be adapted to preserving and/or stabilizing materials collected in an under developed country where refrigeration is impractical and/or unavailable and day time temperatures approach 50° C. Likewise, compositions, systems, and methods may be adapted to preserving and/or stabilizing materials collected in a location where shipping conditions, storage conditions, and/or ambient conditions include temperatures below 20° C. Without being limited to any particular mechanism of action, compositions, systems, and methods of the disclosure may inactivate one or more metal-dependent enzymes and/or one or more metal-independent enzymes present in a test sample (e.g., bodily fluid) containing the macromolecule and/or biomolecule of interest. For example, a divalent metal chelator may bind available metals (e.g., Mg²⁺ and Ca²⁺) to such an extent that metals that remain available to the metal-dependent enzymes (e.g., deoxyribonucleases) are insufficient to support catalysis (i.e., nucleic acid degradation). Again, without being limited to any particular mechanism of action, a chelator enhancing component may inactivate one or more metal independent enzymes found in a bodily fluid. For example, a metal independent enzyme may include a DNA ligase (e.g., D4 DNA ligase), a DNA polymerase (e.g., T7 DNA polymerase), an exonuclease (e.g., exonuclease 2, λ-exonuclease), a kinase (e.g., T4 polynucleotide kinase), a phosphotase (e.g., BAP and CIP phosphotase), a nuclease (e.g., BL31 nuclease and XO nuclease), and an RNA-modifying enzyme (e.g., E. coli RNA polymerase, SP6, TJ, T3 RNA polymerase, and T4 RNA ligase). Without being limited to any particular mechanism of action a purine base and/or a pyrimidine base may bind to a nucleic acid and act as an isomeric target for one or more enzymes that degrade DNA and/or RNA.

According to some specific example embodiments of the disclosure, the yield from PCR amplification of a target nucleic acid (e.g., gonococcal DNA) contacted with a macromolecule stabilizing composition having purine base may be at least about 2-fold higher, about 3-fold higher, about 4-fold higher, about 5-fold higher, about 6-fold higher, about 7-fold higher, about 8-fold higher, about 9-fold higher, and/or 10-fold higher than the yield from PCR amplification of the same target nucleic acid not contacted with a macromolecule stabilizing composition having a purine base. According to some specific example embodiments of the disclosure, the yield from PCR amplification of a target nucleic acid (e.g., gonococcal DNA) contacted with a macromolecule stabilizing composition having a chelator, a chelator enhancing component, and a purine base may be about 2-fold higher, about 3-fold higher, about 4-fold higher, about 5-fold higher, about 6-fold higher, about 7- fold higher, about 8-fold higher, about 9-fold higher, and/or 10-fold higher than the yield from PCR amplification of the same target nucleic acid contacted with a macromolecule stabilizing composition having a chelator and a chelator enhancing component, but lacking a purine base. For example, the yield from PCR amplification of a target nucleic acid (e.g., gonococcal DNA) contacted with a macromolecule stabilizing composition having EDTA (0.1 M), sodium thiocyanate (1 M), and adenine may be about 10-fold higher than the yield from PCR amplification of the same target nucleic acid contacted with a macromolecule stabilizing composition having EDTA (0.1 M) and sodium thiocyanate (1 M), but lacking adenine.

Compositions

A composition for preserving and/or stabilizing a macromolecule and/or biomolecule (a "macromolecule stabilizing composition"), according to some embodiments of the disclosure may include a chelator, a chelator enhancing component, a purine base, and/or a pyrimidine base. For example, a macromolecule stabilizing composition may include a chelator, a chelator enhancing component, and a purine base.

A chelator may include, for example, ethylenediaminetetraacetic acid (EDTA), [ethyl enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and l,2-bis(2- aminophenoxy)ethane-N,N,N',N^l-tetraacetic acid (BAPTA), and/or salts thereof. A chelator, if included, may be present at any desirable concentration. For example, a chelator may be included at a concentration of at least about 0.001 M, at least about 0.005 M, at least about 0.01 M, at least about 0.05 M, and/or at least about 0.1 M. A chelator may be included at a concentration of up to about 0.001 M, up to about 0.005 M, up to about 0.01 M, up to about 0.05 M, and/or up to about 0.1 M. A chelator may be included at a concentration of from about 0.001 M to about 0.1 M. Where two or more chelators are included in a single composition, either the concentration of each chelator or the total concentration of the combined chelators may fall within any of the provided ranges. In some embodiments, a chelator may include EDTA, EGTA, BAPTA, imidazole, iminodiacetate (IDA), bis(5- amidino-2-benzimidazolyl)methane (BABIM), and/or salts thereof. A chelator enhancing component may include, for example, lithium chloride, guanidine, sodium salicylate, sodium perchlorate, sodium thiocyanate, and combinations thereof. As those of ordinary skill in the art will appreciate, guanidine includes guanine, a purine base, and a ribose. A chelator enhancing component, if included, may be present at any desirable concentration. For example, a chelator enhancing component may be included at a concentration of at least about 0.1 M, at least about 0.5 M, at least about 1 M, at least about 1.5 M, at least about 1.75 M, at least about 2 M, at least about 3 M, at least about 4 M, and/or at least about 5 M. A chelator enhancing component may be included at a concentration of up to about 0.1 M, up to about 0.5 M, up to about 1 M, up to about 1.5 M, up to about 1.75 M, and/or up to about 2 M. A chelator enhancing component may be present at a concentration within a range having endpoints defined by any of the foregoing concentrations. For example, a chelator enhancing component may be included at a concentration of from about 0.1 M to about 1.75 M, from about 0.1 M to about 2.0 M, from about 0.1 M to about 3.0 M, from about 0.5 M to about 3.0 M, and/or from about 0.1 M to about 5.0 M.

