A protective agent for stabilizing RNA in a body fluid and use thereof

Abstract

The application discloses a protective agent for stabilizing RNA in body fluid and application thereof. The active component of the protective agent comprises nucleoside triphosphates and a protein denaturant, wherein the nucleoside triphosphates comprise at least one of adenosine triphosphate or guanosine triphosphate. The application finds through experiments that the nucleoside triphosphates can achieve excellent RNA protection effect at a lower concentration, wherein the half protection concentration is about 21 mg / mL, which is obviously lower than that of traditional strong denaturant guanidinium isothiocyanate (128.30 mg / mL). In addition, when the nucleoside triphosphates are used in combination with guanidinium isothiocyanate, a significant synergistic enhancement effect (CI<0.7) is achieved, and RNA degradation can be continuously delayed for up to 384 hours of incubation. The protective agent is not only suitable for the protection of in vitro transcription mRNA, but also suitable for the protection of various RNA types such as viral RNA, and can stably play a role in a complex body fluid environment such as plasma, and can be widely applied to the preservation of clinical samples.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a protective agent for stabilizing RNA in body fluids and its application. Background Technology

[0002] RNA is highly susceptible to degradation by endogenous ribonucleases in bodily fluids (such as plasma, serum, and urine), resulting in poor stability during in vitro detection and storage. This, in turn, affects the accuracy and reproducibility of downstream analyses such as RT-qPCR and sequencing. To inhibit RNase activity, researchers have proposed several RNA protection strategies, such as protective agent systems composed of strong protein denaturants (e.g., guanidine isothiocyanate GITC), reducing agents (e.g., β-mercaptoethanol), or surfactants (e.g., sodium N-lauryl sarcosinate). For example, a mixture of guanidine isothiocyanate, trisodium citrate, sodium N-lauryl sarcosinate, and β-mercaptoethanol can be used to prevent RNA degradation during pretreatment.

[0003] However, RNA protectants in related technologies still have significant shortcomings. First, strong denaturants, such as GITC, typically require high concentrations (e.g., hundreds of mg / mL) to effectively inhibit RNase. This not only increases reagent costs but may also inhibit subsequent RNA capture, reverse transcription, or PCR amplification, affecting detection sensitivity. Second, high concentrations of denaturants can damage the structural integrity of cells or extracellular vesicles, limiting their use in situations where preserving the native state of vesicles is necessary. Furthermore, current protectants mostly rely on a single mechanism (e.g., protein denaturation) and lack the ability to synergistically inhibit multiple pathways of RNA degradation. Therefore, their protective effect remains unsatisfactory under prolonged incubation or complex body fluid environments.

[0004] Therefore, there is an urgent need to develop an RNA protectant that can efficiently inhibit RNase activity at low concentrations, has minimal interference with subsequent detection, and possesses long-term stable protection capabilities, so as to achieve long-term, wide-range, and low-toxicity RNA stability protection in body fluids. Summary of the Invention

[0005] The first aspect of this invention aims to provide a protective agent for stabilizing RNA in body fluids.

[0006] A second aspect of the present invention is to provide a method for stabilizing RNA.

[0007] A third aspect of the present invention aims to provide the use of nucleoside triphosphates or a combination thereof with a protein denaturant in the preparation of RNA protection products.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a protective agent for stabilizing RNA in body fluids, the active ingredients of which include nucleoside triphosphates and protein denaturants, wherein the nucleoside triphosphates include at least one of adenosine triphosphates or guanosine triphosphates.

