Method for enhancing RNA (Ribonucleic Acid) stability and application thereof

Abstract

The invention discloses a method for enhancing RNA (Ribonucleic Acid) stability and application thereof. In the invention, the inventor finds that at least one of purine nucleoside or a derivative thereof and pyrimidine nucleoside or a derivative thereof is contacted with RNA in the same system, so that the enzymatic degradation of the RNA can be inhibited, and the stability of the RNA in the system is enhanced. Therefore, the long-term stability of the RNA can be enhanced on the premise of not influencing the biological activity and translation efficiency of the RNA, and the method is suitable for storage and transportation of the RNA.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a method for enhancing RNA stability and its application. Background Technology

[0002] RNA (ribonucleic acid) has broad application prospects in the biomedical field, such as mRNA vaccines, RNA therapies, and molecular diagnostic reagents. However, the inherent instability of RNA molecules is one of the main obstacles to their practical application. RNA is susceptible to hydrolysis and enzymatic degradation, and usually deactivates rapidly at room temperature, which means that its storage and transportation must rely on costly freezing or ultra-low temperature cold chain systems.

[0003] Currently, methods to improve RNA stability mainly include chemically modifying nucleotides (e.g., using 5-methylcytosine or pseudouracil), optimizing RNA sequences or designing secondary structures (e.g., designing "hyperfolded" mRNA), and encapsulating RNA in complexes using carriers such as lipid nanoparticles (LNPs). In addition, some techniques attempt to dehydrate RNA and encapsulate it in inert atmosphere capsules for room temperature storage. However, these existing technologies still have significant limitations: certain nucleotide modifications (such as 7-deadenine) may significantly inhibit translation efficiency; many carrier complexes remain unstable after lyophilization or at room temperature, and their in vivo delivery efficiency may decrease due to aggregation or dissociation; while physical encapsulation methods may face problems such as complex processes, cumbersome RNA recovery steps, and difficulties in large-scale production.

[0004] Therefore, there is an urgent need in this field to develop a simple and efficient method that can significantly enhance the long-term stability of RNA without affecting its biological activity and translation efficiency, and is suitable for storage and transportation. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a method for enhancing RNA stability and its application. This invention is the first to discover that purine nucleosides, pyrimidine nucleosides, or their derivatives can be used to enhance RNA stability in a system by inhibiting the enzymatic degradation of RNA.

[0006] A first aspect of the present invention provides a method for improving RNA stability, comprising the steps of contacting RNA with at least one of a purine nucleoside or a derivative thereof, or a pyrimidine nucleoside or a derivative thereof.

[0007] In this invention, RNA refers to ribonucleic acid, and each ribonucleotide consists of three parts: ribose, phosphate group and nitrogenous base.

[0008] In some embodiments of the present invention, the RNA includes, but is not limited to, messenger RNA, transfer RNA, ribosomal RNA, non-coding RNA (such as microRNA, small interfering RNA, long non-coding RNA, etc.), guide RNA, and RNA molecules with catalytic functions (ribozymes).

[0009] In some embodiments of the present invention, the purine nucleosides include adenine nucleoside (AMP), guanine nucleoside (GMP), and xanthine nucleoside (IMP).

[0010] In some embodiments of the present invention, the pyrimidine nucleosides include: cytosine nucleoside (CMP) and uridine nucleoside (UMP).

[0011] In some embodiments of the present invention, the derivatives include: chemically modified purine nucleosides or pyrimidine nucleosides, or analogs thereof.

[0012] In some embodiments of the present invention, the chemical modification includes phosphorylation.

[0013] In some embodiments of the present invention, the phosphorylation includes diphosphorylation and triphosphorylation.

[0014] In some embodiments of the present invention, the analogues include: deoxyribose analogues.

[0015] In some embodiments of the present invention, the deoxyribose analogues include: monodeoxyribose analogues and dideoxyribose analogues.

[0016] In some embodiments of the present invention, the derivative is selected from diphosphates, triphosphates, and deoxyribose analogs of purine or pyrimidine nucleosides.

[0017] In some embodiments of the present invention, the derivatives include at least one of: ADP, GDP, CDP, UDP, TDP, ATP or its monodeoxy or dideoxy analogs, GTP or its monodeoxy or dideoxy analogs, CTP or its monodeoxy or dideoxy analogs, and UTP.

