RNA molecule, pharmaceutical composition and use thereof

Abstract

A small non-coding RNA molecule. The small non-coding RNA molecule has an intervention effect on aging at multiple organ levels, can delay and / or ameliorate aging in a subject or extend the life-span of a subject, and provides a new therapy for precision treatment to extend the human healthy life expectancy. The small non-coding RNA molecule can also be used for treating paraquat poisoning, preventing or treating osteoporosis in a subject, improving cognitive function and / or emotional state in a subject, preventing or treating sarcopenia in a subject, ameliorating muscle functional decline caused by aging, improving body composition in a subject, or treating alopecia in a subject.

Description

RNA molecules, pharmaceutical compositions and their applications Technical Field

[0001] This disclosure pertains to the field of pharmaceutical technology and specifically relates to active RNA molecules, pharmaceutical compositions, and applications for anti-aging, treatment of paraquat poisoning, prevention or treatment of osteoporosis in subjects, improvement of cognition and / or mood in subjects, prevention or treatment of sarcopenia in subjects, improvement of age-related muscle function decline, improvement of body composition in subjects, or treatment of hair loss in subjects. Background Technology

[0002] Anti-aging is a perpetual pursuit for humankind. Aging is not merely a change in appearance, but also the root cause of many health problems. As our understanding of the mechanisms of aging deepens, more and more molecular, cellular, and biological processes related to aging are being discovered, leading to the emergence of new anti-aging drugs and therapies, such as nicotinamide mononucleotide, taurine, spermidine, SASP (senescence-associated secretory phenotype) therapy, and stem cell therapy. While these drugs and therapies have shown considerable anti-aging potential, they primarily focus on downstream interventions—intervening in existing aging phenotypes such as metalloproteinases, chemokines, interleukin-6 (IL-6), and interleukin-8 (IL-8)—which cannot meet the growing global challenge of aging. Therefore, there is a need to develop new, precise anti-aging strategies based on upstream genes.

[0003] Chinese patent application CN116585342A discloses an active ingredient containing miRNA and its application. This active ingredient comprises miRNAs of the miR-302 / miR367 family or modified miRNA derivatives of the miR-302 / miR367 family. This active ingredient can reverse aging cells, causing them to return to the cell cycle and proliferate, thus potentially preventing or treating age-related diseases. However, the essence of anti-aging is to achieve anti-aging of multiple organs to extend healthy lifespan, while this patent application only focuses on anti-aging of the liver and does not provide data on life extension in animals treated with its miRNA.

[0004] Chinese patent application CN117257957A discloses the application of miR-660-5p in the preparation of drugs for treating photoaging. Although this patent application develops miR-660-5p therapy to reduce β-galactosidase staining, increase cell viability, increase type I collagen secretion, reduce matrix metalloproteinase 1 secretion, or reduce ROS content, this patent application only focuses on the field of skin aging, and the application scenarios of its products are relatively limited.

[0005] Chinese patent application CN115820641A discloses a novel miRNA inhibitor and its application in alleviating iMSC senescence in vitro. This miRNA inhibitor is a miR-311 inhibitor. While this patent application develops a novel miRNA inhibitor to alleviate iMSC amplification-induced replicative senescence, including reducing the expression of senescence-related marker proteins (p21 and p16INK4A), promoting cell proliferation in the later stages of iMSC senescence, improving mitochondrial membrane potential in the later stages of senescence, and reducing iMSC mitochondrial density, this miR-311 inhibitor only achieves anti-aging at the in vitro cellular level and fails to achieve the goal of in vivo anti-aging.

[0006] Transport RNA-derived small RNAs (tRNA-derived small RNAs, tRNA-derived small RNA fragments, tsRNAs) are a class of non-coding small RNA molecules, approximately 16-40 nt in length, produced by the cleavage of tRNA under various stress conditions. Excessive production of tsRNAs due to aging interferes with normal gene expression regulation, triggers increased intracellular oxidative stress, increases the incidence of DNA damage, thereby affecting cellular function and overall physiological processes, and accelerating the decline of cellular and tissue function. Leucine-derived tRNA fragments (tRFs) mediated by TRMT6 / 61A can damage hematopoietic stem cell function by activating programmed necrosis. During brain aging, large amounts of glutamate tRFs abnormally accumulate in mitochondria, leading to impaired translation of mitochondrial proteins and destruction of cristae structure, ultimately damaging glutamate synthesis and accelerating brain aging and the pathological progression of Alzheimer's disease. Therefore, antisense oligonucleotide antagonists targeting tsRNAs with pro-aging functions hold promise for development into new therapies for precision treatment to extend human healthy lifespan. Summary of the Invention

[0007] The purpose of this disclosure is to address the shortcomings of existing anti-aging drugs, such as limited application scenarios and inability to achieve multi-organ anti-aging, by providing a non-coding small RNA molecule that enables applications in both in vitro and in vivo anti-aging scenarios. This molecule has interventional effects on aging at the multi-organ level, providing a new therapy for precision treatment to extend human healthy lifespan. The disclosure also aims to provide an RNA molecule for treating paraquat poisoning, preventing or treating osteoporosis in subjects, improving cognition and / or mood in subjects, preventing or treating sarcopenia in subjects, improving age-related muscle function decline, improving body composition in subjects, or treating hair loss.

[0008] In view of the shortcomings of the prior art, this disclosure provides tsRNAs with pro-aging function, an anti-aging RNA molecule, a pharmaceutical composition, and their applications.

[0009] In one aspect, this disclosure provides a tsRNA with pro-aging function, which is a small non-coding RNA derived from tRNA-Sec-NCA, comprising a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO:1 or a fragment thereof, or a modified sequence of said sequence or fragment thereof, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity. The sequence shown in SEQ ID NO:1 is as follows:

[0010] 5'P-GCCCGGAUGAUCCUCAGUGGUCUGGGGUGCAGGCU-OH 3' (named tRF-Sec-NCA).

[0011] The experiments disclosed herein demonstrate that tRF-Sec-NCA significantly inhibits cell proliferation and that tRF-Sec-NCA intervention significantly induces cell senescence. These results indicate that tRF-Sec-NCA induces the occurrence and development of cell senescence. Cells treated with tRF-Sec-NCA showed significantly increased levels of senescence factors P21, P16, and IL6, and significantly shortened relative telomere length. These experimental results suggest that tRF-Sec-NCA can serve as a biomarker for cell senescence.

[0012] On the other hand, this disclosure provides a kit for diagnosing aging, the kit being used to detect the content of the aforementioned small non-coding RNA in a sample; the kit includes: reverse transcription primers for the small non-coding RNA and detection primers.

[0013] On the other hand, this disclosure provides an application of the aforementioned small non-coding RNA in the preparation of diagnostic reagents for aging, wherein the reagents determine the risk level of aging in a subject by detecting the content of small non-coding RNA in the subject's biological sample.

[0014] On the other hand, this disclosure provides a detection method for predicting or diagnosing aging risk, which includes: taking blood leukocytes from a subject, measuring the aforementioned small non-coding RNA, and when the mass spectrometry response / RNA mass (ng) is greater than 800, the subject has aging risk.