[0009] The protective agent for stabilizing RNA in body fluids according to embodiments of the present invention has at least the following beneficial effects: The protective agent of this invention exhibits high RNA protection efficiency and low concentration of active components, while also possessing the advantage of low cytotoxicity. Experiments have shown that nucleoside triphosphates (such as ATP or GTP) can achieve excellent RNA protection at low concentrations, with a half-maximal protective concentration (PC50) of approximately 21 mg / mL, compared to a high PC50 of 128.30 mg / mL for the traditional strong denaturant GITC, indicating that the protective efficiency of ATP / GTP is approximately six times that of GITC. At a concentration of 32 mg / mL, ATP and GTP increased mRNA residue by approximately 130-fold and 1200-fold, respectively. Furthermore, notably, cell viability assays show that the active components of the protective agent (such as ATP and GTP), within the concentration range required for RNA protection, exhibit lower toxicity to human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma cell line (A549) than GITC, demonstrating its excellent safety profile.

[0010] In some embodiments of the present invention, the final concentration of the nucleoside triphosphate is 1~100 mg / mL.

[0011] Preferably, the final concentration of the nucleoside triphosphate is 1~64 mg / mL. For example, it can be 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 64 mg / mL, etc.

[0012] More preferably, the final concentration of the nucleoside triphosphate is 16-32 mg / mL.

[0013] In some embodiments of the present invention, the protein denaturing agent includes at least one of guanidine hydrochloride and guanidine isothiocyanate.

[0014] Preferably, the protein denaturant is guanidine isothiocyanate.

[0015] This invention reveals that the combined use of nucleoside triphosphates (such as ATP or GTP) and guanidine isothiocyanate (GITC) exhibits a significant synergistic enhancement effect on RNA protection. Combination index (CI) analysis showed that the CI for GITC combined with ATP was 0.65, and the CI for GITC combined with GTP was 0.68, both significantly less than 1, indicating a synergistic effect. Furthermore, in a long-term incubation experiment lasting 384 hours, the combined use of GTP and GITC further reduced the RNA degradation rate and significantly prolonged the stable preservation time of RNA compared to the single-use group. In practical applications, lower doses of GITC can be used to further reduce system toxicity while maintaining the protective effect.

[0016] In some embodiments of the present invention, the final concentration of the protein denaturant is 1 to 1000 mg / mL.

[0017] Preferably, the final concentration of the protein denaturing agent is 100-800 mg / mL. For example, it can be 100, 125, 150, 180, 200, 300, 400, 500, 600, 700 or 800 mg / mL, etc.

[0018] In some embodiments of the present invention, the RNA includes at least one of mRNA, tRNA, rRNA, and sRNA.

[0019] The protective agent of this invention is not only applicable to in vitro transcribed mRNA, but also to various RNA types such as viral RNA, and can function rapidly and stably in complex body fluid environments such as plasma. It can be widely used in the collection, transportation and storage of clinical samples (containing RNA).

[0020] A second aspect of the invention provides a method for stabilizing RNA, comprising adding a nucleoside triphosphate and a protein denaturing agent to a body fluid containing RNA, wherein the nucleoside triphosphate includes at least one of adenosine triphosphate or guanosine triphosphate.

[0021] The method according to embodiments of the present invention has at least the following beneficial effects: This invention significantly inhibits the activity of endogenous ribonucleases in a short time (e.g., 15 minutes) by adding low concentrations of nucleoside triphosphates (such as ATP or GTP) to body fluids (such as plasma). The protective effect is comparable to that of the traditional strong denaturant GITC and shows a good linear relationship. In particular, at a concentration of 32 mg / mL, the residual mRNA can be increased by 130 to 1200 times, and the required half-maximal protective concentration (about 21 mg / mL) is much lower than that of GITC (about 128 mg / mL), resulting in higher protective efficiency.

[0022] In some embodiments of the present invention, the RNA includes RNA from bodily fluids or RNA that has undergone purification.

[0023] In some embodiments of the present invention, the body fluid is selected from plasma, serum, urine, saliva or extracellular vesicle suspension.

[0024] In some embodiments of the present invention, the protein denaturing agent includes at least one of guanidine hydrochloride and guanidine isothiocyanate.

[0025] Preferably, the protein denaturant is guanidine isothiocyanate.

