WO2025015224A2 - Treatment of samples containing nucleic acids - Google Patents

Abstract

Methods for treating biological samples containing nucleic acids using high ionic strength and temperature. The sample may be separated into two or more phases to segregate various components of the sample for enzymatic reactions to detect sequences of interest. Kits for performing the method of the invention are also provided.

Description

Treatment of Samples Containing Nucleic Acids

Federally Sponsored Research and Development

[0001] This invention was made with government support under grant 1R43 AG065039, awarded by the National Institutes of Health. The government has certain rights in the invention.

Technical Field

[0002] This invention relates to laboratory methods, and, more particularly, to the treatment of samples containing nucleic acids in preparation for enzymatic reactions.

Background of the Invention

[0003] RNA is notoriously fragile. Compared to DNA, it is a snowflake that begins to deteriorate at room temperature, and degrades more rapidly when exposed to temperatures 50°C or higher. RNA is also vulnerable to being digested by RNase enzymes, which are present in cells; even clean laboratory surfaces can contain trace amounts of RNase. This has led gene expression researchers and healthcare laboratory personnel to take elaborate precautions to preserve the RNA in a sample, particularly by not exposing samples with RNA to high temperatures. These precautions also include the avoidance of storing RNA without addition of RNase inhibitors or preservatives, or at temperatures above ultra-low temperatures (e.g, -85°C). Summary

[0004] The present invention provides methods for treating samples containing nucleic acids, such as DNA and RNA. In one embodiment, the ionic strength of the sample is raised, and the sample is heated. The sample may be separated into two or more phases, such as a solid phase and a liquid phase, to segregate various components of the sample. Enzymatic reactions can be performed on the sample, as an unseparated phase or in one or more phases, which can lead to analysis of the nucleic acids. Kits for performing the method of the invention are also provided.

Detailed Description of the Invention

[0005] Storing samples at room temperature, without addition of RNase inhibitors or preservatives, just dried on a surface, is not a method that investigators would choose to preserve the integrity of RNA for subsequent research quality molecular assay. Though more stable, storing DNA on a surface at room temperature, dried, without preservatives or additions is also not typical for research-quality assays. Typically, if a sample is stored on filter paper, then the RNA or DNA is extracted (removed) from the filter paper before assay, eliminating interference, as in the case of whole blood spotted on filter paper. Also, typically the filter paper used for storing samples of DNA and RNA is first treated with chemicals, such as widely used Flinders® Technology Associates (FTA) cards, which lyse the cells and protect the DNA or RNA from degradation until it is purified, or others such as GenSaver™ DNA cards. [0006] The present invention provides methods for treating samples containing nucleic acids, such as DNA and RNA. Examples of DNA include nuclear or mitochondrial DNA, modified DNA, or cDNA that is reverse transcribed from RNA, or DNA containing synthetic or unnatural (nonnaturally occurring) bases. Examples of RNA include such as mRNA, rRNA, tRNA, miRNA, snRNAs (small nuclear RNAs), siRNAs (e.g., small interfering RNAs, small inhibitory RNAs, and synthetic inhibitory RNAs), antisense RNAs, circular RNAs, or long noncoding RNAs, or RNA containing unnatural bases, or modified RNA. The sample can also contain proteins, peptides, and other agents to be detected or measured.

[0007] The samples can be biological in origin, taken from the environment, or artificially created. Samples can be taken from any organism. Examples of samples include an animal sample, a bacterial sample, a sample from a body fluid, such as from cerebrospinal fluid, a blood sample, cells or cell-free fractions isolated from a sample, an environmental sample, a human sample, a plant sample, blood plasma, a sample containing microorganisms, a sample obtained from a plant, a sample comprising an RNA vaccine, a tissue sample, a viral sample, such as a sample containing viral RNA, and a sample of whole blood, or a sample from cells derived from whole blood. Samples derived from blood include fractions of a blood sample following centrifugation, such as those containing plasma, buffy coat (containing leukocytes and platelets), erythrocytes, as well as nucleic acids purified from such fractions such as cell-free DNA or RNA.