[0015] On the other hand, this disclosure provides an RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, wherein the aforementioned RNA molecule is a small non-coding RNA antagonist, which is an antisense oligonucleotide of the aforementioned tRF-Sec-NCA or a fragment thereof, comprising a sequence having at least 80% sequence identity with the tRF-Sec-NCA antagonist sequence or a fragment thereof shown in SEQ ID NO:2, or a modified sequence of said sequence or a fragment thereof, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, wherein the sequence shown in SEQ ID NO:2 is as follows:

[0016] 5'P-AGCCUGCACCCCAGACCACUGAGGAUCAUCCGGGC-OH 3' (named tRF-Sec-NCA antagonist).

[0017] In some implementations, the RNA molecule is a truncated fragment (Table 1) containing at least the 3' end of the tRNA-Sec-NCA antagonist, with a length of 8-35 nt, and functions the same or substantially the same as the tRF-Sec-NCA antagonist.

[0018] In some preferred embodiments, the sequence of the small non-coding RNA antagonist is shown in SEQ ID NO:2.

[0019] In another aspect, this disclosure provides a conjugate or a pharmaceutically acceptable salt thereof comprising: (i) the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, and (ii) a ligand conjugated to the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, wherein at least one nucleotide of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate is conjugated to a ligand.

[0020] In another aspect, this disclosure provides a composition comprising the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0021] On the other hand, this disclosure provides the use of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition in the preparation of a medicament for delaying and / or improving aging or prolonging lifespan of a subject, for preventing or treating osteoporosis in a subject, for improving cognition and / or mood in a subject, for preventing or treating sarcopenia in a subject, for improving age-related muscle function decline, for improving body composition in a subject, or for improving hair loss in a subject.

[0022] In another respect, this disclosure provides a method for delaying and / or improving aging of a subject or prolonging the subject's lifespan, comprising administering to the subject an effective amount of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0023] In another respect, this disclosure provides a method for treating paraquat poisoning, comprising administering to a subject an effective amount of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0024] In another respect, this disclosure provides a method for preventing or treating osteoporosis in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0025] In another respect, this disclosure provides a method for improving the cognition and / or mood of a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0026] In another respect, this disclosure provides a method for preventing or treating sarcopenia in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0027] On the other hand, this disclosure provides a method for improving age-related muscle function, comprising administering to a subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0028] In another aspect, this disclosure provides a method for improving the body composition of a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0029] In another respect, this disclosure provides a method for improving hair loss in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0030] The beneficial technical effects of this disclosure are:

[0031] (1) Chinese patent application CN116585342A mainly uses miRNA to intervene in the aging process, but does not specify the target of miRNA. Therefore, the mechanism of action of this patent is unclear, and it is impossible to explain its effect on normal genes, and it is also difficult to rule out potential genotoxicity. The nucleic acid molecule disclosed in this paper is completely targeted by the tRF-Sec-NCA molecule, which is expressed at higher rates with aging, and it has been demonstrated that the tRF-Sec-NCA molecule can induce aging. Therefore, the aging intervention strategy disclosed in this paper is more precise.

[0032] (2) The miRNA molecule described in Chinese patent application CN116585342A mainly targets liver aging. However, since the miRNA molecule is expected to enter various organs via blood circulation after intravenous injection, its effects on other organs are unknown. The tRF-Sec-NCA antagonist disclosed in this paper can upregulate and downregulate aging factors in major organs in a paraquat acute mouse model, achieving anti-aging effects on multiple organs. Therefore, the anti-aging strategy disclosed in this paper is more comprehensive.

[0033] (3) The tRF-Sec-NCA antagonist disclosed herein can also be used to prevent or treat osteoporosis in subjects, to improve subjects’ cognition and / or mood, to prevent or treat subjects’ sarcopenia, to improve age-related muscle function decline, to improve subjects’ body composition, or to improve subjects’ hair loss.

[0034] The small non-coding RNA antagonist disclosed herein is a nucleic acid preparation with a simple structure, easy preparation method, and diverse administration methods. Furthermore, the small non-coding RNA antagonist disclosed herein overcomes the limitations of current anti-aging drugs, exhibiting significant anti-aging effects. It can reduce the level of aging factors in senescent cells and prolong telomere length, providing a new treatment strategy for the development of new drugs for anti-aging, prevention or treatment of osteoporosis, improvement of cognition and / or mood, prevention or treatment of sarcopenia, improvement of age-related muscle function decline, improvement of body composition, or improvement of hair loss, and laying the corresponding theoretical foundation. Attached Figure Description

[0035] Figure 1 shows the expression of tRF-Sec-NCA in the kidneys (A), leukocytes (B), and human leukocytes (C, D) of mice of different ages. *P<0.05

[0036] Figure 2 shows that tRF-Sec-NCA transfection promotes senescence in TCMK-1 and 2BS cells. (A) CCK-8 assay showed that tRF-Sec-NCA transfection significantly inhibited the proliferation rate of both cell types. (B) β-galactosidase staining results showed that tRF-Sec-NCA transfection significantly increased the number of senescent cells in both cell types.

[0037] Figure 3 shows that tRF-Sec-NCA transfection significantly increased senescence factors and shortened telomere length in TCMK-1 and 2BS cells. *P<0.05; ***P<0.001; ****P<0.0001.

[0038] Figure 4 shows the in vitro anti-aging effect of tRF-Sec-NCA antagonists. (A) Intervention with tRF-Sec-NCA antagonists significantly accelerated the proliferation of two types of senescent cells, TCMK-1 and 2BS; (B) Intervention with tRF-Sec-NCA antagonists significantly reduced the number of senescent cells.

[0039] Figure 5 shows that transfection with the tRF-Sec-NCA antagonist significantly reduced senescence factors and prolonged telomere length in TCMK-1 and 2BS senescent cells. *P<0.05; ***P<0.001; ****P<0.0001.

[0040] Figure 6 shows the anti-aging effect of tRF-Sec-NCA antagonists in an acute paraquat mouse model. (A) Intervention with tRF-Sec-NCA antagonists significantly prolonged the survival rate of mice; (B) Intervention with tRF-Sec-NCA antagonists significantly reduced the levels of aging factors in various organs of mice. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0041] Figure 7 shows the anti-aging effects of tRF-Sec-NCA antagonists in naturally aging mice. (A) Hair loss was significantly reduced after tRF-Sec-NCA antagonist intervention in aged mice; (B) Lifespan was significantly increased after tRF-Sec-NCA antagonist intervention in aged mice; (C) Body weight was significantly reduced after 9 months of tRF-Sec-NCA antagonist intervention in aged mice; (D) Bone mineral density was significantly increased after 8 months of tRF-Sec-NCA antagonist intervention in aged mice; (E) Fat percentage was significantly reduced after 8 months of tRF-Sec-NCA antagonist intervention in aged mice; (F) Lean meat percentage was significantly increased after 8 months of tRF-Sec-NCA antagonist intervention in aged mice. *P<0.05; **P<0.01.

[0042] Figure 8 shows the intervention effect of tRF-Sec-NCA antagonists on the healthy lifespan of naturally aging mice. (A) After intervention with tRF-Sec-NCA antagonists, the movement trajectory and distance in the open field of aged mice significantly increased; (B) After intervention with tRF-Sec-NCA antagonists, the movement trajectory and maze alternation rate in the Y maze of aged mice significantly increased; (C) After intervention with tRF-Sec-NCA antagonists, the grip strength of aged mice significantly increased; (D) After intervention with tRF-Sec-NCA antagonists, the dwell time on the rotundus of aged mice significantly increased. *P<0.05; **P<0.01; ***P<0.001.