[0026] This invention reveals that combining nucleoside triphosphates (such as ATP or GTP) with GITC exhibits a synergistic effect (CI < 0.7) and can sustainably delay RNA degradation during incubation up to 384 hours. Furthermore, the nucleoside triphosphates used in this invention show significantly lower toxicity to various cell types (such as HUVEC and A549) within the effective protective concentration range compared to GITC, combining the advantages of high efficiency, low toxicity, long-lasting effect, and suitability for complex body fluid environments.

[0027] In some embodiments of the present invention, the final concentration of the nucleoside triphosphate is 1~100 mg / mL.

[0028] Preferably, the final concentration of the nucleoside triphosphate is 1~64 mg / mL. For example, it can be 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 64 mg / mL, etc.

[0029] More preferably, the final concentration of the nucleoside triphosphate is 16-32 mg / mL.

[0030] In some embodiments of the present invention, the final concentration of the protein denaturant is 1 to 1000 mg / mL.

[0031] Preferably, the final concentration of the protein denaturing agent is 100-800 mg / mL. For example, it can be 100, 125, 150, 180, 200, 300, 400, 500, 600, 700 or 800 mg / mL, etc.

[0032] In some embodiments of the present invention, the temperature of the RNA in the stabilizing body fluid is -20 to 38°C.

[0033] Preferably, the temperature of the RNA in the stabilizing body fluid is 4~37℃. For example, it can be 4, 5, 10, 15, 18, 25, 28, 30, 32, 36 or 37℃, etc.

[0034] A third aspect of the invention provides the use of a composition of nucleoside triphosphate and a protein denaturant in the preparation of an RNA protection product, wherein the nucleoside triphosphate includes at least one of adenosine triphosphate or guanosine triphosphate.

[0035] In some embodiments of the present invention, at least one of the protein denaturing agents guanidine hydrochloride and guanidine isothiocyanate is selected.

[0036] Preferably, the protein denaturant is guanidine isothiocyanate.

[0037] In some embodiments of the present invention, the final concentration of the nucleoside triphosphate is 1~100 mg / mL.

[0038] Preferably, the final concentration of the nucleoside triphosphate is 1~64 mg / mL. For example, it can be 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 64 mg / mL, etc.

[0039] More preferably, the final concentration of the nucleoside triphosphate is 16-32 mg / mL.

[0040] In some embodiments of the present invention, the final concentration of the protein denaturant is 1 to 1000 mg / mL.

[0041] Preferably, the final concentration of the protein denaturing agent is 100-800 mg / mL. For example, it can be 100, 125, 150, 180, 200, 300, 400, 500, 600, 700 or 800 mg / mL, etc.

[0042] In some embodiments of the present invention, the mass concentration ratio of the nucleoside triphosphate to the protein denaturant is 1:5~30.

[0043] In some embodiments of the present invention, the product includes reagents, lyophilized powders, chips, or reagent kits.

[0044] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of the effect of different concentrations of nucleoside triphosphates on mRNA stability in Example 1 of the present invention are shown, where a is ATP and b is GTP; Figure 2 This is the detection result of the inhibitory effect of nucleoside triphosphate or guanidine isothiocyanate on RNA degradation at different concentrations within a short period of time in Example 2 of the present invention; Figure 3The results of the detection of the long-term protective effect of nucleoside triphosphates and / or guanidine isothiocyanate on RNA in Example 3 of the present invention; Figure 4 The results of the half-maximal protective concentration (WMC) of nucleoside triphosphate or guanidine isothiocyanate against RNA in Example 4 of this invention are shown, where a is GITC, b is ATP, and c is GTP. Figure 5 The results of the detection of the synergistic effect of the combination of nucleoside triphosphate and guanidine isothiocyanate on RNA protection in Example 5 of the present invention are shown, where a is the combination of ATP and GTP, b is the combination of GITC and ATP, and c is the combination of GITC and GTP. Figure 6 The results of cytotoxicity testing of nucleoside triphosphate and / or guanidine isothiocyanate in Example 6 of the present invention are shown, where a is the cytotoxicity test result on human umbilical vein endothelial cells and b is the cytotoxicity test result on human lung adenocarcinoma cell lines. Detailed Implementation

[0046] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0047] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0048] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items. For example, A and / or B includes (A and B) and (A or B).