A purine base may include adenine, guanine, and combinations thereof. A purine base may also include analogs and/or variants (e.g., methyladenine, methylguanine, ethyladenine, ethylguanine). A purine base may also include structurally similar analogs and/or variants such as inosine, caffeine, uric acid, theobromine, theophylline, 2- aminopurine, 6-aminopurine, hypoxanthine (6-oxy purine), and xanthine (2,6-dioxy purine). A purine base may include a salt (e.g., adenine hemisulfate salt, adenine hydrochloride). A purine base, if included, may be present at any desirable concentration. For example, a purine base may be included at a concentration of at least about 0.1 M, at least about 0.25 M, at least about 0.5 M, at least about 0.75 M, at least about 1 M, at least about 1.5 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 4 M, at least about 5 M, at least about 6 M, and/or at least about 7 M. A purine base may be included at a concentration of up to about 0.1 M, up to about 0.25 M, up to about 0.5 M, up to about 0.75 M, up to about 1 M, up to about 1.5 M, up to about 2 M, up to about 2.5 M, up to about 3 M, up to about 4 M, up to about 5 M, up to about 6 M, and/or up to about 7 M. A purine base, if included, may be present at a concentration within a range having endpoints defined by any of the foregoing concentrations. For example, a purine base may be included at a concentration of from about 0.1 M to about 1.0 M, from about 0.1 M to about 2.0 M, from about 0.1 M to about 5.0 M, from about 0.1 M to about 1.75 M, from about 0.5 M to about 2.0 M, from about 0.75 M to about 3 M, and/or from about 0.1 M to about 7 M.

A pyrimidine base may include, for example, cytosine, thymine, uracil, and combinations thereof. A pyrimidine base may also include analogs and/or variants {e.g., methylcytosine, methylthymine, methyluracil, ethylcytosine, ethylthymine, ethyluracil). A pyrimidine base may also include structurally similar analogs and/or variants such as orotic acid, thiamine, 5-fluorouracil, 6-azauracil, pyrazine, and/or pyridazine. A pyrimidine base may include a salt {e.g., pyrimidine salt, 2-piperazinopyrimidine salt ). A pyrimidine base, if included, may be present at any desirable concentration. For example, a pyrimidine base may be included at a concentration of at least about 0.1 M, at least about 0.25 M, at least about 0.5 M, at least about 0.75 M, at least about 1 M, at least about 1.5 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 4 M, at least about 5 M, at least about 6 M, and/or at least about 7 M. A pyrimidine base may be included at a concentration of up to about 0.1 M, up to about 0.25 M, up to about 0.5 M, up to about 0.75 M, up to about 1 M, up to about 1.5 M, up to about 2 M, up to about 2.5 M, up to about 3 M, up to about 4 M, up to about 5 M, up to about 6 M, and/or up to about 7 M. A pyrimidine base, if included, may be present at a concentration within a range having endpoints defined by any of the foregoing concentrations. For example, a pyrimidine base may be included at a concentration of from about 0.1 M to about 1.0 M, from about 0.1 M to about 2.0 M, from about 0.1 M to about 5.0 M, from about 0.1 M to about 1.75 M, from about 0.5 M to about 2.0 M, from about 0.75 M to about 3 M, and/or from about 0.1 M to about 7 M.

In some embodiments, a macromolecule stabilizing composition may include an amount of a divalent metal chelator selected from EDTA, EGTA BAPTA, and salts thereof; and an amount of at least one chelator enhancing component selected from lithium chloride, guanidine, sodium salicylate, sodium perchlorate, and sodium thiocyanate. The amount of a divalent metal chelator may be generally in the range of from about 0.001 M to about 0.1 M. The amount of a chelator enhancing component may be generally in the range of from about 0.1 M to about 2.0 M. The amount of chelator in a composition maybe, for example, at least about 0.01 M. The amount of chelator enhancing component in a composition may be, for example, at least about 1 M. A macromolecule stabilizing composition , in some embodiments, may further include an amount of at least one enzyme inactivating component such as manganese chloride, sarkosyl, or sodium dodecyl sulfate, generally in the range of about 0-5% molar concentration. In some embodiments, a macromolecule stabilizing composition may include a purine base, a pyrimidine base, or both a purine base and a pyrimidine base. For example, a composition may include a chelator, a chelator enhancing component, and a purine base (e.g. , adenine). In some embodiments, a macromolecule stabilizing composition may include only (a) a chelator, (b) a chelator enhancing component, and (c) a purine base and/or a pyrimidine base. A macromolecule stabilizing composition, in other embodiments, may include one or more solvents (e.g., aqueous and/or organic), buffers, salts, surfactants, oxidizing agents, reducing agents, and/or other reagents.