[0043] Figure 9 shows the intervention effects of tRF-Sec-NCA antagonists on aging-related biochemical indicators in naturally aged mice. (A) Plasma IL6 expression level significantly decreased after 4 months of tRF-Sec-NCA antagonist intervention in aged mice; (B) Expression levels of aging factors P16, P21, and IL6 in blood leukocytes significantly decreased after 4 months of tRF-Sec-NCA antagonist intervention in aged mice; (C) Telomere length significantly increased in some organs after 9 months of tRF-Sec-NCA antagonist intervention in aged mice; (D) Expression level of aging factor P16 significantly decreased in some organs after 9 months of tRF-Sec-NCA antagonist intervention in aged mice; (E) Expression level of aging factor P21 significantly decreased in some organs after 9 months of tRF-Sec-NCA antagonist intervention in aged mice; (F) Expression level of aging factor IL6 significantly decreased in some organs after 9 months of tRF-Sec-NCA antagonist intervention in aged mice. *P<0.05; **P<0.01; ***P<0.001.

[0044] Figure 10 shows the plasma concentration-time curve of tRF-Sec-NCA antagonist after tail vein injection in mice via LNP delivery. Detailed Implementation

[0045] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0046] As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise.

[0047] As used herein, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0048] As used herein, the term “about” when referring to an index value or range allows for a certain degree of variation in that value or range, such as within 10% or 5% of the value or range defined therein.

[0049] As used in this article, the term "ribonucleic acid" or "RNA" describes a molecule composed of nucleotides, which consist of a nucleobase, a ribose, and a phosphate group. RNA is usually a single-stranded molecule and can perform a variety of functions. Specific ribonucleic acids include messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), small hairpin RNA (shRNA), and microRNA (miRNA), etc. RNA synthesis can usually be carried out by chemical synthesis and enzymatic synthesis (Michelson, AM, and Todd, ARNucleotides part XXXII. Synthesis of a dithymidine dinucleotide containing a 3':5'-internucleotidic linkage. J. Chem. Soc. (Resumed), 1955, 2632–2638. Wang Jing et al. Research progress on related technologies for enzymatic synthesis of RNA. Biotechnology Bulletin. 2016, 32(3):44-51).

[0050] As used herein, the term "transfer RNA" refers to an RNA polymer, typically 70 to 100 nucleotides in length, that delivers amino acids to ribosomes during protein synthesis for addition to the growing peptide chain. In this document, "transfer RNA" refers to both traditional tRNA molecules and tRNA molecules with one or more modifications. The terms "transfer RNA," "tRNA," and "transfer ribonucleic acid" are used interchangeably throughout this document.

[0051] As used herein, the term "transfer RNA-derived small RNA" refers to a class of non-coding small RNA molecules, approximately 16-40 nt in length, produced by the cleavage of tRNA under various stress conditions. In this document, the terms "transfer RNA-derived small RNA," "tsRNA," and "tsRNAs" are used interchangeably.

[0052] As used herein, the term "non-coding" refers to the partial or complete sequence of a nucleic acid molecule that does not encode the protein it expresses. Non-coding sequences include, but are not limited to, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions.

[0053] In this article, "anti-aging" includes two aspects: one is to delay and / or improve the aging of the subjects; the other is to extend the lifespan of the subjects.

[0054] As used in this article, the term "aging" refers to the irreversible changes that occur gradually over time in the structure and function of an organism. Aging involves cellular senescence, organ senescence, and system senescence.

[0055] As used in this article, the term “cellular senescence” refers to the process by which cells irreversibly cease dividing and enter a state of permanent growth arrest without undergoing cell death. The term “cellular senescence” is selected from aging hallmarks that are currently recognized as aging hallmarks, including: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, stem cell exhaustion and altered intercellular communication, disabled macroautophagy, chronic inflammation and dysbiosis (López-Otín, Carlos, et al. Hallmarks of aging: An expanding universe. Cell, 2023, 186(2):243-278).

[0056] The term "organ aging" as used in this article refers to the functional decline and degeneration of various organs in the body, such as the heart, liver, spleen, lungs, kidneys, brain, thymus, pancreas, skin, and bones, over time. Organ aging not only includes the characteristics of cellular aging, but also changes in the physiological structure and function of organs (Hainan Bao, et al. Biomarkers of aging. Science China. Life sciences. 2023 May, 66(5):893–1066).

[0057] The term "physiological system aging" as used in this article refers to the decline in motor, cognitive, metabolic, transport, respiration, absorption, excretion, and reproductive capabilities caused by the functional degeneration of physiological systems, including the musculoskeletal, nervous, endocrine, cardiovascular, lymphatic, respiratory, digestive, urinary, reproductive, immune, skin, and sensory systems. Systemic aging encompasses the connections between various organs and represents a higher-dimensional manifestation.

[0058] As used in this article, the term "lifespan" refers to the normal length of time an organism's life is lived.

[0059] As used in this article, natural aging refers to the inevitable physiological and biochemical changes that occur in organisms as they age. Unlike induced aging models, natural aging is a slow, continuous process that every organism experiences.

[0060] As used in this article, the term "delay" refers to the slowing and / or halting of an individual's transition from their current level of youth or aging to a more advanced level of aging through effective interventions. This "delay" can occur at any stage of an individual's life cycle, extending the stability of physiological functions and structures by influencing and modulating aging-related biological mechanisms, thereby mitigating the process of further decline.

[0061] As used herein, the term "improvement" refers to the reduction of an individual's degree of aging through effective interventions, or the restoration and / or reversal of an individual from a more severe degree of aging to a milder degree of aging or a younger state. Such "improvement" can occur at any stage of an individual's life cycle, by influencing and modulating age-related biological mechanisms to alleviate and / or reverse age-related physiological and functional changes, thereby improving an individual's overall health, quality of life, and physiological function.

[0062] The terms “patient,” “individual,” and “subject” are used interchangeably to refer to human or animal patients, individuals, and subjects, and include living organisms that use compounds or pharmaceutical compositions or by methods as provided herein to delay and / or improve aging or prolong life, prevent or treat osteoporosis, improve cognition and / or mood, prevent or treat sarcopenia, improve age-related muscle function decline, improve body composition, or improve hair loss. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammals. However, it should be understood that “patient” does not mean the presence of symptoms. In some embodiments, the patient is a human being. In some embodiments, the subject is a human being.

[0063] According to the methods provided herein, an effective amount of one or more drugs provided herein (e.g., tRNA-Sec-NCA antagonists or pharmaceutically acceptable salts thereof, conjugates thereof or pharmaceutically acceptable salts thereof, or compositions comprising thereof) is administered to a subject. An “effective amount” is an amount sufficient to achieve the stated purpose (e.g., to achieve the purpose of administration to delay and / or improve one or more symptoms of aging in the subject, or to prolong the subject’s lifespan). Examples of an “effective amount” include, for example, an amount sufficient to help delay and / or improve one or more symptoms of aging in the subject, which may also be referred to as a “therapeutic effective amount.” For example, for a given parameter, a therapeutic effective amount will show an increase or decrease of at least about 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease in a “fold.” For example, a therapeutic effective amount may have an effect of at least about 1.2 times, 1.5 times, 2 times, 5 times, or more relative to a control.