[0049] In this invention, the purity of adenosine triphosphate (ATP) is ≥98%; the purity of guanosine triphosphate (GTP) is ≥98%. The solvent for the ATP or GTP working solution is phosphate-buffered saline (PBS) with a pH of 7.4. Human umbilical vein endothelial cells are cultured using endothelial cell-specific medium (ECM, containing 5% fetal bovine serum and 1% endothelial growth factor); human lung adenocarcinoma cell lines are cultured using DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin).

[0050] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0051] Example 1 This embodiment uses a mixture of in vitro transcribed mKate2 mRNA and total cell protein extract as samples to detect the effect of ATP / GTP on RNA stability. The specific experiments are as follows.

[0052] 1. Experimental Methods 0.5 μg of mKate2 mRNA obtained from in vitro transcription was added to 20 μL of ATP or GTP working solution (PBS solvent, pH 7.4), followed by 40 μL of total cell protein extraction solution, bringing the total reaction volume to 60 μL. The initial concentration of mKate2 mRNA was approximately 8.33 ng / μL, and the final concentrations of ATP or GTP were set to 0, 1, 2, 4, 16, and 32 mg / mL, respectively. The mixtures were incubated at 37°C for 6 h, purified and eluted using an RNA purification kit, and the residual mRNA content in each group was detected by RT-qPCR.

[0053] Specifically, 1 μL of the treated sample was added to a 20 μL reverse transcription reaction system, which included 5 μL of reverse transcription mix and 14 μL of water. The mixture was reacted at 37°C for 15 min, followed by inactivation of the reverse transcriptase at 85°C for 5 s. After reverse transcription, 1 μL of cDNA was added to a 20 μL PCR reaction system. This system included 10 μL of PCRMix, 0.4 μL of forward primer, 0.4 μL of reverse primer, and 8.2 μL of water. The PCR amplification program was: 95°C pre-denaturation for 30 s; followed by 40 cycles, each consisting of 95°C for 10 s and 65°C for 30 s. The residual mRNA in the 0 mg / mL ATP-treated group was set as 1, and the relative fold increases of residual mRNA in different ATP-treated groups were calculated.

[0054] 2. Experimental Results The results are as follows Figure 1 As shown, compared with the groups without ATP or GTP, both ATP and GTP exhibited concentration-dependent RNA protective effects. With increasing concentration, the relative mRNA content significantly increased. At a concentration of 32 mg / mL, the residual mRNA level in the ATP-treated group was approximately 130-fold higher than that in the 0 mg / mL group, and approximately 1200-fold higher in the GTP-treated group.

[0055] The above results indicate that nucleoside triphosphates (such as ATP or GTP) can significantly enhance RNA stability.

[0056] Example 2 This embodiment uses a mixture of SARS-CoV-2 RNA standards with different copy numbers and sterile New Zealand rabbit plasma as test samples to detect the effects of nucleoside triphosphates (such as ATP and GTP) and guanidine isothiocyanate (GITC) on RNA protection. The specific experiments are as follows.