In some embodiments, a macromolecule stabilizing composition may have a pH of from about 4.5 to about 8.0. A macromolecule stabilizing composition may be formulated such that upon being combined with the sample to be preserved and/or stabilized (e.g., a bodily fluid), the mixture has a pH of from about 4.5 to about 8.0. In some embodiments, a suitable buffer may be selected from Good buffers (e.g., HEPES), potassium acetate, sodium phosphate, potassium bicarbonate , tris(hydroxyamino)methane (Tris), and combinations thereof. For example, a buffer may include potassium acetate, sodium acetate, potassium phosphate, sodium phosphate, Tris, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, 3-(N-morpholino)propane sulfonic acid (MOPS) buffer, 2-[(2-amino-2- oxoethyl)amino]ethanesulfonic acid (ACES) buffer, N-(2-acetamido)2-iminodiacetic acid buffer (ADA), 3-[(l,l-dimethyl-2-hydroxyethyl)amino]-2-propanesulfonic acid (AMPSO) buffer, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, Bicine (N,N- bis(2-hydroxyethylglycine) buffer, bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bis-Tris) buffer, 3-(cyclohexylamino)-l-propanesulfonic acid (CAPS) buffer, 3- (cyclohexylamino)-2-hydroxy-l-propanesulfonic acid (CAPSO) buffer, 2-(N- cyclohexylamino)ethanesulfonic acid (CHES) buffer, 3-[N,N-bis(2-hydroxyethyl)amino]-2- hydroxy-propanesulfonic acid (DIPSO) buffer, N-(2-hydroxyethylpiperazine)-N'-(3- propanesulfonic acid) (HEPPS) buffer, N-(2-hydroxyethyl)piperazine-N'-(2- hydroxypropanesulfonic acid) (HEPPSO) buffer, 2-(N-morpholine)ethanesulfonic acid (MES) buffer, triethanolamine buffer, imidazole buffer, glycine buffer, ethanolamine buffer, phosphate buffer, 3-(N-morpholine)-2-hydroxypropanesulfonic acid (MOPSO) buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, piperazine-N,N'-bis(2- hydroxypropanesulfonic acid) (POPSO) buffer, N-tris[(hydroxymethyl)methyl]-3- aminopropanesulfonic acid (TAPS) buffer, 2-hydroxy-3-[tris(hydroxvmethyl)methylamino]- 1-propanesulfonic acid (TAPSO) buffer, N-[Tris(hydroxymethyl)methyl]-2- aminoethanesulfonic acid (TES) buffer, N-[Tris(hydroxymethyl)methyl]glycine (tricine) buffer, 2-amino-2-methyl- 1,3 -propanediol buffer, 2-amino-2 -methyl- 1-propanol buffer, and combinations thereof. A surfactant, in some embodiments, may include a detergent. A detergent may include, for example, a nonionic detergent. A nonionic detergent may include polyoxyethylene (20) sorbitan monolaurate, octyl-phenoxypolyethoxyethanols, nonyl- phenoxypolyethoxyethanols, octyl flucopyranosides, dodecyl maltopyranosides, heptyl thioglucopyranosides, big CHAP detergents, Genapol X-80, Pluronic detergents, polyoxyethylene esters of alkylphenols (e.g., Triton), and/or derivatives and analogues thereof.

A macromolecule stabilizing composition, according to some embodiments, may be prepared and/or used as a solid, liquid, or a gas.

Systems

A system, according to some embodiments of the disclosure, may include a macromolecule stabilizing composition and a sample storage container. For example, a system may include a container configured and arranged to receive a sample containing the macromolecule(s) and/or biomolecule(s) to be preserved and/or stabilized. A container may be configured and arranged to contact the sample with a macromolecule stabilizing composition. In a simple example, a macromolecule stabilizing composition formulated as a solid (e.g., tablet, powder, or hydrogel) may be deposited in the bottom of a small tube. Upon placing a sample (e.g., a liquid sample) in the tube, the macromolecule stabilizing composition may contact and mix with the sample milieu. The sample may be contacted (e.g., mixed) with a macromolecule stabilizing composition at the same time it is placed in a container or at some time thereafter. In some embodiments of the disclosure, a system may include a macromolecule stabilizing composition further including a lipid, surfactant, and/or detergent. For example, a macromolecule stabilizing composition may be comprised in a micelle, a liposome, a vesicle, and/or a membrane-bound space. A system, according to some embodiments, may include a macromolecule stabilizing composition and instructions for use. In some embodiments, a system may include a macromolecule stabilizing composition, a sample storage container, and instructions for use. A system may also include a shippable container configured to contain a sample storage container and its contents.

Methods

A method of preserving and/or stabilizing a macromolecule and/or biomolecule (a "macromolecule stabilizing method"), according to some embodiments of the disclosure, may include contacting the macromolecule with a macromolecule stabilizing composition. For example, a bodily fluid comprising a macromolecule may be contacted with a macromolecule stabilizing composition having a chelator, a chelator enhancing component, and a purine base (e.g., adenine).

The present disclosure also relates to methods for improving the signal response of a molecular assay of a test sample, including contacting the test sample with a macromolecule stabilizing composition to produce a preserved and/or stabilized test sample ("preserved test sample"), isolating and/or purifying a molecular analyte of interest from the test sample, and performing a molecular assay on the isolated and/or purified molecular analyte of interest. Without being limited to any particular mechanism of action, improved signal response in a nucleic acid assay may be due in part to enhanced hybridization as a result of the use of a macromolecule stabilizing composition of the present disclosure.