[0064] As used herein, the term "administration" generally refers to administering a compound or composition to a subject or system to achieve drug delivery to the subject or system. Those skilled in the art will appreciate the various routes that may be used for administration to a subject (e.g., a person) where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial infusion), oral, skin (which may be or include, for example, one or more of topical to the dermis, intradermal, intradermal, transdermal, etc.), intestine, stomach, intramuscular, intranasal, intraperitoneal, in a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by tracheal infusion), etc. In many embodiments provided in this disclosure, administration is oral. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a fixed number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses separated by time) and / or periodic (e.g., individual doses separated by the same time period) administration. In some embodiments, administration may involve continuous administration (e.g., infusion) for at least a selected period of time.

[0065] In this article, the life extension rate is calculated using the following formula:

[0066] Life extension rate = (average lifespan of the treatment group - average lifespan of the control group) / average lifespan of the control group × 100%

[0067] In one aspect, this disclosure provides an RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, wherein the RNA molecule comprises a sequence having at least 80% sequence identity with the tRF-Sec-NCA antagonist sequence or a fragment thereof shown in SEQ ID NO:2, or a modified sequence of the aforementioned sequence or a fragment thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, wherein the sequence shown in SEQ ID NO:2 is as follows:

[0068] 5'P-AGCCUGCACCCCAGACCACUGAGGAUCAUCCGGGC-OH 3'.

[0069] In some embodiments, the RNA molecule contains at least a truncated fragment of the 3' end of the tRF-Sec-NCA antagonist sequence shown in SEQ ID NO:2, with a length of 8-35 nt, and the RNA molecule has the same or substantially the same function as the tRF-Sec-NCA antagonist.

[0070] In some implementations, the RNA molecule is as shown in SEQ ID NO:2.

[0071] In some embodiments, the pharmaceutically acceptable salt is selected from carboxylates, alkali metal salts, ammonium salts, alkaline earth metal salts, salts formed with organic bases, and other pharmaceutically acceptable salts.

[0072] In some alternative embodiments, the salt is an alkali metal salt, preferably a sodium or potassium salt.

[0073] In some alternative embodiments, the salt is an alkaline earth metal salt, preferably a magnesium or calcium salt.

[0074] In some alternative implementations, the salt is an ammonium salt.

[0075] In some implementations, the RNA molecule contains at least one modified nucleotide.

[0076] In some embodiments, the tRF-Sec-NCA antagonist and its metabolite molecules described above comprise one, two, or more modified nucleotides. In some embodiments, the modified nucleotides comprise base modifications and / or ribose modifications.

[0077] In some embodiments, the modified nucleotides include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, 2'-O-methylpseudouridine, β,D-galactosonucleotide, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylinosine, 1-methylguanosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 5-methyluridine, 3-methyl Cytidine, N4-methylcytosine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, β,D-mannose-Q nucleoside, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxyaminomethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-thiomethyl-N6-isopentene Brutal adenosine, N-((9-β-D-furanosyl-2-thiomethylpurine-6-yl)carbamoyl)threonine, N-((9-β-D-furanosylpurine-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid-methyl ester, uridine-5-oxyacetic acid, wybutoxosine, pseudouridine, Q nucleoside, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine Urate, 5-thiouridine, N-((9-β-D-ribofuranopurine-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methyluridine, Wybutosine, 3-(3-amino-3-carboxy-propyl)uridine, N6-acetyladenosine, and 2-methylthio-N6-methyladenosine, one, two, or more of these.

[0078] Existing technologies have demonstrated that 2'-O-methylguanosine can significantly enhance the spatial structural stability of RNA-derived tRNA from non-pathogenic E. coli. Leu(CAA) The anti-colon cancer activity of 5'-tRF (Kai-Yue Cao, Yu Pan, Tong-Meng Yan, Zhi-Hong Jiang. Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli. International Journal of Biological Macromolecules 2020, 142:355-365). On the other hand, 5-methyluridine can also significantly increase the 3'-tRNA derived from Ganoderma lucidum by enhancing the spatial structural stability of RNA. Ile(GAU)The anticancer activity of half (Fei Ren, Kai-Yue Cao (Co-first author), Rui-Ze Gong, Peng Tao, Yi Xiao, Zhi-Hong Jiang. The role of post-transcriptional modification on a new tRNAIle(GAU) identified from Ganoderma lucidumin its fragments'cytotoxicity on cancer cells. International Journal of Biological Macromolecules 2023,229:885-895).

[0079] In some alternative implementations, the RNA molecule contains at least one 2'-modified nucleotide.

[0080] In some alternative embodiments, the 2'-modified nucleotide is selected from one or more of the following: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxynucleotides.

[0081] In some alternative embodiments, the 2'-modification is selected from the following: 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluorine, 2'-O-methoxyethyl.

[0082] In some alternative implementations, the RNA molecule has a 5'(E)-vinylphosphonate nucleotide at its 5' end.

[0083] In some implementations, the oligonucleotide contains at least one modified nucleotide inter-bond.

[0084] In some alternative implementations, at least one modified nucleotide inter-bond is a phosphate thioester bond.

[0085] In some alternative implementations, the RNA molecule contains two phosphate thioester nucleotide bonds at its 3' end.

[0086] In some embodiments, the RNA molecule is a sequence having a 5' hydroxyl group and a 3' phosphate group or a sequence having a 5' hydroxyl group and a 3' hydroxyl group.

[0087] In some implementations, the RNA molecule is a double-stranded RNA molecule that serves as a template.

[0088] In some embodiments, the double-stranded RNA molecule also includes a 3' overhang, preferably a 3' overhang consisting of two nucleotides. Providing a 3' overhang improves the stability of the RNA molecule.

[0089] In another aspect, this disclosure provides a conjugate or a pharmaceutically acceptable salt thereof comprising: (i) the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, and (ii) a ligand conjugated to the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, wherein at least one nucleotide of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate is conjugated to a ligand.

[0090] In some alternative implementations, the ligand comprises carbohydrates, amino sugars, cholesterol, peptides, or lipids.

[0091] In some alternative implementations, the ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0092] In some alternative implementations, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0093] In some alternative implementations, the ligand is L96.

[0094] In another aspect, this disclosure provides a pharmaceutical composition comprising the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier, diluent and / or excipient.

[0095] In some alternative embodiments, the composition is in the form of an oral, intravenous, subcutaneous, or intramuscular injection.

[0096] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. The term "pharmaceutically acceptable" means that when the molecular bulk, molecular fragment, or composition is properly administered to an animal or human, it does not produce adverse, allergic, or other adverse reactions. Specific examples of substances that can serve as pharmaceutically acceptable carriers or components thereof include phosphoric acid, citric acid, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); antibacterial agents (e.g., octadecyl dimethylbenzene ammonium chloride, hexachlorocyclohexane quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol); low molecular weight (less than about 10 kDa) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic... Polymers, such as polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including, for example, glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions; metal complexes; and / or nonionic surfactants (e.g., including TWEENTM, PLURONICS™, or polyethylene glycol). Furthermore, depending on the formulation method, commonly used fillers, diluents, binders, humectants, disintegrants, and / or surfactants can be appropriately selected by those skilled in the art. The pharmaceutical composition may be in solid, semi-solid, or liquid form, preferably in liquid form.