[0057] 1. Experimental Methods 0.5 μL of SARS-CoV-2 RNA standards at different concentrations were added to 39.5 μL of sterile New Zealand rabbit plasma (containing plasma endogenous nucleases), and mixed well to form a 40 μL sample system. Then, 20 μL of different treatment solutions prepared with PBS were added to each system, bringing the total volume to 60 μL, as shown in the following groups: (1) ATP group: Add ATP working solution to make the final ATP concentration 32 mg / mL; (2) GTP group: Add GTP working solution to make the final GTP concentration 32 mg / mL; (3) GITC group: Add GITC working solution to make the final concentration of GITC 473 mg / mL (guanidine isothiocyanate, CAS number 593-84-0); where 473 mg / mL is the commonly used optimal working concentration of GITC for treating nucleic acid samples; (4) PBS control group: Add an equal volume of PBS as a blank control to eliminate the influence of solvent and sample volume changes on the detection results.

[0058] After each group of samples was added, it was incubated at 25°C for 15 min to evaluate the effects of different treatment conditions on the stability of SARS-CoV-2 RNA standards in plasma and subsequent detection capabilities.

[0059] One-step RT-qPCR was used for detection. After purification and elution using a viral RNA purification kit, 1 μL of the above samples was added to a 20 μL one-step RT-qPCR reaction system, resulting in SARS-CoV-2 RNA standard copy numbers of 6000, 1500, 375, 94, 23, 6, and 1.5 copies in each reaction system. The 20 μL reaction system consisted of: 10 μL One Step SYBR Green Mix, 1 μL One Step SYBR Green Enzyme Mix, 0.4 μL ROX Reference Dye, 0.4 μL forward primer, 0.4 μL reverse primer, and 6.8 μL water.

[0060] The amplification program was as follows: reverse transcription was performed at 50℃ for 15 min; pre-denaturation was performed at 95℃ for 30 s; followed by 40 cycles: 95℃ for 10 s, 65℃ for 30 s. The changes in Ct values ​​for each group were detected and recorded.

[0061] 2. Experimental Results The results are as follows Figure 2 As shown, the ATP and GTP treatment groups exhibited similar Ct value trends to the GITC treatment group across different viral copy number ranges, showing a good linear relationship. No effective amplification signal was obtained in the PBS control group, presumably due to the lack of protective reagents, leading to RNA degradation in plasma. These results indicate that nucleoside triphosphates can effectively inhibit RNA degradation by endogenous plasma nucleases within a short time, demonstrating protective effects comparable to common protein denaturing agents (such as guanidine isothiocyanate).

[0062] Example 3 This embodiment uses a mixture of SARS-CoV-2 RNA standard and sterile New Zealand rabbit plasma as the test sample to further explore the effects of nucleoside triphosphate and guanidine isothiocyanate on long-term RNA protection. The specific experiments are as follows.

[0063] 1. Experimental Methods 0.5 μL of SARS-CoV-2 RNA standard at a concentration of 10000 copies / μL was mixed with 19.5 μL of sterile New Zealand rabbit plasma (containing plasma endogenous nucleases), and then 40 μL of different treatment solutions prepared with PBS were added for grouped treatment. ATP group: ATP was added to a final concentration of 32 mg / mL; GTP group: GTP was added to a final concentration of 32 mg / mL; Guanidine isothiocyanate group: Guanidine isothiocyanate (GITC) was added to a final concentration of 473 mg / mL.

[0064] GTP+GITC group: GTP and guanidine isothiocyanate were added to a final concentration of 32 mg / mL and a final concentration of 473 mg / mL.

[0065] After mixing, the samples were incubated at 25°C for 1.5, 3, 6, 12, 24, 48, 96, 192, and 384 h to evaluate the long-term protective efficacy of ATP, GTP, and GITC against SARS-CoV-2 RNA standards.

[0066] After incubation, the samples were purified and eluted using a viral RNA purification kit, and detected by one-step RT-qPCR. Specifically, 1 μL of the treated sample was added to a 20 μL reaction system, which included 10 μL One-Step SYBR Green Mix, 1 μL One-Step SYBR Green Enzyme Mix, 0.4 μL ROX Reference Dye, 0.4 μL forward primer, 0.4 μL reverse primer, and 6.8 μL water. The reaction program was: 50℃ for 15 min; 95℃ for 30 s; followed by 40 cycles: 95℃ for 10 s, 65℃ for 30 s. The residual mRNA in the 0 h treatment group was set as 100%, and the percentage of residual mRNA in each treatment group at different time points was calculated.