The present disclosure further relates to methods for improving hybridization of nucleic acids, including contacting a test nucleic acid with a macromolecule stabilizing composition to form a test solution and contacting the test solution with a target nucleic acid under conditions that permit test nucleic acid - target nucleic acid hybridization. As will be understood by those skilled in the art, other equivalent or alternative compositions, systems, and methods for preserving and/or stabilizing a macromolecule and/or biomolecule according to embodiments of the present disclosure can be envisioned without departing from the essential characteristics thereof. For example, a macromolecule stabilizing composition may be formulated as a powder, granule, tablet, capsule, liquid, syrup, paste. A macromolecule stabilizing composition may be deposited in a sample container by any available method. For example, a macromolecule stabilizing composition may be coated (e.g., sprayed or spray-dried) onto an inner surface of a sample container before a macromolecule-containing sample is introduced. A macromolecule stabilizing composition may also be simply placed in a sample container in a solid or liquid form. Alternatively, a macromolecule stabilizing composition may be kept in a separate container and only contacted with a sample after the sample has been placed in a sample container. These equivalents and alternatives along with obvious changes and modifications are intended to be included within the scope of the present disclosure. Accordingly, the foregoing disclosure is intended to be illustrative, but not limiting, of the scope of the disclosure as illustrated by the following claims. Some specific embodiments of the disclosure may be understood, by referring, at least in part, to the following specific example embodiments. These examples illustrate some, but not all, aspects of some embodiments of the disclosure and additional variations will be apparent to one skilled in the art having the benefit of the present disclosure.

EXAMPLE 1

Figure 1 is a bar graph of DNA concentration in urine preserved and/or stabilized in accordance with an embodiment of the disclosure. The number of transformants in ten types of urine specimens were tested using a GTT, counted hourly, and then summarized. The standard Gonostat protocol (see Example 2, infra) was employed, and the preservative used was IM guanidine HC1/0.01M EDTA. A count of two hundred colonies demonstrates total preservation of a specimen. The number of gonococcal transformants in the preserved urine remained relatively constant approaching two hundred, throughout the four hours of the test. No significant difference in level of preservation was observed among the different types of urine specimens. Therefore, the example composition tested provided nearly total protection for DNA in urine. EXAMPLE 2

Figure 2 is a graph of eight day GTT serial data on urine preserved and/or stabilized in accordance with an embodiment of the disclosure. 1 pg of gonococcal DNA was spiked into 9 mL of fresh human urine and 1 mL of aqueous a macromolecule stabilizing composition containing IM sodium perchlorate and 0.01M EGTA. 300 μL was spotted onto a lawn of the Gonostat organism at 24 hour intervals for eight days. The plates contained BBL

Chocolate II agar and were incubated at 37° C for 24 hours before readings were taken. The number of colonies observed throughout the eight-day testing period ranged from a low count of one hundred eighty-eight to a high count of one hundred ninety-seven. Thus, embodiments of the disclosure may preserve and/or stabilize DNA in urine for a significantly longer period of time than previously provided.

EXAMPLE 3

Figure 3 is a graph comparing PCR results in unpreserved and preserved (preserved and/or stabilized) normal urine according to an embodiment of the disclosure. A MOMP template to Chlamydia trachomatis was used and amplified using a standard PCR protocol.

200 copies of the MOMP target were spiked into 9 mL of fresh human urine containing IM sodium perchlorate and 0.01M BAPTA. PCR was done each hour for eight hours total, hi the unprotected urine, approximately three PCR absorbances were measured one hour after the addition of DNA to the urine. The number of PCR absorbances approached zero by the sixth hour. By contrast, in the preserved and/or stabilized specimen, in excess of three PCR absorbances were measured at the one hour testing. However, approximately three PCR absorbances were still observed by the sixth hour. Therefore, embodiments of the disclosure may preserve and/or stabilize sufficient DNA and nucleic acid sequences to permit PCR testing well beyond the testing limits of unpreserved urine. The results shown in the Figure are consistent for all types of DNA in a urine specimen. EXAMPLE 4

The reagents and methods of the disclosure may be used for preserving other bodily fluids and excretions, such as blood serum. Figure 4 is a graph of eight day serial data on preserved and/or stabilized serum according to an embodiment of the disclosure. The protocol used was similar to Example 3, except fresh human serum was used. The number of transformant colonies observed throughout the eight-day testing period ranged from a high count of one hundred ten at the one day measurement to a low count of approximately ninety- two at the seven day measurement. In fact, the test results actually showed an increase in transformant colonies between days seven and eight. Thus, some embodiments of the disclosure preserve and/or stabilize DNA in serum for a significantly longer period of time than previously attainable.

EXAMPLE 5

Figure 5 is a graph of DNA concentration in preserved and/or stabilized serum according to an embodiment of the disclosure. The serum was preserved and/or stabilized with a macromolecule stabilizing composition comprising IM guanidine HC1/0.01M EDTA.

The protocol used was similar to Example 3, except fresh human serum was used, and the duration time of the study was ten hours. In excess of 120 transformants were measured at the time gonococcal DNA was added to the serum. Approximately 100 transformants were counted at the six hour measurement. However, by the tenth hour, testing indicated that the concentration of biologically active DNA in the preserved serum had increased to approximately 110 transformant colonies.

EXAMPLE 6 An example embodiment of a method 10 for preserving DNA is illustrated diagrammatically in Figure 6. This protocol is described in Table 1, below and has been observed to produce high yields of DNA/RNA suitable for such testing methods as PCR, restriction fragment length polymorphisms assay (RFLP), and nucleic acid probes using urine specimens. TABLE 1

EXAMPLE 7

Preservation of DNA in Simulated Clinical Specimens In the following experiment, simulated clinical urine specimens were produced and tested for the presence of gonococcal DNA. The chemicals listed in Table 2, below, were added, at the concentrations previously described, to urine specimens from healthy adults, as was EDTA.