[0097] In some embodiments, the pharmaceutical composition further comprises a nucleic acid stabilizer. Examples of stabilizing agents for stabilizing and maintaining nucleic acids include cationic compounds, detergents, dissociative salts, ribonuclease inhibitors, chelating agents, and mixtures thereof. Stabilizers may include, for example, crosslinking fixatives such as paraformaldehyde or precipitants such as ethanol. Stabilizers can function by forming covalent bonds between cellular molecules or by precipitating some intracellular molecules or by other methods. In some embodiments, the stabilizer includes a cell lysis buffer. Cell permeation buffers are also known in the art and may contain detergents that permeate the cell membrane, allowing probes and dyes to pass through the membrane. Examples of detergents used in cell lysis buffers include, but are not limited to, Triton X-100, saponins, NP-40, etc. The concentrations of the cell lysis and permeation agents are adjusted for a given end use. Cell lysis and permeation may not be optimal when present at too low a concentration. Undesirable cell damage may occur at too high a concentration. Conventional, empirically based procedures can be performed to determine the preferred route in each case. In some embodiments, stabilizers include chloroform, phenol, and TRIzol. However, in a more preferred embodiment, the stabilizer is an easily removable or cytotoxic component, and most preferably a pharmaceutically acceptable component.

[0098] In some embodiments, the pharmaceutical composition is packaged and delivered in the form of plasmids, viral vectors, liposomes, dendritic macromolecules, inorganic nanoparticles, or cell-penetrating peptides. The tRF-Sec-NCA antagonist can be packaged directly or as a precursor. The plasmids and viral vectors may contain selection markers (e.g., enrichment-friendly tags, such as his tag; or detection-friendly tags, such as GFP) and an origin of replication matching the cell type specified by the cloning vector, while the expression vector contains regulatory elements necessary to influence expression in designated target cells. The viral vector can be a bacteriophage, lentivirus, retrovirus, adenovirus, or adeno-associated virus. The liposomes can be cationic or neutral liposomes, prepared or modified by known methods; for example, the addition of polyethylene glycol (PEG) to modify liposomes can effectively prevent the aggregation of liposomal vectors and increase their stability.

[0099] In another aspect, this disclosure provides the use of an RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof or a pharmaceutically acceptable salt thereof, or a composition thereof, in the preparation of a medicament for delaying and / or improving aging or prolonging lifespan of a subject, for treating paraquat poisoning, for preventing or treating osteoporosis in a subject, for improving cognition and / or mood in a subject, for preventing or treating sarcopenia in a subject, for improving age-related muscle function decline, for improving body composition in a subject, or for improving hair loss in a subject.

[0100] In another respect, this disclosure provides a method for delaying and / or improving aging of a subject or prolonging the subject's lifespan, comprising administering to the subject an effective amount of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0101] In another respect, this disclosure provides a method for treating paraquat poisoning in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0102] In another aspect, this disclosure provides a method for preventing or treating osteoporosis in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the osteoporosis is senile osteoporosis.

[0103] In another respect, this disclosure provides a method for improving the cognition and / or mood of a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0104] In another respect, this disclosure provides a method for preventing or treating sarcopenia in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0105] On the other hand, this disclosure provides a method for improving age-related muscle function decline, comprising administering to a subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof.

[0106] In another aspect, this disclosure provides a method for improving the body composition of a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, improving the body composition of a subject includes reducing the subject's fat percentage and / or increasing the subject's lean meat percentage.

[0107] On the other hand, this disclosure provides a method for improving hair loss in a subject, comprising administering to the subject an effective amount of the aforementioned RNA molecule, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or a conjugate thereof, or a pharmaceutically acceptable salt thereof, or a composition thereof. In some embodiments, the hair loss is age-related hair loss.

[0108] In some implementations, aging is selected from one or more of cellular aging, organ aging, and physiological system aging.

[0109] In some embodiments, cellular senescence is selected from one or more of epithelial cells, connective tissue cells, muscle cells, nerve cells, blood cells, germ cells, stem cells, immune cells, sensory cells, and glandular cells.

[0110] In some implementations, organ aging is selected from one or more organs such as the heart, liver, spleen, lungs, kidneys, brain, thymus, pancreas, skin, and bones.

[0111] In some implementations, systemic aging is selected from one or more of the following systems: musculoskeletal system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, reproductive system, immune system, skin system, and sensory system.

[0112] In some implementations, the subjects are selected from both mammals and non-mammals.

[0113] In some implementations, the subjects are mammals.

[0114] In some implementations, the subjects are selected from humans, cattle, dogs, monkeys, goats, sheep, dairy cows, deer, rats, mice, and other mammals.

[0115] In some implementations, the subject is a human being.

[0116] In some implementations, the subject's age is at least approximately 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years.

[0117] In some implementations, the subjects are between approximately 16 and 95 years old, or between approximately 18 and 95 years old.

[0118] In some implementations, the subjects are between approximately 50 and 65 years old, or between approximately 60 and 75 years old, or between approximately 70 and 85 years old, or between approximately 80 and 95 years old.

[0119] In some implementations, the subjects are adults aged 25-60.

[0120] In some implementations, the subjects are young people aged 18 to 25.

[0121] In some implementations, the subjects are elderly people aged 60 or older.

[0122] In some implementations, the subjects are elderly people aged 65 and over.

[0123] In some embodiments, the drug comprises at least about 1 μg of RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition.

[0124] In some embodiments, the drug comprises at least about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 9 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 100 μg, 150 μg, 250 μg, 500 μg, 750 μg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 6 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 150 mg, 250 mg, or 500 mg of RNA molecules, pharmaceutically acceptable salts thereof, hydrates or solvates thereof, or conjugates thereof, or pharmaceutically acceptable salts thereof, or compositions thereof.

[0125] In some embodiments, the composition is administered at a dose of at least about 0.2 μg / kg of RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or at a dose in which the active ingredient comprises at least about 0.2 μg / kg of RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts.

[0126] In some embodiments, the composition is administered at a dose of at least about 50 μg / kg to 10 mg / kg of RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or at a dose in which the active ingredient comprises at least about 50 μg / kg to 10 mg / kg of RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts.

[0127] In some embodiments, the concentrations are at least about 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 105 μg / kg, 110 μg / kg, 115 μg / kg, 120 μg / kg, 125 μg / kg, 130 μg / kg, 135 μg / kg, 140 μg / kg, 145 μg / kg, 150 μg / kg, and 180 μg / kg. The following dosages of RNA molecules were administered at pharmaceutically acceptable levels: g, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, and 10 mg / kg. Salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or containing at least about 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 105 μg / kg, 110 μg / kg, 115 μg / kg, 120 μg / kg, 125 μg / kg, 130 μg / kg, 135 μg / kg, 140 μg / kg, or 145 μg. / kg, 150μg / kg, 180μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 350μg / kg, 400μg / kg, 450μg / kg, 500μg / kg, 550μg / kg, 600μg / kg, 650μg / kg, 700μg / kg, 750μg / kg, 800μg / kg, 850μg / kg, 900μg / kg, 950μg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 5mg / kg, 10mg / kg, a dosage composition of RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts.

[0128] In some embodiments, the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition may be applied via an enteral route, such as an oral route, for example, in tablets or capsules, or via a parenteral route, such as in the form of an injectable solution or suspension, or via a topical route, such as in the form of a lotion, gel, ointment or cream, or in the form of a nasal or suppository.

[0129] In some embodiments, RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions, are formulated for oral administration.

[0130] In some embodiments, the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition, is a food, beverage, feed composition, or nutritional supplement.