[0067] 2. Experimental Results The results are as follows Figure 3 As shown, both ATP and GTP alone can delay RNA degradation, while GITC can maintain stability for a longer period. The combined use of GTP and GITC can further reduce the RNA degradation rate, indicating that nucleoside triphosphates have the ability to protect RNA stability for a long time. Furthermore, the experiment found that GITC's protective ability over a longer period is superior to that of ATP / GTP alone, presumably because of the relatively higher concentration of GITC and the fact that GITC is a protein denaturant, while ATP / GTP mainly act as inhibitors.

[0068] Example 4 This embodiment uses a mixture of SARS-CoV-2 RNA standard and sterile New Zealand rabbit plasma as the test sample to explore the protective ability of different concentrations of nucleoside triphosphate and guanidine isothiocyanate on RNA. The specific experiments are as follows.

[0069] 1. Experimental Methods 0.5 μL of SARS-CoV-2 RNA standard at a concentration of 10,000 copies / μL was mixed with 39.5 μL of sterile New Zealand rabbit plasma (containing endogenous nucleases), and then 20 μL of ATP / GTP / GITC working solution (pH 7.4, PBS) was added simultaneously, bringing the total reaction volume to 60 μL. The final concentrations of ATP / GTP were 8, 16, 24, 32, and 64 mg / mL, and the final concentrations of GITC were 30, 60, 120, 240, and 480 mg / mL, respectively. After mixing, the samples were incubated at 25°C for 2 h to evaluate the protective efficacy of different concentrations of ATP, GTP, and GITC against the SARS-CoV-2 RNA standard. An equal volume of PBS was used as a positive control group instead of sterile New Zealand rabbit plasma.

[0070] After incubation, the samples were purified and eluted using a viral RNA purification kit, and detected by one-step RT-qPCR. Specifically, 1 μL of the treated sample was added to a 20 μL reaction system, which included 10 μL One-Step SYBR Green Mix, 1 μL One-Step SYBR Green Enzyme Mix, 0.4 μL ROX Reference Dye, 0.4 μL forward primer, 0.4 μL reverse primer, and 6.8 μL water. The reaction program was: 50℃ for 15 min; 95℃ for 30 s; followed by 40 cycles: 95℃ for 10 s, 65℃ for 30 s. Using the RNA content of the PBS positive control group as 100%, the percentage of residual mRNA in different treatment groups was calculated. The half-maximal protective concentration (PC50) was calculated using dose-response curve fitting.

[0071] 2. Experimental Results The results are as follows Figure 4 As shown, the calculated concentrations of small molecules required for GITC to protect half of the RNA from degradation are 128.30 mg / mL, ATP is 21.40 mg / mL, and GTP is 21.05 mg / mL.

[0072] The above results indicate that nucleoside triphosphates can achieve RNA protection effects comparable to traditional strong denaturants at relatively low concentrations.

[0073] Example 5 This embodiment uses a mixture of SARS-CoV-2 RNA standard and sterile New Zealand rabbit plasma as the test sample to further explore whether the combined use of nucleoside triphosphate and guanidine isothiocyanate (GITC) has a synergistic effect on RNA protection. The specific experiment is as follows.

[0074] 1. Experimental Methods 0.5 μL of SARS-CoV-2 RNA standard at a concentration of 10,000 copies / μL was mixed with 19.5 μL of sterile New Zealand rabbit plasma (containing plasma endogenous nucleases), and then 40 μL of ATP / GTP / GITC mixed working solution (ATP+GTP, ATP+GITC, GTP+GITC) at pH 7.4 with PBS as solvent was added simultaneously, bringing the total reaction volume to 60 μL. This resulted in final ATP / GTP concentrations of 4, 8, 12, 16, 24, and 32 mg / mL, and final GITC concentrations of 30, 60, 120, 240, and 480 mg / mL, respectively. After mixing, the samples were incubated at 25°C for 2 h. The synergistic effect of the combined use of ATP, GTP, and GITC on RNA protection was evaluated using the combination index (CI). An equal volume of PBS was used as a positive control group instead of sterile New Zealand rabbit plasma.