[0018] In some embodiments of the present invention, the derivatives are selected from: ADP, GDP, CDP, UDP, TDP, ATP or their monodeoxy or dideoxy analogs, GTP or their monodeoxy or dideoxy analogs, CTP or their monodeoxy or dideoxy analogs, and UTP.

[0019] In some embodiments of the present invention, the purine nucleosides or their derivatives, pyrimidine nucleosides or their derivatives include: ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, dTTP, ADP, GDP, AMP, GMP, etc.

[0020] A second aspect of the invention provides the use of at least one of purine nucleosides or derivatives thereof, or pyrimidine nucleosides or derivatives thereof, in improving RNA stability.

[0021] In some embodiments of the present invention, the definitions of purine nucleosides or their derivatives, pyrimidine nucleosides or their derivatives are the same as those described above.

[0022] In some embodiments of the present invention, the improvement in stability includes: inhibiting the enzymatic degradation of RNA.

[0023] In some embodiments of the present invention, the enzymatic degradation includes at least one of the following: (1) Degradation based on exonucleases or their analogues; (2) Degradation based on endonucleases or their analogues; (3) Degradation based on RNA helicases or their analogues; (4) Degradation based on pathway enzymes or their analogues in the mRNA degradation pathway.

[0024] In some embodiments of the present invention, the exonuclease or its analogues include at least one of Xrn1, Xrn2, RNase J, RNase PH, PNPase, RNase II or RBN-1, and RNase R.

[0025] In some embodiments of the present invention, the exonuclease analogue includes, but is not limited to, exonucleosomes.

[0026] In this invention, the exonucleosome refers to a large complex composed of multiple protein subunits that performs 3'→5' ribonuclease activity in eukaryotes and archaea, and is responsible for degrading various RNA substrates such as mRNA, rRNA and non-coding RNA.

[0027] In some embodiments of the present invention, the exosome includes, but is not limited to: eukaryotic exosomes, archaea exosomes, and yeast cytoplasmic exosomes (such as EXO-10).

[0028] In some embodiments of the present invention, the endonuclease or its analogues include at least one of RNase A, RNase T1, RNase III, Cas protein, tRNA cutting enzyme, ribozyme, Smg6, RNase E, RNase P, and Ire1.

[0029] In some embodiments of the present invention, the RNA helicase or its analogues include, but are not limited to, UPF proteins, such as UPF1 protein.

[0030] In some embodiments of the present invention, the pathway enzymes or analogues of the mRNA degradation pathway include, but are not limited to: deadenylate enzymes, uncapping enzymes, YTHDF2 protein, etc.

[0031] In some embodiments of the present invention, the purine nucleoside or its derivative, or the pyrimidine nucleoside or its derivative, inhibits the enzymatic degradation of RNA by binding to an enzyme.

[0032] In some embodiments of the present invention, the enzymatic degradation includes: enzymatic degradation based on RNase R or RNase A.

[0033] The beneficial effects of this invention are: This invention is the first to discover that purine nucleosides, pyrimidine nucleosides, or their derivatives can be used to enhance the stability of RNA in a system by inhibiting the enzymatic degradation of RNA. Attached Figure Description

[0034] Figure 1 The graph shows the changes in mRNA content after incubation with different concentrations of RNase A on mKate2 mRNA protected by 0.25 mg / mL ATP, GTP, and CTP.

[0035] Figure 2 The graph shows the changes in mRNA content after incubation of mKate2 mRNA protected by 2 mg / mL ATP, GTP, and CTP with different concentrations of RNase A.

[0036] Figure 3 This graph shows the change in mRNA content after incubation with cell lysate following incubation of ATP- and GTP-protected mKate2 mRNA.

[0037] Figure 4 The graph shows the changes in mRNA content after incubation with RNase A following the protection of different concentrations of ATP with mKate2 mRNA.

[0038] Figure 5 The graph shows the changes in mRNA content after incubation with RNase R of mKate2 mRNA protected by different concentrations of ATP.

[0039] Figure 6 The differential power (DP) is the power of the mixture of ATP and RNase A.

[0040] Figure 7 The graph shows the enthalpy change (ΔH) after mixing ATP with RNase A in different molar ratios.

[0041] Figure 8 The differential power (DP) is the power of the mixture of ATP and RNase R.

[0042] Figure 9 The graph shows the enthalpy change (ΔH) after mixing ATP with RNase R in different molar ratios.

[0043] Figure 10 The differential power (DP) is the result of mixing GTP and RNase A.