A suspension of gonococci was immediately added to each urine specimen. The added gonococci were an ordinary strain of N. gonorrhoeae, 49191, which was grown overnight on GC agar medium at 37° C in a 5% CO₂ atmosphere. The N. Gonorrhoeae colonies were picked and suspended in GC buffer. A 1/10 volume of a suspension containing approximately 10 Colony forming units (cfu) per mL was added to the urine. As a positive control, the suspension of gonococci was also added to Hepes buffer. All simulated clinical specimens and the Hepes controls were tested at time zero, i.e., when the chemicals and gonococci were added. The specimens and controls were also tested after storage at room temperature for six days. This six day period was selected to approximate the maximum time expected between collecting, mailing, and testing patient specimens.

With the exception of urine samples containing Sodium dodecyl sulfate (SDS) and sarkosyl, the simulated specimens and Hepes controls were processed as follows: 1. A 10 mL quantity was centrifuged at 4000 rpm for 30 minutes.

2. The supernatant was decanted, and the pellet was suspended in 1 mL phosphate buffer.

3. The suspension was heated for 10 minutes in a water bath at 60° C.

4. After cooling, the suspension was used in the GTT. The simulated urine specimens containing SDS-EDTA or sarkosyl-EDTA were processed as follows:

1. Approximately a 2 1/2 volume (approximately 25 mL) of 95% ethyl alcohol was added to the tube with the urine and macromolecule stabilizing composition. The contents were mixed by inverting the tube several times. 2. The mixture was centrifuged at 4000 rpm for 30 minutes.

3. The pellet was suspended in 10 mL of 70% alcohol and centrifuged.

4. The pellet was then suspended in 1 mL phosphate buffer.

5. The suspension was heated for 10 minutes in a water bath at 60° C.

6. After cooling, the suspension was used in the GTT. The inoculated urine was stored at room temperature for 6 days prior to testing. The formulations that preserved and/or stabilized (+) or did not preserve and/or stabilize (-) gonococcal DNA in the inoculated urine for six days to approximately the same degree as in the Hepes buffer control are indicated. Although the results of the Gonostat™ assay may be semi-quantitated, the tests were not designed to rank the relative efficacy of the macromolecule stabilizing compositions. Thus, the results given in Table 2 indicate whether or not the particular chemical preserved and/or stabilized DNA in urine over a six day period to same degree as in the Hepes buffer. TABLE 2

The 92% sensitivity exhibited with male urine specimens is comparable to the culture results reported in the literature. In addition, the 88% sensitivity exhibited with female urine specimens exceeds the previously-reported levels.

While a preferred embodiment of the disclosure is directed to the preservation of gonococcal DNA, it will be readily apparent to one skilled in the art that the disclosure is adaptable for use in preserving other types of DNA, such as that of Haemophilus influenzae and Bacillus subtilis. Some embodiments of the disclosure may also be used to preserve and/or stabilize RNA contained in bodily fluid samples. Such preserved RNA may be used for RNA transcriptase and reverse transcriptase assays for viral segments and human gene sequence testing.

Furthermore, although a macromolecule stabilizing composition may be added to a bodily fluid, e.g., a urine specimen, a urine specimen may also be added to a macromolecule stabilizing composition without detriment to the efficacy of preservation/stabilization. Optimal preservation of the DNA may be achieved by adding a single macromolecule stabilizing composition of the disclosure to a specimen. EXAMPLE 8

PCR Detection of Penicillinase-producing./Vgmer;^'α gonorrhea

The PCR signal-enhancing effect of a macromolecule stabilizing composition of the disclosure is demonstrated by the following example. Four varieties of TEM-encoding plasmids are found in PPNG. These are the 6.7 kb (4.4 Mda) Asian type, the 5.1 kb (3.2 Mda) African type, the 4.9 kb (3.05-Mda) Toronto type and the 4.8 kb (2.9-Mda) Rio Type. This PCR assay for PPNG takes advantage of the fact that the TEM-I gene is located close to the end of the transposon Tn2; by the use of one primer in the TEM-I gene and the other in a sequence beyond the end of Tn2, and common to all four plasmids, a PCR product only from plasmids and not from TEM-I encoding plasmids was obtained. (Table 3, below) The conditions associated with this protocol were modified to include the macromolecule stabilizing composition in the hybridization and the treated probe was mixed with the 761-bp amplification product per standard PCR protocol. The results were read at A₄₅₀ nm.

Materials and Reagents

BBL chocolate 11 agar plates

Sterile Tris Buffer 10 mM Tris (pH 7.4), 1 mM EDTA 0.5-mL Gene Amp reaction tubes Sterile disposable Pasteur pipette tips

Aerosol-resistant tips PCR master mix: 50 mM KCl 2 mM MgCl 50 μM each of

Deoxyribonucleoside triphosphate; 2.5 U of Taq Polymerase (Perkin Elmer); 5% glycerol;

50 pmol each of primers PPNG-L and PNG-R (per 100 μL reaction) Denaturation solution

IM Na 5X Denhardt's solution Prehybridization Solution

5X SSC(IX SSC is 0.015 M NaCl plus 0.015 M sodium citrate); 5X Denhardt's solution; 0.05% SDS;

0.1% Sodium Ppi, and

100 μg of sonicated salmon sperm DNA per mL. Hybridization Solution

Same as prehybridization solution but without Denhardt's solution and including 200 μL of macromolecule stabilizing composition 1.