[0008] Use of the method can be for research, for monitoring and/or identifying exposure, monitoring and/or identifying diseases or conditions, or for determining risk of disease, condition, or change of condition such as risk of schizophrenic break. Among other uses for the method are testing samples for drug presence, side effects, or efficacy, such as for drugs used for obesity, drug addiction, neurological conditions, or other diseases. Subjects can self-collect a fingerstick sample and spot it on filter paper; since the sample is sufficiently stable and not considered a biohazard, it can be mailed or otherwise transported to a testing site. Tests using this method can be carried out on samples from the general population as a screen, or self-collected to help enable subjects to decide whether they need to see a physician — such as to indicate they may have Alzheimer’s disease, and should see a health professional for further evaluation, diagnosis, and possible treatment. Other conditions include environmental exposure to chemicals, stress, addiction, obesity, altitude sickness, and monitoring the efficacy of medication for weight reduction or appetite suppression.

[0009] Such samples are sometimes provided with added agents to stabilize the sample or sample components. Stabilizing agents include antioxidants, natural or modified carbohydrates, citrate salts, dextrose, EDTA, enzyme inhibitors, heme, hemoglobin, heparin, polyols such as glycerol, reducing agents, and thrombin, as well as cell-stabilizing or preservation reagents. However, the presence of such agents can be undesirable during subsequent steps of the method. Samples can also be stabilized against changes in pH with compatible buffering agents.

[0010] In an embodiment, the sample is treated to raise the ionic strength of the sample. The ionic strength of the sample can be raised to at least 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.9M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2. IM, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3.0M, 3.25M, 3.5M, 3.75M, 4.0M, 4.5M, 5.0M, 5.5M, 6.0M, or any range between the aforementioned ionic strengths. [0011] The increased ionic strength can be provided by any water-soluble salt, such as a combination of cations and anions. Examples of cations include sodium (Na⁺), calcium (Ca²⁺), magnesium (Mg²⁺), and potassium (K⁺). Examples of anions include chloride (CT), bicarbonate (HCO³ ), carbonate (CO?^2-), sulfate (SO4²/). More than one cation and/or more than one anion can be combined to obtain the desired ionic strength in the sample. Typical methods for increasing the ionic strength of a sample include adding a solution with a higher ionic strength, dialysis of a sample in a solution having the desired ionic strength, ion-exchange chromatography or elution, and buffer exchange using a spin column. The solution can contain other components such as compatible buffering agents and/or inhibitors to undesired enzyme activity.

[0012] Samples can also be added to filter paper or other surface and dried, with storage at room temperature, or refrigerated, or frozen until assayed. Such samples can be stored in a container with a desiccant added to the container.

[0013] At various stages of the method, the sample can be in various containers, for example a test tube or a multi -well plate such as a PCR plate. The containers can be sealed to prevent contamination, reduce evaporation, or to enable various steps to be performed at different pressures. If desired, a layer of relatively nonreactive liquid such as mineral oil can be added to the container to serve as a barrier between the sample and the atmosphere. [0014] In the method of the invention, a sample can be heated at least once to a temperature of 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, 65°C, 66°C, 68°C, 70°C, 72°C, 74°C, 75°C, 76°C, 78°C, 80°C, 82°C, 84°C, 85°C, 86°C, 88°C, 90°C, 92°C, 94°C, 95°C, 96°C, 98°C, 100°C, 102°C, 103°C, 104°C, 105°C, 106°C, 108°C, or even 110°C or higher, depending on the sample, ionic strength, pressure, and duration of heating. Useful temperatures include the aforementioned temperatures as an upper limit or any range between the aforementioned temperatures. Surprisingly, nucleic acids or representative sets thereof can remain stable at the upper temperature ranges to proceed to further steps and analysis.

[0015] The duration of the heating can be at least or up to (or lasting longer than) 30 sec, 45 sec, 1 min, 1.5 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 22 min, 24 min, 25 min, 27 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour, 1.5h, 2h, 2.5h, 3h, or more, including ranges between the aforementioned durations.