[0044] Figure 11 The graph shows the enthalpy change (ΔH) after mixing GTP and RNase A in different molar ratios.

[0045] Figure 12 The differential power (DP) is the result of mixing GTP and RNase R.

[0046] Figure 13 The graph shows the enthalpy change (ΔH) after mixing GTP with RNase R in different molar ratios.

[0047] Figure 14 The differential power (DP) is the result of mixing CTP and RNase A.

[0048] Figure 15 The graph shows the enthalpy change (ΔH) after mixing CTP with RNase A in different molar ratios.

[0049] Figure 16 The differential power (DP) is the result of mixing CTP and RNase R.

[0050] Figure 17 The graph shows the enthalpy change (ΔH) after mixing CTP with RNase R in different molar ratios. Detailed Implementation

[0051] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0052] Example 1 In this embodiment, mKate2 (far-red fluorescent protein reporter gene) mRNA is used as an example to demonstrate a method for enhancing RNA stability. It should be understood that the use of mKate2 mRNA as an example is not intended to limit the scope of RNA to which this invention can be applied; the method of this invention can be applied to any type of RNA.

[0053] The specific experimental steps are as follows: mKate2 mRNA was co-incubated with gradient concentrations of the endonuclease RNase A at 25 °C for 2 hours. The co-incubation solution was a PBS buffer containing different NTPs, specifically PBS buffer (pH 7.2-7.4) containing ATP, GTP, or CTP at a final concentration of 0.25 mg / mL. A PBS buffer without NTPs was used as a control.

[0054] After incubation, the mRNA concentration in the system was detected using qPCR.

[0055] The primers used are: Upstream primer: 5'-GGGGTGAACTTCCCATCCAA-3' (SEQ ID NO: 1); Downstream primer: 5'-TGTCCACATAGTAGACGCCG-3' (SEQ ID NO: 2).

[0056] The results are as follows Figure 1 As shown.

[0057] It was found that the concentration of RNase A required to degrade half of the RNA was 0.3461 ng / mL. However, after adding NTP, the concentration of RNase A required to degrade half of the RNA increased by more than two times. After adding ATP, GTP, and CTP, the concentrations were 0.7543, 0.9425, and 0.7252 ng / mL, respectively. This indicates that NTP can enhance the stability of RNA and prevent RNA from being degraded by the endonuclease RNase A.

[0058] Example 2 In this embodiment, mKate2 (far-red fluorescent protein reporter gene) mRNA is used as an example to demonstrate a method for enhancing RNA stability. It should be understood that the use of mKate2 mRNA as an example is not intended to limit the scope of RNA to which this invention can be applied; the method of this invention can be applied to any type of RNA.

[0059] In this embodiment, the experimental method is the same as in Example 1, except that the final NTP concentration in the PBS buffer was adjusted from 0.25 mg / mL to 2 mg / mL.

[0060] After incubation, the mRNA concentration in the system was detected using qPCR.

[0061] The results are as follows Figure 2 As shown.

[0062] It can be found that the concentration of RNase A required to degrade half of the RNA is 0.3461 ng / mL, while after the addition of NTP, the concentration of RNase A required to degrade half of the RNA increases by more than 5 times. After the addition of ATP, GTP and CTP, the concentrations are 3.619, 1.800 and 1.983 ng / mL, respectively, indicating that NTP can enhance the stability of RNA.

[0063] Example 3 In this embodiment, mKate2 (far-red fluorescent protein reporter gene) mRNA is used as an example to demonstrate a method for enhancing RNA stability. It should be understood that the use of mKate2 mRNA as an example is not intended to limit the scope of RNA to which this invention can be applied; the method of this invention can be applied to any type of RNA.

[0064] In this embodiment, cell lysis buffer is used instead of RNase A as a conditional factor leading to RNA degradation.

[0065] The specific experimental steps are as follows: SW480 cells were treated with cell lysis buffer (purchased from Cell Signaling Technology) at a ratio of 10 million SW480 cells / 1 mL of cell lysis buffer, centrifuged at 14,000 g at 4°C, and the supernatant was collected. mKate2 mRNA was incubated with the supernatant (50 μL of supernatant per 100 ng mRNA) at 37°C for 18 hours. NTP was added to the supernatant. In this example, ATP was added at final concentrations of 0, 1, 2, 4, 16, and 32 mg / mL, respectively, and the pH of the system after addition was 7.2-7.4.