[0075] After incubation, the samples were purified and eluted using a viral RNA purification kit, and detected by one-step RT-qPCR. Specifically, 1 μL of the treated sample was added to a 20 μL reaction system, which included 10 μL One-Step SYBR Green Mix, 1 μL One-Step SYBR Green Enzyme Mix, 0.4 μL ROX Reference Dye, 0.4 μL forward primer, 0.4 μL reverse primer, and 6.8 μL water. The reaction program was: 50℃ for 15 min; 95℃ for 30 s; followed by 40 cycles: 95℃ for 10 s, 65℃ for 30 s. The half-maximal protective concentration (PC50) was calculated by dose-response curve fitting, with the RNA content of the PBS positive control group as 100%.

[0076] Where CI (combination index) = D1 / (D X )1+D2 / (D X )2; (D X )1 and (D X PC50 represents the dose required to produce a 50% effect when drugs 1 and 2 are used alone. D1 and D2 represent the doses of drugs 1 and 2 required to produce the same effect when used in combination, respectively. CI < 1 indicates synergistic effect, CI = 1 indicates additive effect, and CI > 1 indicates antagonistic effect. Within the range of CI < 1, a smaller value usually indicates a stronger synergistic effect.

[0077] 2. Experimental Results The results are as follows Figure 5The results showed that the CI was 1.07 for ATP and GTP in combination; 0.65 for GITC and ATP in combination; and 0.68 for GITC and GTP in combination, where CI < 1 indicates a synergistic effect.

[0078] The above results indicate a synergistic enhancement effect between nucleoside triphosphates and GITC.

[0079] Example 6 This embodiment tests the cytotoxicity of nucleoside triphosphates and GITC, and the specific experiments are as follows.

[0080] 1. Experimental Methods In this experiment, human umbilical vein endothelial cells (HUVEC) and human lung adenocarcinoma cell line (A549) were selected as cell models. The effects of different concentrations of GITC, ATP, and GTP on cell survival rate were detected by cell viability assay. The specific steps are as follows: (1) Cell culture and inoculation HUVEC and A549 cells were cultured in incubators at 37°C, 5% CO2, and saturated humidity. Cells were passaged every 2-3 days. Cells in the logarithmic growth phase were harvested, digested with trypsin, and then the digestion was terminated with serum-containing medium. The cells were centrifuged at 1000 rpm for 5 min, collected, and resuspended in complete medium.

[0081] The cells were then seeded into 96-well plates, 100 μL per well. The 96-well plates were pre-cultured in an incubator for 24 h to allow the cells to adhere completely before use.

[0082] (2) Cell grouping treatment Different concentrations of GITC, ATP, and GTP were added according to concentration gradients of 0.63, 1.25, 2.50, 5.00, 10.00, 20.00, 40.00, and 80.00 mM, with three replicates for each concentration. A negative control group (containing only complete culture medium) and a blank control group were also included. The 96-well plates were returned to the incubator for another 24 h of incubation.

[0083] (3) Cell viability detection and statistics Cell viability was assessed using the CCK-8 assay. After incubation, 10 μL of CCK-8 solution was added to each well, and incubation continued for 1–4 h. The absorbance (OD value) at 450 nm was then measured using a microplate reader, and cell viability and IC50 were calculated. 50 value.

[0084] Cell viability (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%.