[0016] In addition, the sample can be heated to a first temperature for a first duration, and then heated or cooled to a second temperature for a second duration. The sample can be heated or cooled to a third temperature for a third duration, and so on. The temperature can also be ramped up or ramped down from one temperature to another over a period of time. If desired, the sample can be maintained or stored at a different temperature. [0017] As a step in the method, the sample can optionally be separated into at least two phases by chemical or mechanical means. The separation can isolate substantial amounts of desired or undesirable components into one phase or another. This can be performed using conventional laboratory equipment such as a manual or automated micropipette, a mechanical sipper, and a range of automated laboratory devices for handling liquids.

[0018] Useful means of separating a sample into phases includes centrifugation to two or more separate components of differing solubility or density. For example, centrifugation can separate a precipitate from a supernatant, or from another liquid or semi-liquid of a different density. An example of a semi-liquid (or semi-solid) phase is a blob. As used herein, a “blob” is a physical phase of a sample having no fixed or definite shape but remaining separate from an adjacent liquid phase. The separation can be due to the blob having a higher surface tension than the surrounding liquid phase. When a blob has a greater density than the surrounding liquid, it may come to rest at the bottom of the container. A particular blob is a “glob”, which has an essentially globular shape. Another separation means is filtration where a sample is passed through a physical filter to capture or entrap components on the filter or to binding agents attached to the filter, while allowing a filtrate phase to pass through. Yet another means of separating phases is to allow certain components to adhere to a solid phase, such as a bead or solid surface like filter paper, and allowing other components to be solubilized, extracted, or otherwise separated from the solid phase into another phase, such as a liquid phase. The filter paper can be treated or untreated, such as Whatman® qualitative filter paper (e.g., Grades 1, 2, 3, or 4) or Protein Saver Cards. Treated filter papers include FTA® cards or filter paper treated with chaotropic salts, to fix and store nucleic acids. The treatment can be in the form of protein denaturants impregnated into the paper matrix to cause lysis of cells. Other treatments include use of chelating agents and free-radical traps.

[0019] The detection of nucleic acids and nucleic acid sequences under such conditions may be promoted when occurring in one phase compared to another. Without being bound by a particular explanation or mechanism, the adhesion of nucleic acids or other components of interest to filter papers and other solid supports may promote or increase accessibility to enzymes in subsequent steps by immobilization and/or denaturation of nucleic acids, whether partial or complete. Such adhesion may result in improved or expanded detection of nucleic acid sequences compared to the original sample, for example by increased accessibility of nucleotides for base-pair hybridization.

[0020] Various enzymatic reactions can be performed on the sample or on a phase of the sample containing components of interest, such as nucleic acids. The substrates of the enzymes can be single- or double-stranded DNA, RNA, or DNA:RNA hybrids or DNA-RNA chimeric molecules. Particular enzymatic reactions include ligation, amplification, such as with a polymerase, cleavage, and hydrolysis of nucleic acids. The cleavage reactions can be sequence- or site-specific. Nucleic acids can be degraded using exonucleases or endonucleases, and they can degrade single-stranded, double-stranded nucleic acids, including double-strands having singlestranded overhangs. The activity of certain enzymes can be inhibited by the presence of undesirable components such as heme, hemoglobin, IgG, lactoferrin, and DNA from other cells.

[0021] After a degradation or denaturation step, additional steps may be added. For example, enzymatic-based assays can be carried out to measure the RNA or DNA, such as conversion of RNA to cDNA and then performing PCR, or such as an isothermal reaction. Other steps can be performed, such as nuclease protection or hybridization-based assays on the RNA, DNA, or cDNA.