[0131] In some embodiments, the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition, is a liquid, syrup, tablet, lozenge, gummy, capsule, powder, gel or film.

[0132] In some embodiments, the use of an RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or conjugate or its pharmaceutically acceptable salt, or composition in combination with a second therapeutic agent in the preparation of a medicament for delaying and / or improving aging of a subject or prolonging the lifespan of a subject.

[0133] In some embodiments, the second therapeutic agent is selected from one or more dietary supplements or drugs with anti-aging effects, such as rapamycin, β-nicotinamide mononucleotide (NMN), metformin, urolithiasis A, vitamin C, taurine, spermidine, quercetin, resveratrol, α-ketoglutarate, and ergothioneine.

[0134] In some embodiments, compared with a control group that has not been administered RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions, subjects who have been administered RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions have a lifespan extended by at least 10 days, 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, 20 years, 30, 40 years, 50 years, 60 years, 70 years, or 80 years.

[0135] In some embodiments, compared with a control group not treated with RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions, subjects treated with RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions experienced a lifespan extension of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, or 90% or longer.

[0136] In some embodiments, compared with a control group not treated with RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions, subjects treated with RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts, or compositions experienced a delay or improvement in aging symptoms of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 100%, 200%, or higher.

[0137] In some implementations, the improvement is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more higher than the control level.

[0138] In some embodiments, the composition is administered daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or annually at a dose of at least about 0.2 μg / kg, or at a dose of at least about 0.2 μg / kg of RNA molecules, their pharmaceutically acceptable salts, hydrates or solvates, or conjugates or their pharmaceutically acceptable salts.

[0139] In some embodiments, the composition is administered once, twice, or three times daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or annually at a dose of at least about 0.2 μg / kg, consisting of an RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvation, or a conjugate thereof.

[0140] On the other hand, this disclosure provides a small non-coding RNA with pro-aging function, comprising a sequence having at least 80% sequence identity with the sequence or a fragment thereof of tRF-Sec-NCA shown in SEQ ID NO:1, or a modified sequence of the sequence or a fragment thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, wherein the sequence shown in SEQ ID NO:1 is as follows:

[0141] 5'P-GCCCGGAUGAUCCUCAGUGGUCUGGGGUGCAGGCU-OH 3'.

[0142] On the other hand, this disclosure provides a kit for diagnosing aging, the kit being used to detect the content of the aforementioned small non-coding RNA in a sample; the kit includes: reverse transcription primers for the small non-coding RNA and detection primers.

[0143] On the other hand, this disclosure provides an application of the aforementioned small non-coding RNA in the preparation of diagnostic reagents for aging, wherein the reagents determine the risk level of aging in a subject by detecting the content of small non-coding RNA in the subject's biological sample.

[0144] On the other hand, this disclosure provides a detection method for predicting or diagnosing aging risk, which includes: taking blood leukocytes from a subject, measuring the aforementioned small non-coding RNA, and when the mass spectrometry response / RNA mass (ng) is greater than 800, the subject has aging risk.

[0145] While the aging process is difficult to reverse, the rise of various anti-aging therapies in recent years has increased the possibilities for extending lifespan. With the rapid development of the anti-aging field, the understanding of aging mechanisms has also provided more development strategies for precision anti-aging. tsRNA plays an important biological role in various physiological processes, but its relationship with aging remains unclear. This disclosure finds that tRF-Sec-NCA levels increase significantly with aging in mice and humans, providing direct evidence that this RNA molecule induces aging. This indicates that tRF-Sec-NCA is a novel anti-aging target. The tRF-Sec-NCA antagonist disclosed in this paper exhibits significant anti-aging effects in vitro and in vivo, especially in a paraquat-induced acute mouse model, significantly reducing aging factors in multiple organs while achieving a 159% lifespan extension rate. This demonstrates that the tRF-Sec-NCA antagonist can delay and / or improve aging or extend lifespan in subjects. Notably, this lifespan extension rate exceeds that of all currently reported anti-aging therapies. The pharmacokinetic analysis of this disclosure identified 13 fragments produced by the degradation of the tRF-Sec-NCA antagonist in plasma. Since these fragments are stably present in plasma, it is speculated that they may also possess anti-aging effects. In aged rats, intervention with the tRF-Sec-NCA antagonist of this disclosure resulted in significant increases in bone mineral density, fat percentage, and lean muscle percentage; significantly increased movement trajectory and distance in the open field; significantly increased movement trajectory and maze alternation rate in the Y-maze; significantly increased grip strength; and significantly increased dwell time on the rotundus.

[0146] The present disclosure is further described below with reference to the embodiments and accompanying drawings. It should be understood that the embodiments are only used to further illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0147] In the following examples, tRF-Sec-NCA (SEQ ID NO:1) and tRF-Sec-NCA antagonist (SEQ ID NO:2) were both chemically synthesized by Suzhou Beixin Biotechnology Co., Ltd.

[0148] Example 1: tRF-Sec-NCA expression is upregulated with aging

[0149] Kidneys and leukocytes were extracted from wild-type male Balb / c mice aged 6 months and 20 months (leukocytes from each group were mixed before extraction) and analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). Specifically, sRNA was isolated and enriched using the mirVana™ miRNA isolation kit (Thermo, USA) according to the manufacturer's instructions, and the abundance of tRF-Sec-NCA was analyzed using UHPLC-MS / MS. The analytical methods are as follows:

[0150] Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analysis was performed using an Agilent UHPLC 1290 system (Agilent, USA), equipped with an Agilent Ultrahigh Definition 6545Q-TOF mass spectrometer. Chromatographic separation was performed using an ACQUITY UPLC OST C18 column (2.1 mm inner diameter, 100 mm column length, 1.7 μm particle size, Waters, USA). Mobile phase A consisted of 100 mM hexafluoroisopropanol and 15 mM triethylamine, while mobile phase B consisted of 50% methanol dissolved in mobile phase A. The gradient elution program was as follows: 0–1.5 min, mobile phase B maintained at 2%; 1.5–8.3 min, mobile phase B changed from 2% to 28%; 8.3–16.5 min, mobile phase B changed from 28% to 34%. Ion source parameters: gas flow temperature maintained at 320 °C, voltage at 3.5 kV, sheath gas flow rate at 12 L / min, and sheath gas temperature at 350 °C.

[0151] The statistical results are shown in Figure 1. tRF-Sec-NCA (SEQ ID NO:1) was significantly upregulated in mouse kidneys (Figure 1A) and mouse leukocytes (Figure 1B) with age of the mice.

[0152] Example 2: The potential of tRF-Sec-NCA as an aging biomarker

[0153] Small RNA (sRNA) was extracted from leukocytes of healthy individuals of different ages collected clinically and detected according to the method in Example 1. Statistical results are shown in Figure 1C, where tRF-Sec-NCA (SEQ ID NO:1) was significantly upregulated with age. To verify this trend, leukocytes from another group of individuals were collected for analysis, and the results showed that tRF-Sec-NCA (SEQ ID NO:1) exhibited a similar trend in the newly collected samples (Figure 1D). These results indicate that tRF-Sec-NCA (SEQ ID NO:1) has the potential to become a biomarker for clinical aging detection, with a higher risk of aging when its mass spectrometry response / RNA mass (ng) is greater than 800.