1 mL DNA/RNA macromolecule stabilizing composition (IM guanidine HC1/0.01M EDTA) Avidin-HRP peroxidase complex (Zymed) Magnetic microparticles (Seradyne) TABLE 3

Function Name Nucleotide sequence 5' to 3'

Primer PPNG-L AGT TAT CTA CAC GAC GG (SEQ ID NO:1)

Primer PPNG-R GGC GTA CTA TTC ACT CT (SEQ ID NO:2)

Probe PPNG-C GCG TCA GAC CCC TAT CTA TAA ACT C (SEQ ID NO:3)

Methods

Sample preparation: 2 colonies were picked from a chocolate agar plate. Colonies were suspended in deionized water just prior to setting up PCR. The master mix was prepared according to the recipe above. 5 μL of the freshly prepared bacterial suspension was added to

95 μL of master mix. The DNA was liberated and denatured in a thermocycler using three cycles of 3 min at 94° C and 3 min at 55° C. The DNA was amplified in the thermal cycler by using a two step profile: a 25 s denaturation at 95° C and a 25 s annealing at 55° C for a total of thirty cycles. The time was set between the two temperature plateaus to enable the fastest possible annealing between the two temperatures. 15 pmol of labeled (avidin-HRP complex) detection probe PPNG-C was added to the hybridization solution bound to magnetic micro particles with and without the macromolecule stabilizing composition at 37°

C for 1 hour. The control and treated probes were then added to the amplification product and the reaction was colorimetrically detected at A₄₅₀ nm. The signal obtained from the hybridization probes treated with a macromolecule stabilizing composition of the disclosure was found to be significantly higher than the untreated probes.

EXAMPLE 9 Compositions, systems, and methods in accordance with some embodiments of the disclosure may increase the signal obtained with a nucleic acid testing method, such as a polymerase chain reaction (PCR), LCx, and genetic transformation testing (GTT). For example, compositions, systems, and methods may enhance hybridization in such nucleic acid testing methods as the PCR. Figure 7 illustrates the improvement in hybridization obtained a specific example embodiment of a macromolecule stabilizing composition disclosed herein on the hybridization of penicillinase-producing Neisseria gonorrhea (PPNG) DNA and PPNG-C probe. The PCR protocol was the same as described in Example 10. EXAMPLE 10

Figure 8 and Figure 9 further illustrate the efficacy of specific example embodiments of compositions, systems, and methods of the disclosure in improving the results obtained with nucleic acid testing methods, in this case, a branched DNA assay (Chiron). In the tests run in Figure 8, a bDNA assay was used to assess the protective effect of the macromolecule stabilizing compositions. DNA sequences from the hepatitis C virus were spiked into serum and plasma. The protected serum and plasma were mixed with 9 mL of serum or plasma and 1 mL of macromolecule stabilizing composition. The following formulations were used: 1) IM guanidine HC1/0.01M EDTA, 2) IM sodium perchlorate/O.OlM BAPTA, 3) IM sodium thiocyanate/O.OlM EGTA, and 4) IM lithium chloride/O.OlM EGTA. The formulations were stored for seven days at 4° C. bDNA assay relies on hybridization; it can be seen from clearly the absorbance results that the target sequences were not only protected against degradation, but the more than doubling of the absorbance results indicates an enhancement of hybridization/annealing of the target sequences.

Figure 9 illustrates a serum v. plasma study. 50 μL samples of fresh human plasma, and 1 mL samples of fresh human serum were protected with IM guanidine HC1/0.01M EDTA and the bDNA assay was run on these samples after the samples were stored at 20° F for 48 hours. Results were compared to unprotected samples. It can be seen clearly from the absorbance results that the target sequences were not only protected against degradation, but the more than doubling of the absorbance results indicates an enhancement of hybridization/annealing of the target sequences.

EXAMPLE 11

Heme compounds such as methemoglobin have been observed to interfere with PCR amplification of nucleic acids. For example, Figure 10 shows the results of a series of PCR assays performed according to Example 10, wherein the template, fresh human serum, was spiked with increasing amounts of methemoglobin. As shown, the absorbance decreases as a function of methemoglobin concentration. At the highest concentrations, no absorbance {i.e., amplification) was observed at all.

Macromolecule stabilizing compositions of the disclosure, according to some embodiments, may remove the interference with heme compounds, e.g., methemoglobin, on PCR assays run on blood serum. Figure 11 illustrates the improvement {i.e., increased amplification as measured by absorbance (A_45O)) obtained by adding to the serum sample a macromolecule stabilizing composition comprising 1 M sodium thiocyanate and 0.1 M

EDTA. Like the control (Figure 10), serum samples were spiked with increasing amounts of methemoglobin, to a concentration of 10 dl/mL. Serial PCR assays were run over a four hour period.