[0022] Kits for performing the method of the invention are also provided. The kits can contain one or more of the components used to perform the method of the invention. For example, a kit can contain a high ionic strength solution, an exonuclease in a storage buffer, and a polymerase in a storage buffer. Kit components can contain one or more inhibitors such as active-site binding molecules and peptides, substrate analogs, benzopurpurin B, diethyl pyrocarbonate, guanidinium thiocyanate, peptidomimetic inhibitors, polymerization or cross-linking agents, polypeptide ribonuclease inhibitors (RI), RNA ecz/re™ (Ambion), and a vanadyl ribonucleoside. Further components of the kit can include a detergent, a reaction container, and nucleic acids. Useful nucleic acids include RNA, DNA, such as primers for polymerase amplification. Other components can include treated or untreated filter paper.

[0023]

[0024] Example 1: Detection of RNA sequences from heat-treated samples

[0025] A sample of human whole blood was obtained by fingerstick, then spotted on filter paper and air-dried. Paper containing the dried blood was collected and suspended in buffer, then layered with mineral oil to minimize evaporation. The sample was heated at various temperatures for varying times. The RNA in the sample was then assayed (in triplicate) as previously described in U.S. Patent 9,856,521 using a whole-transcriptome set of probes representing over 20,000 genes, including probes for over 1000 exon junctions. A probe was considered detected if greater than 15 read counts were detected via sequencing. Results are shown as a percentage of the maximum number of probe sequences detected in the sample (average of three replicates).

[0026] In one series, the sample was not heated (detecting 82% of max of whole-transcriptome assay) as a control, or heated for 10 minutes at 80°C (87%) or at 95°C (100%). The corresponding percentages of max for the exon-junction probes were 75%, 46%, and 100%.

[0027] In another series, the sample was not heated as a control (82% of max), or heated at 95°C for 5 minutes, (82%), for 10 minutes (100%), or for 15 minutes (100%). The corresponding percentages of max for the exonjunction probes were 75%, 89%, 100%, and 94%.

[0028] These results demonstrate that the heating step does not degrade the detection of RNA in the sample, but surprisingly, the heating enables detection of greater numbers of the gene sequences and exon junctions present in the dried blood sample. Without being bound by a particular explanation or mechanism, the rising temperature and high ionic strength are believed to unfold access to significant gene sequences that are present in the sample but otherwise difficult for detector oligos to hybridize with at conventional temperatures, thereby allowing enzymatic detection of the sequences to proceed with a more complete representation of the sample.

[0029] [0030] Example 2: Detection of gene expression signatures associated with Alzheimer’s Disease and Parkinson’s Disease

[0031] Patient fingerstick blood samples were collected, heat-treated at 95°C for 10 minutes and analyzed as in Example 1, enabling detection of RNA expression. The expression profiles of de-identified samples were correlated with clinician diagnoses of Alzheimer’s Disease (AD), Parkinson’s Disease (PD), or asymptomatic. When available, /5-amyloid PET scores and DaTscan (dopamine transporter scan) results were considered as factors.