[0066] After incubation, the mRNA concentration in the system was detected using qPCR.

[0067] The results are as follows Figure 3 As shown.

[0068] It was observed that as the NTP concentration increased, the concentration of mKate2 mRNA detectable by qPCR also increased, with an increase of up to 121-fold compared to the group without NTPs. This indicates that NTPs can enhance RNA stability and reduce nuclease degradation in the cellular contents obtained from RNA lysis.

[0069] Example 4 In this embodiment, mKate2 (far-red fluorescent protein reporter gene) mRNA is used as an example to demonstrate a method for enhancing RNA stability. It should be understood that the use of mKate2 mRNA as an example is not intended to limit the scope of RNA to which this invention can be applied; the method of this invention can be applied to any type of RNA.

[0070] In this embodiment, the experimental method is the same as in Example 3, except that the specific selection of NTP is adjusted, and ATP is replaced with an equal concentration of GTP.

[0071] After incubation, the mRNA concentration in the system was detected using qPCR.

[0072] The results are as follows Figure 3 As shown.

[0073] It was observed that as the NTP concentration increased, the concentration of mKate2 mRNA detectable by qPCR also increased, with an increase of up to 1458-fold compared to the group without NTPs. This indicates that NTPs can enhance RNA stability and reduce RNA degradation by nucleases in cell lysates.

[0074] Example 5 In this embodiment, mKate2 (far-red fluorescent protein reporter gene) mRNA is used as an example to demonstrate a method for enhancing RNA stability. It should be understood that the use of mKate2 mRNA as an example is not intended to limit the scope of RNA to which this invention can be applied; the method of this invention can be applied to any type of RNA.

[0075] In this embodiment, the experimental method is the same as in Example 1, except that the concentration of the endonuclease RNase A is further increased to 50 ng / mL, and the final concentration of NTP is also changed.

[0076] The specific experimental steps are as follows: mKate2 mRNA was co-incubated with 50 ng / mL of RNase A at 25 °C for 2 hours. The co-incubation solution was a PBS buffer (pH 7.2-7.4) containing different NTPs, specifically PBS buffers containing ATP at final concentrations of 0, 0.031, 0.125, 0.5, 2, 8, 32, and 90 mg / mL.

[0077] After incubation, the mRNA concentration in the system was detected using qPCR.

[0078] The results are as follows Figure 4 As shown.

[0079] It can be observed that as the NTP concentration increases, the concentration of mKate2 mRNA detectable by qPCR also increases. Compared with the group without NTP, the concentration increases by up to 9618 times, indicating that NTP can enhance RNA stability and reduce RNA degradation by the endonuclease RNase A.

[0080] Example 6 In this embodiment, mKate2 (far-red fluorescent protein reporter gene) mRNA is used as an example to demonstrate a method for enhancing RNA stability. It should be understood that the use of mKate2 mRNA as an example is not intended to limit the scope of RNA to which this invention can be applied; the method of this invention can be applied to any type of RNA.

[0081] In this embodiment, the experimental method is the same as in Example 1, except that the concentration of the endonuclease RNase A is further increased to 4 µg / mL, and the final concentration of NTP is changed.

[0082] The specific experimental steps are as follows: mKate2 mRNA was co-incubated with 4 µg / mL exonuclease RNase R at 25 °C for 2 hours. The co-incubation solution was a PBS buffer (pH 7.2-7.4) containing different NTPs, specifically PBS buffers containing ATP at final concentrations of 0, 0.5, 2, 4, 8, 16, 32, and 64 mg / mL.

[0083] After incubation, the mRNA concentration in the system was detected using qPCR.

[0084] The results are as follows Figure 5 As shown.

[0085] It was observed that as the NTP concentration increased, the concentration of mKate2 mRNA detectable by qPCR also increased, with the concentration increasing by up to 114 times compared to the group without NTPs. This indicates that NTPs can enhance RNA stability and reduce RNA degradation by the endonuclease RNase A.

[0086] The differential power (DP) of the mixture of ATP and RNase A was further determined using isothermal titration calorimetry (ITC).