EXAMPLE 12

An example composition including a divalent metal chelator and a chelator enhancing component had a surprising and synergistic effect on protecting hepatitis B sequences in serum. Specifically, a hepatitis B template was contacted with a test composition {e.g., IM sodium perchlorate/O.OlM EGTA) at room temperature for up to 36 hours (sampled at 2 hour intervals). Samples were subjected to PCR amplification using MD03 and MD06 primers using the sample PCR protocol as described in Example 10. A representation of the results obtained is provided in Figures 12A-12F. Collectively, these figures show that preservation and/or amplification of hepatitis B sequences is increased when specific example embodiments of macromolecule stabilizing compositions of the present disclosure are used compared to the addition of EGTA or sodium perchlorate individually. EXAMPLE 13

Figure 13 illustrates a (relatively modest) preservative effect on gonococcal DNA in urine stored at room temperature and subsequently subjected to PCR detection provided by the individual addition of components of the reagents of the present disclosure, i.e., divalent metal chelators 0.01M BAPTA (Figure 13A), 0.01M EDTA (Figure 13B), 0.01M EGTA (Figure 13C); and chelator enhancing components IM sodium perchlorate (Figure 13D), IM salicylic acid (Figure 13E), IM guanidine HCl (Figure 13F), IM sodium thiocyanate (Figure 13G), and lithium chloride (Figure 13H). The number of transformants in ten types of urine specimens were tested using a GTT, counted hourly, and then summarized. The standard Gonostat protocol (see Example 2, infra) was employed and illustrated a synergistic effect obtained by the combination of divalent metal chelators and chelator enhancing components in protecting gonococcal DNA in urine stored at room temperature and subsequently subjected to PCR detection.

EXAMPLE 14

Compositions comprising purine bases or pyrimidine bases (1 M) were prepared either with or without sodium thiocyanate (1 M) and EDTA (0.1 M). Fresh samples of human urine were collected, spiked with 1 pg of gonococcal DNA, combined with one of the recited compositions, and incubated at room temperature. Aliquots were removed after 8 hours and tested by PCR for the presence of amplifiable gonococcal DNA. The PCR protocol was the same as described in Example 10. As illustrated in Figure 14, compositions with sodium thiocyanate, EDTA, and a purine or pyrimidine base stabilized gonococcal DNA in urine more effectively than compositions with a purine or pyrimidine base alone.

EXAMPLE 15

Compositions comprising sodium thiocyanate, EDTA, and/or adenine were prepared.

Fresh samples of human urine were collected, spiked with 1 pg of gonococcal DNA, combined with one of the recited compositions, and incubated at room temperature. Aliquots were removed after 8 hours and tested by PCR for the presence of amplifiable gonococcal

DNA. The PCR protocol was the same as described in Example 10. As illustrated in Figure

15A, compositions with sodium thiocyanate, EDTA, and adenine generally stabilized gonococcal DNA in urine more effectively than compositions with fewer than all three components. The only exception observed was where the composition comprised sodium thiocyanate and EGTA.

EXAMPLE 16

Compositions comprising sodium perchlorate, lithium chloride, guanidine HCl, guanidine thiocyanate, EDTA, EGTA, BAPTA, and/or adenine were prepared. Fresh samples of human urine were collected, spiked with 1 pg of gonococcal DNA, combined with one of the recited compositions, and incubated at room temperature. Aliquots were removed after 8 hours and tested by PCR for the presence of amplifiable gonococcal DNA. The PCR protocol was the same as described in Example 10. As illustrated in Figure 15B, compositions with a chelator, a chelator enhancing component, and adenine stabilized gonococcal DNA in urine more effectively than compositions with fewer than all three components.

EXAMPLE 17

Compositions comprising sodium thiocyanate, guanidine HCl, EDTA, EGTA, BAPTA, and/or adenine were prepared. Fresh samples of human urine were collected, spiked with 1 pg of gonococcal DNA, combined with one of the recited compositions, and incubated at room temperature. Aliquots were removed after 8 hours and tested by PCR for the presence of amplifiable gonococcal DNA. The PCR protocol was the same as described in

Example 10. As illustrated in Figure 16, compositions with a chelator, a chelator enhancing component, and adenine stabilized gonococcal DNA in urine more effectively than compositions with just one of these components. Therefore, the present disclosure is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this disclosure as illustrated, in part, by the appended claims.

Claims

CLAIMS What is claimed is:

1. A macromolecule stabilizing composition, said composition comprising:

(a) a chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), l,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and salts thereof;

(b) at least one chelator enhancing component selected from the group consisting of guanidine, lithium chloride, sodium salicylate, sodium perchlorate, and sodium thiocyanate; and

(c) a base selected from the group consisting of a purine base and a pyrimidine base.

2. A macromolecule stabilizing composition according to claim 1, wherein the concentration of the chelator is from about 0.001M to about 0.1M.

3. A macromolecule stabilizing composition according to claim 1, wherein the concentration of the at least one chelator enhancing component is from about 0.1 M to about

2M.

4. A macromolecule stabilizing composition according to claim 1 , wherein the concentration of the base is from about 0.1 M to about 5 M.

5. A macromolecule stabilizing composition according to claim 1, wherein the macromolecule stabilizing composition is formulated as an aqueous solution.

6. A macromolecule stabilizing composition according to claim 1, wherein the at least one chelator enhancing component is selected from the group consisting of sodium perchlorate, sodium thiocyanate, and lithium chloride.

7. A macromolecule stabilizing composition according to claim 1, wherein the at least one chelator enhancing is present in an amount of about IM.

8. A macromolecule stabilizing composition according to claim 1, wherein the divalent metal chelator is present in an amount of about 0.01M.

9. A macromolecule stabilizing composition according to claim 1, wherein the base is present in an amount of about 1 M.

10. A macromolecule stabilizing composition according to claim 1 further comprising at least one enzyme inactivating component selected from the group consisting of manganese chloride, sarkosyl, and sodium dodecyl sulfate.