[0154] Example 3: tRF-Sec-NCA induces senescence in young cells

[0155] tRF-Sec-NCA (SEQ ID NO:1) was transfected into young mouse glomerular epithelial cells TCMK-1 (Qingqi (Shanghai) Biotechnology Development Co., Ltd.) and human embryonic kidney fibroblasts 2BS (China Center for Type Culture Collection, catalog number GDC0303) via Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA). After treatment for 72 hours, the results were detected using the CCK-8 assay and β-galactosidase staining method, as detailed below:

[0156] Cell culture: The TCMK-1 and 2BS cell lines were cultured in DMEM (Gibco) and MEM (Gibco) media containing 10% FBS (Gibco) and 1% penicillin / streptomycin, respectively. All cell lines were cultured at 37°C under a humid atmosphere containing 5% CO2.

[0157] CCK-8 assay for cell viability: Two cell lines in logarithmic growth phase were seeded into 96-well plates with 5,000 cells per well and three replicates per group. Cells were allowed to adhere for 24 hours before treatment. The culture medium was removed, and 100 μL of 50 nM and 25 nM tRF-Sec-NCA solutions containing Lipofectamine RNAiMAX transfection reagent were added to each well for 6 days. Each day, 10 μL of CCK-8 reagent was added to three wells and incubated for approximately 2 hours. The absorbance was measured at 450 nm using a microplate reader.

[0158] Cell viability percentage (%) = (Absorbance value of drug-treated group - Absorbance value of control group) / Absorbance value of control group × 100%

[0159] β-galactosidase staining: Two cell lines in logarithmic growth phase were seeded into 6-well plates and incubated at 200,000 cells per well, allowing them to adhere for 24 hours before treatment. The culture medium was removed, and TCMK-1 cells were treated with 50 nM tRF-Sec-NCA solution, while 2BS cells were treated with 25 nM tRF-Sec-NCA solution (transfection reagents were the same as above). After 48 hours, the cell culture medium was aspirated, and the cells were treated with a β-galactosidase staining kit (Beyotime Biotechnology Co., Ltd., China) according to the manufacturer's instructions. The staining was observed and photographed under a regular light microscope.

[0160] The CCK-8 assay results, shown in Figure 2A, indicate that tRF-Sec-NCA significantly inhibited cell proliferation. The β-galactosidase staining results, shown in Figure 2B, show that tRF-Sec-NCA intervention significantly induced cellular senescence. These results suggest that tRF-Sec-NCA induces the development and progression of cellular senescence.

[0161] Example 4: Effects of tRF-Sec-NCA on senescence factors and telomeres in young cells

[0162] 50 nM and 25 nM tRF-Sec-NCA (SEQ ID NO:1) solutions were transfected into young mouse glomerular epithelial cells TCMK-1 and human embryonic kidney fibroblasts 2BS via Lipofectamine RNAiMAX transfection, respectively. After 72 hours of treatment, senescence factors and telomere length in the cells were measured as follows:

[0163] Sensitivity factor assay: Total RNA was extracted using the TRIzol method according to the manufacturer's protocol, and reverse transcription was performed using the Thermo Scientific Maxima SYBR Green / ROX qPCR Master Mix Reverse Transcription Kit (Thermo Fisher Scientific, USA). The expression levels of sensitivity factors P21, P16 and IL6 were determined using real-time quantitative PCR.

[0164] Telomere relative length determination: DNA was extracted using a genomic DNA mini-extraction kit (Beyotime Biotechnology Co., Ltd., China) according to the manufacturer's protocol, and the expression levels of tel1b and 36B4 were determined using real-time quantitative PCR. The tel1b / 36B4 (T / S) ratio is the telomere relative length.

[0165] As shown in Figure 3, the senescence factors P21, P16, and IL6 were significantly increased in TCMK-1 and 2BS cells treated with tRF-Sec-NCA, and their relative telomere length was significantly shortened.

[0166] Example 5: Evaluation of the in vitro anti-aging activity of tRF-Sec-NCA antagonists

[0167] The tRF-Sec-NCA antagonist (SEQ ID NO:2) was transfected into senescent mouse glomerular epithelial cells TCMK-1 and human embryonic kidney fibroblasts 2BS via Lipofectamine RNAiMAX transfection. The results were detected using the CCK-8 assay and β-galactosidase staining method, as detailed below:

[0168] Establishment of a cell senescence model: TCMK-1 cells were cultured in DMEM medium (Gibco) containing 40 mmol / L D-Gal for 72 hours to construct a TCMK-1 cell senescence model; 2BS cells were continuously cultured to 42 generations to become naturally senescent cells.

[0169] Cell viability assay using the CCK-8 assay: Senescent TCMK-1 and 2BS cells were seeded into 96-well plates with 5,000 cells per well and incubated in triplicate for each group. Cells were allowed to adhere for 24 hours before treatment. The culture medium was removed, and the cells were treated with 20 nM and 10 nM tRF-Sec-NCA antagonist solutions containing the Lipofectamine RNAiMAX transfection reagent (100 μL per well), respectively. Senescent TCMK-1 and 2BS cells were treated for 6 days. Each day, 10 μL of CCK-8 reagent was added to three wells, and the cells were incubated for approximately 2 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability percentage (%) = (Absorbance of treated group - Absorbance of control group) / Absorbance of control group × 100%

[0170] Cellular β-galactosidase staining: Senescent TCMK-1 and 2BS cells were seeded in 6-well plates at a density of 200,000 cells per well and allowed to adhere for 24 hours before treatment. The culture medium was removed, and senescent TCMK-1 cells were treated with 20 nM tRF-Sec-NCA antagonist solution, while senescent 2BS cells were treated with 10 nM tRF-Sec-NCA solution (transfection reagents were the same as above). After 48 hours, the cell culture medium was aspirated, and the cells were treated with a β-galactosidase staining kit (Beyotime Biotechnology Co., Ltd., China) according to the manufacturer's instructions. The cells were observed and photographed under a regular light microscope.

[0171] The CCK-8 assay results, shown in Figure 4A, indicate that the tRF-Sec-NCA antagonist significantly increased cell proliferation. The β-galactosidase staining results, shown in Figure 4B, show that the tRF-Sec-NCA antagonist significantly interfered with cell senescence. These results suggest that the tRF-Sec-NCA antagonist inhibits the occurrence and development of cell senescence.

[0172] Example 6: Effects of tRF-Sec-NCA antagonists on senescence factors and telomeres in senescent cells

[0173] 20 nM and 10 nM tRF-Sec-NCA antagonist solutions (SEQ ID NO:2) were transfected into young mouse glomerular epithelial cells TCMK-1 and human embryonic kidney fibroblasts 2BS via Lipofectamine RNAiMAX transfection. After 72 hours of treatment, senescence factors and telomere length in the cells were measured as follows:

[0174] Sensitivity factor assay: Total RNA was extracted using the TRIzol method according to the manufacturer's protocol, reverse transcription was performed using the Thermo Scientific Maxima SYBR Green / ROX qPCR Master Mix Reverse Transcription Kit (Thermo Fisher Scientific, USA), and the expression levels of sensitivity factors P21 and IL6 were determined using real-time quantitative PCR.

[0175] Telomere relative length determination: DNA was extracted using a genomic DNA mini-extraction kit (Beyotime Biotechnology Co., Ltd., China) according to the manufacturer's protocol, and the expression levels of tel1b and 36B4 were determined using real-time quantitative PCR. The tel1b / 36B4 (T / S) ratio is the telomere relative length.