[0032] A set of 68 genes was identified: ACTB, ALAS2, ARHGAP9, ARHGDIB, B2M, BNIP3L, C9orf78, CRIP1, CSF3R, CTDSP1, DCAF12, DDX5, DEF A3, FCGR3A, FCMR, FPR1, FTL, GABARAPL2, GADD45B, GNAS, GNLY, GPX1, GYPC, HBA1, HBG1, HDGF, HLA-A, HLA-C, IFIT1, IGF2R, IGLV3-21, ITGB2, LST1, LYZ, MAP2K3, MG AM, MMP25, MNDA, MTRNR2L9, MYO IF, NCF2, NEAT1, NKG7, NPRL3, PDZK1IP1, PGGHG, PIP4K2A, PRF1, PROK2, PSMF1, RFK, RNASET2, S100A6, S100A9, SI OOP, SEC62, SLC2A3, SORL1, TENT5C, T0MM7, TRBC1, TRIM22, TRIM58, UBB, VSIR, YBX1, YBX3, and YWHAZ. Subsets of the 68 genes or supersets that include some or all of the 68 genes can be used as a correlate with pathways associated with AD or PD, for example, subsets of genes associated with pathways for neutrophil-mediated immunity, neutrophil activation involved in an immune response, or neutrophil degranulation. Use of a subset of those genes can be used to classify patients with AD, and a different but partially overlapping subset of genes can be used to classify patients with PD. The detection of the genes can be used for drug discovery to identify compounds or treatments that modulate the expression of the genes, related pathways, or conditions. [0033] The invention therefore provides methods for classifying an Alzheimer’s Disease (AD) or Parkinson’s Disease (PD) state by detecting expression of one or more of genes of the gene signature. The invention also provides methods for monitoring an AD or PD state by performing the method on samples taken over time from a patient suspected of having AD or PD, or for the early detection of AD or PD from patients not yet having symptoms that reach the level required for the diagnosis of AD or PD, or from those having biomarkers of AD or PD but not yet exhibiting symptoms or having symptoms (such as mild cognitive impainnent) that lie below a defined threshold or do not completely fulfil a standardized set of diagnostic criteria. Such biomarkers include tau protein (such as total tau or phosphorylated tau 181), neurofilament light chain (NfL) or /?-amyloid in bodily fluids such as cerebrospinal fluid (CSF), saliva, urine, blood, or plasma, for example. Other criteria include results from structural (e.g, MRI, CT), functional (e.g., FDG-PET), and molecular compound imaging, for example imaging for amyloid plaque. The invention includes performing the methods on patients, analyzing results according to an algorithm or machine learning or other artificial intelligence, and reporting results to an end user. These methods can be computer-implemented using a variety of computer systems including mobile and other hardware devices, applications and other software, and server- or internet-connected systems. The invention also provides kits of nucleic acid probes for detecting the expression of one or more of genes in the gene signature. [0034] The headings provided above are intended only to facilitate navigation within the document and should not be used to characterize the meaning of one portion of text compared to another. Skilled artisans will appreciate that additional embodiments are within the scope of the invention. The invention is defined only by the following claims; limitations from the specification or its examples should not be imported into the claims.

Claims

Claims

1. A method for treating a sample containing RNA, comprising

(a) raising the ionic strength of the sample to at least 0. IM;

(b) heating the sample to at least 50 °C; and

(c) perfonning a polymerase reaction on the sample.

2. The method of claim 1, wherein the sample is selected from the group consisting of an animal sample, a bacterial sample, a blood sample, an environmental sample, extracted RNA, extracted DNA, a human sample, a plant sample, blood plasma, a tissue sample, a sample comprising an RNA vaccine, a sample containing viral RNA, and a sample of whole blood.

3. The method of claim 1, wherein the sample contains a stabilization agent.

4. The method of claim 1, wherein step (a) is performed by adding a solution.

5. The method of claim 4, wherein the solution comprises a detergent.

6. The method of claim 1, wherein heating step (b) is sustained for longer than 5 minutes.

7. The method of claim 1, further comprising the step of

(c) separating the sample into at least two phases, wherein at least one phase contains RNA.

8. The method of claim 7, wherein the phases in separating step (c) are solid and liquid.

9. The method of claim 7, wherein the phases in separating step (c) are a semiliquid glob and a supernatant.

10. The method of claim 7, wherein the phases are separated by centrifugation.

11. The method of claim 1, further comprising the step of performing reverse transcription.

12. The method of claim 1, further comprising the step of ligating nucleic acids.

13. The method of claim 1, further comprising the step of adding a nuclease.

14. The method of claim 13, wherein the nuclease is capable of digesting DNA.

15. The method of claim 1, further comprising the step of detecting the presence of RNA.

16. The method of claim 1, further comprising the step of sequencing DNA.

17. A kit comprising a high ionic strength solution, an exonuclease in a storage buffer, and a polymerase in a storage buffer.

18. The kit of claim 17, further comprising a detergent.

19. The kit of claim 17, further comprising a reaction container.

20. The kit of claim 17, further comprising DNA.

21. The kit of claim 17, further comprising primers for polymerase amplification.