[0087] Intra-conjugative temperature regulation (ITC) was performed under isothermal conditions on a Microcal Peaq-ITC Automated system. Specifically, RNase A and ATP were dissolved in PBS, with RNase A concentration of 10 µM and ATP concentration of 500 µM. The raw heat flux data were baseline-corrected and integrated using the instrument software. The net binding isotherm was obtained by subtracting background contributions such as ligand dilution heat and buffer baseline from the heat generated by each injection. A one-site binding model was then used for nonlinear fitting. The binding constant Kd, binding enthalpy change ΔH, and binding stoichiometry n were obtained from the fitting, and the free energy ΔG and entropy term ΔS were calculated accordingly. All fittings were performed using Malvern Peaq-ITC Analysis software.

[0088] The results are as follows Figure 6 As shown.

[0089] The DP (differential power)-time curve of ITC is the most original and intuitive data form in ITC experiments. It reflects the change of compensation power that the instrument needs to apply over time in order to maintain a constant sample cell temperature.

[0090] Figure 6 The results demonstrate the change in the thermal effect over time caused by the injection of ATP solution into RNase A. If a binding reaction occurs, heat will be released or absorbed. The results show that the injection of ATP solution into RNase A generates heat, requiring the instrument to apply negative compensation power; therefore, the peak in the graph appears downward.

[0091] The enthalpy change (ΔH) of ATP mixed with RNase A in different molar ratios was also obtained using the above method.

[0092] The results are as follows Figure 7 As shown.

[0093] It can be found that the binding constant (Kd) of ATP mixed with RNase A is 84.6 μM.

[0094] The differential power (DP) and enthalpy change (ΔH) of various combinations of ATP and RNase R, GTP and RNase A, GTP and RNase R, CTP and RNase A, and ADP and RNase A were determined using the same method.

[0095] The results are as follows Figure 8-17 As shown.

[0096] It can be found that the binding constant (Kd) of ATP with RNase R is 90 μM, the binding constant (Kd) of GTP with RNase A is 16.3 μM, the binding constant (Kd) of GTP with RNase R is 34.8 μM, the binding constant (Kd) of CTP with RNase A is 61.4 μM, and the binding constant (Kd) of ADP with RNase A is 119 μM.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for improving RNA stability, comprising the following steps: Contact RNA with at least one of purine nucleosides or their derivatives, or pyrimidine nucleosides or their derivatives.

2. The method according to claim 1, characterized in that, The purine nucleosides include: adenine nucleoside (AMP), guanine nucleoside (GMP) and xanthine nucleoside (IMP). The pyrimidine nucleosides include: cytosine nucleoside (CMP) and uridine nucleoside (UMP).

3. The method according to claim 1 or 2, characterized in that, The derivatives include: chemically modified purine nucleosides or pyrimidine nucleosides, or analogs thereof; Preferably, the chemical modification includes: phosphorylation; Preferably, the analogues include deoxyribose analogues.

4. The method according to claim 3, characterized in that, The derivatives include: Diphosphates, triphosphates, and deoxyribose analogs of purine or pyrimidine nucleosides; Preferably, the derivatives include at least one of: ADP, GDP, CDP, UDP, ATP or its monodeoxy or dideoxy analogs, GTP or its monodeoxy or dideoxy analogs, CTP or its monodeoxy or dideoxy analogs, and UTP.

5. The use of at least one of purine nucleosides or their derivatives, or pyrimidine nucleosides or their derivatives, in improving RNA stability; Preferably, the purine nucleoside or its derivative, pyrimidine nucleoside or its derivative are defined as described in any one of claims 1-4.

6. The application according to claim 5, characterized in that, The improvement of RNA stability includes: inhibiting the enzymatic degradation of RNA.

7. The application according to claim 6, characterized in that, The enzymatic degradation includes at least one of the following: (1) Degradation based on exonucleases or their analogues; (2) Degradation based on endonucleases or their analogues; (3) Degradation based on RNA helicases or their analogues; (4) Degradation based on pathway enzymes or their analogues in the mRNA degradation pathway.

8. The application according to claim 7, characterized in that, The exonuclease or its analogues include at least one of Xrn1, Xrn2, RNase J, RNase PH, PNPase, RNase II or RBN-1, and RNase R.

9. The application according to claim 7, characterized in that, The endonuclease or its analogues include at least one of RNase A, RNase T1, RNase III, Cas protein, tRNA-cleaving enzyme, ribozyme, Smg6, RNase E, RNase P, and Ire1.

10. The application according to claim 6, characterized in that, The purine nucleosides or their derivatives, pyrimidine nucleosides or their derivatives, inhibit the enzymatic degradation of RNA by binding to enzymes.

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