[0085] 2. Experimental Results The results are as follows Figure 6 As shown, in HUVEC cells, the IC50 of GITC was 8.52 mM, or 1.01 mg / mL, and the IC50 of ATP was... 50 The IC50 of GTP was 3.20 mM (i.e., 1.63 mg / mL). 50 The IC50 of GITC was 3.00 mM (1.57 mg / mL); in A549 cells, the IC50 of GITC was 25.02 mM (2.96 mg / mL), and the IC50 of ATP was... 50 The IC50 of GTP was 26.11 mM (i.e., 13.24 mg / mL). 50 The concentration was 15.81 mM (i.e., 8.27 mg / mL).

[0086] The above results indicate that nucleoside triphosphates have lower overall toxicity than the traditional high-concentration GITC system within the concentration range required to achieve RNA protection.

[0087] In summary, this invention provides a protective agent for stabilizing RNA in body fluids and its application. This invention discovers that using nucleoside triphosphates (such as ATP or GTP) as the active ingredient, alone or in combination with guanidine isothiocyanate (GITC), can inhibit ribonuclease-mediated RNA degradation. Specifically, experiments show that ATP or GTP at low concentrations (approximately 21 mg / mL half-maximal protective concentration) can achieve RNA protection effects comparable to the traditional strong denaturant GITC (approximately 128 mg / mL half-maximal protective concentration); at a concentration of 32 mg / mL, ATP and GTP increased mRNA residue by approximately 130-fold and 1200-fold, respectively. Furthermore, when GTP is used in combination with GITC, the combination index CI < 1, exhibiting a synergistic enhancement effect, and can continuously delay RNA degradation during incubation up to 384 hours. In addition, cell viability experiments show that the nucleoside triphosphate concentration required to achieve RNA protection is significantly less toxic to HUVEC and A549 cells than the high-concentration GITC system (e.g., in A549 cells, the IC50 of GITC is significantly lower). 50 The concentration was 2.96 mg / mL, while the IC50 of ATP and GTP was... 50 The concentrations were 13.24 mg / mL and 8.27 mg / mL, respectively. The RNA protectant of this invention has advantages such as high protective efficiency, low effective concentration, significant synergistic effect, low cytotoxicity, and applicability to various bodily fluid environments (such as plasma), and has excellent application prospects in the field of in vitro RNA protection and detection.

[0088] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A protective agent for stabilizing RNA in body fluids, characterized in that, The active ingredients include nucleoside triphosphates and protein denaturants, wherein the nucleoside triphosphates include at least one of adenosine triphosphate or guanosine triphosphate.

2. The protective agent according to claim 1, characterized in that, The final concentration of the nucleoside triphosphate is 1~100 mg / mL.

3. The protective agent according to claim 1 or 2, characterized in that, The protein denaturing agent includes at least one of guanidine hydrochloride and guanidine isothiocyanate.

4. The protective agent according to claim 3, characterized in that, The protein denaturant is guanidine isothiocyanate.

5. The protective agent according to claim 4, characterized in that, The final concentration of the protein denaturant is 1~1000 mg / mL.

6. A method for stabilizing RNA, characterized in that, This includes adding nucleoside triphosphates and protein denaturing agents to body fluids containing RNA, wherein the nucleoside triphosphates include at least one of adenosine triphosphate or guanosine triphosphate.

7. The method according to claim 6, characterized in that, The protein denaturing agent includes at least one of guanidine hydrochloride and guanidine isothiocyanate.

8. The method according to claim 7, characterized in that, The final concentration of the nucleoside triphosphate is 1~100 mg / mL; And / or, the final concentration of the protein denaturant is 1~1000 mg / mL.

9. The use of a composition of nucleoside triphosphate and a protein denaturant in the preparation of RNA protection products, wherein the nucleoside triphosphate includes at least one of adenosine triphosphate or guanosine triphosphate.

10. The application according to claim 9, characterized in that, The mass concentration ratio of the nucleoside triphosphate to the protein denaturant is 1:5~30.

Images