12. A macromolecule stabilizing composition according to claim 1 further comprising a buffer.

13. A macromolecule stabilizing composition according to claim 11, wherein the buffer comprises a compound selected from the group consisting of potassium acetate, sodium acetate, potassium phosphate, sodium phosphate, tris(hydroxyamino)methane, N-(2- hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 3-(N-morpholino)propane sulfonic acid, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, N-(2-acetamido)2-iminodiacetic acid, 3- [(l,l-dimethyl-2-hydroxyethyl)amino]-2-propanesulfonic acid, N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid, N,N-bis(2-hydroxyethylglycine, bis-(2-hydroxyethyl)imino- tris(hydroxymethyl)methane, 3-(cyclohexylamino)-l-propanesulfonic acid, 3- (cyclohexylamino)-2 -hydroxy- 1 -propanesulfonic acid, 2-(N-cyclohexylamino)ethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid, N-(2- hydroxyethylpiperazine)-N' -(3 -propanesulfonic acid), N-(2-hydroxyethyl)piperazine-N'-(2- hydroxypropanesulfonic acid), 2-(N-morpholine)ethanesulfonic acid, triethanolamine buffer, imidazole, glycine, ethanolamine, 3-(N-morpholine)-2-hydroxypropanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), N-tris[(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, 2-hydroxy-3- [tris(hydroxymethyl)methylamino] - 1 -propanesulfonic acid, N- [Tris(hydroxymethyl)methyl] - 2-aminoethanesulfonic acid, N-[Tris(hydroxymethyl)methyl]glycine, 2-amino-2 -methyl- 1,3- propanediol, 2-amino-2 -methyl- 1-propanol, and combinations thereof.

14. A method of preserving or stabilizing a macromolecule, said method comprising: contacting a macromolecule with a macromolecule stabilizing composition comprising

(a) a chelator selected from the group consisting of etbylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), l,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and salts thereof;

15. A method according to claim 14, wherein the concentration of the chelator is from about 0.00 IM to about 0.1 M.

16. A method according to claim 14, wherein the concentration of the at least one chelator enhancing component is from about 0.1 M to about 2M.

17. A method according to claim 14, wherein the concentration of the base is from about 0.1 M to about 5 M.

18. A method according to claim 14, wherein the macromolecule stabilizing composition is formulated as an aqueous solution.

19. A method according to claim 14, wherein the at least one chelator enhancing component is selected from the group consisting of sodium perchlorate, sodium thiocyanate, and lithium chloride.

20. A method according to claim 14, wherein the macromolecule stabilizing composition further comprises at least one enzyme inactivating component selected from the group consisting of manganese chloride, sarkosyl, and sodium dodecyl sulfate.

21. A method according to claim 14, wherein the macromolecule stabilizing composition further comprises a buffer.

22. A macromolecule stabilizing composition according to claim 21, wherein the buffer comprises a compound selected from the group consisting of potassium acetate, sodium acetate, potassium phosphate, sodium phosphate, tris(hydroxyamino)methane, N-(2- hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), 3-(N-morpholino)propane sulfonic acid, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid, N-(2-acetamido)2-iminodiacetic acid, 3- [(l,l-dimethyl-2-hydroxyethyl)amino]-2-propanesulfonic acid, N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid, N,N-bis(2-hydroxyethylglycine, bis-(2-hydroxyethyl)imino- tris(hydroxymethyl)methane, 3-(cyclohexylamino)-l-propanesulfonic acid, 3- (cyclohexylamino)-2 -hydroxy- 1 -propanesulfonic acid, 2-(N-cyclohexylamino)ethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid, N-(2- hydroxyethylpiperazine)-N'-(3-propanesulfonic acid), N-(2-hydroxyethyl)piperazine-N'-(2- hydroxypropanesulfonic acid), 2-(N-morpholine)ethanesulfonic acid, triethanolamine buffer, imidazole, glycine, ethanolamine, 3-(N-morpholine)-2-hydroxypropanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), N-tris[(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, 2-hydroxy-3- [tris(hydroxymethyl)methylamino]- 1 -propanesulfonic acid, N-[Tris(hydroxymethyl)methyl]- 2-aminoethanesulfonic acid, N- [Tris(hydroxymethyl)methyl] glycine, 2-amino-2-methyl-l,3- propanediol, 2-amino-2-methyl-l-propanol, and combinations thereof.

23. A method according to claim 14, wherein the macromolecule is a nucleic acid selected from the group consisting of DNA, RNA, mRNA, and cDNA.

24. A method according to claim 23 wherein the nucleic acid is eukaryotic DNA.

25. A method according to claim 14, wherein the macromolecule is present in a bodily fluid obtained from a human subject.

26. A method according to claim 25, wherein the bodily fluid comprises a material selected from the group consisting of blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat.

27. A system for preserving a macromolecule in a sample, said system comprising: a sample container configured and arranged to receive and contain a sample comprising the macromolecule; and a macromolecule stabilizing composition comprising

28. A system according to claim 27 further comprising user instructions.

29. A system according to claim 27, wherein the sample container contains the macromolecule stabilizing composition.

30. A system according to claim 27, wherein the macromolecule stabilizing composition further comprises a solid, a liquid, or a hydrogel.

31. A system according to claim 27, wherein the sample container comprises at least one inner surface and at least one outer surface.

32. A system according to claim 27, wherein the macromolecule stabilizing composition is a coating on the at least one inner surface.

33. A system according to claim 27, wherein the sample container comprises at least one vesicle.

34. A system according to claim 27, wherein the sample container comprises at least one liposome.

35. A system according to claim 27, wherein the sample container comprises at least one micelle.