[0176] As shown in Figure 5, the senescence factors P21 and IL6 were significantly reduced in TCMK-1 and 2BS senescent cells treated with tRF-Sec-NCA antagonists, and the relative telomere length was significantly prolonged.

[0177] Example 7: Anti-aging effect of tRF-Sec-NCA antagonist on paraquat-acute aging mice

[0178] Wild-type Balb / c female 6-week-old mice were purchased from Zhuhai Beston Biotechnology Co., Ltd. and kept at 25°C in a pathogen-free laboratory with free access to food and water. Animal experiments were conducted according to the institution's animal care guidelines and the committee-approved protocol. To establish an acute aging model, mice were intraperitoneally injected with paraquat (Sigma, 50 mg / kg), and 2 and 6 hours after modeling, were intravenously injected with high-dose (2.5 mg / kg) and low-dose (1.25 mg / kg) tRF-Sec-NCA antagonist solution encapsulated in LNP (SDR8002, Shengdi Biotechnology). The control group received the same dose of LNP solution via intravenous injection.

[0179] Mice were observed every 12 hours to calculate survival rates. Hearts, livers, spleens, lungs, and kidneys were collected from deceased mice. Tissue samples were taken from the high-dose group mice, and total RNA was extracted using the TRIzol method according to the manufacturer's protocol. Reverse transcription was performed using the Thermo Scientific Maxima SYBR Green / ROX qPCR Master Mix Reverse Transcription Kit (Thermo Fisher Scientific, USA), and the expression levels of aging factors P21 and IL-6 were determined using real-time quantitative PCR.

[0180] As shown in Figure 6A, the tRF-Sec-NCA antagonist (SEQ ID NO:2) significantly prolonged the survival rate of acute model mice in a dose-dependent manner. The lifespan extension rate was 51.9% in the low-dose group (P<0.05) and 167.3% in the high-dose group (P<0.01). Figure 6B shows that the high-dose tRF-Sec-NCA antagonist significantly reduced the expression of aging factors P21 and IL6 in most organs, further demonstrating its anti-aging activity.

[0181] Example 8: The intervention effect of tRF-Sec-NCA antagonists on the natural lifespan of naturally aging mice.

[0182] Wild-type male C57 mice, aged 16 months, were purchased from Beijing Micro-Xuan Technology Co., Ltd., and kept at 25°C in a pathogen-free laboratory with free access to food and water. Animal experiments were conducted according to the institution's animal care guidelines and the committee-approved protocol. Mice were injected weekly via tail vein with an LNP-encapsulated tRF-Sec-NCA antagonist solution. Mouse weight was recorded weekly.

[0183] As shown in Figure 7A, after 4 months of administration, aged mice treated with the tRF-Sec-NCA antagonist (SEQ ID NO:2) showed significantly less hair loss compared to the LNP control group. Figure 7B shows that aged mice treated with the tRF-Sec-NCA antagonist had a 34.7% increased half-life compared to the LNP control group. Figure 7C shows that after 9 months of administration, aged mice treated with the tRF-Sec-NCA antagonist experienced a 5% decrease in body weight, with no significant differences in food intake or sleep, indicating that the antagonist has no obvious toxic side effects. Figure 7D shows that after 8 months of administration, aged mice treated with the tRF-Sec-NCA antagonist had significantly increased bone density compared to the LNP control group. Figure 7E shows that after 8 months of administration, aged mice treated with the tRF-Sec-NCA antagonist had a significantly lower fat percentage compared to the LNP control group. Figure 7F shows that aged mice treated with the tRF-Sec-NCA antagonist had a significantly higher lean meat percentage compared to the LNP control group.

[0184] Example 9: The intervention effect of tRF-Sec-NCA antagonists on the healthy lifespan of naturally aging mice.

[0185] Wild-type male C57 mice, aged 16 months, were purchased from Beijing Weixuan Technology Co., Ltd., and kept at 25°C in a pathogen-free laboratory with free access to food and water. Animal experiments were conducted according to the institution's animal care guidelines and the committee-approved protocol. Mice were injected intravenously with an LNP-encapsulated tRF-Sec-NCA antagonist solution (2.5 mg / kg, once weekly). At 7 months of administration, the mice's behavior was assessed using an open field test apparatus, a Y-maze test apparatus, a grasping strength test apparatus, and a rotarod test apparatus, according to the manufacturer's protocol.

[0186] As shown in Figure 8A, aged mice treated with tRF-Sec-NCA antagonists exhibited significantly increased movement trajectories and distances in the open field compared to the LNP control group, indicating that tRF-Sec-NCA antagonists can significantly improve age-related decline in motor activity and anxiety. Figure 8B shows that aged mice treated with tRF-Sec-NCA antagonists exhibited significantly increased movement trajectories and maze alternation rates in the Y-maze compared to the LNP control group, indicating that tRF-Sec-NCA antagonists can significantly improve age-related decline in short-term working memory. Figure 8C shows that aged mice treated with tRF-Sec-NCA antagonists exhibited significantly increased grip strength compared to the LNP control group, indicating that tRF-Sec-NCA antagonists can significantly improve age-related decline in muscle grip strength. Figure 8D shows that aged mice treated with tRF-Sec-NCA antagonists exhibited significantly increased dwell time on the rotarod compared to the LNP control group, indicating that tRF-Sec-NCA antagonists can significantly improve age-related decline in muscle and nerve strength.

[0187] Example 10: The intervention effect of tRF-Sec-NCA antagonists on aging factors in naturally aging mice

[0188] Wild-type male C57 mice, aged 16 months, were purchased from Beijing Micro-Xuan Technology Co., Ltd., and kept at 25°C in a pathogen-free laboratory with free access to food and water. Animal experiments were conducted according to the institution's animal care guidelines and the committee-approved protocol. Mice were injected via tail vein with an LNP-encapsulated tRF-Sec-NCA antagonist solution (2.5 mg / kg, once weekly). At 4 months post-administration, the expression level of the aging factor IL-6 in mouse plasma was determined using the Mouse IL-6 ELISA Kit (Hangzhou Linke Biotechnology Co., Ltd., China), according to the manufacturer's protocol. Simultaneously, total RNA was extracted from mouse blood leukocytes using a magnetic bead-based tissue / cell / blood total RNA extraction kit (Tiangen Biotech Co., Ltd., China), and reverse transcription was performed using the Thermo Scientific Maxima SYBR Green / ROX qPCR Master Mix reverse transcription kit (Thermo Fisher Scientific, USA). The expression levels of aging factors P21 and IL-6 were determined using real-time quantitative PCR. Nine months after drug administration, DNA was extracted using a genomic DNA mini-extraction kit (Beyotime Biotechnology Co., Ltd., China) according to the manufacturer's protocol, and the expression levels of tel1b and 36B4 were determined using real-time quantitative PCR. The tel1b / 36B4 (T / S) ratio represents the relative telomere length. RNA was extracted from various organs using the mirVana™ miRNA isolation kit (Thermo, USA), and the expression levels of telomeres, aging factors P16, P21, and IL-6 were determined using real-time quantitative PCR.

[0189] Figure 9A shows that treatment with the tRF-Sec-NCA antagonist at a dose of 2.5 mg / kg for 4 months significantly decreased the level of the aging factor IL6 in mouse plasma. Figure 9B shows that treatment with the tRF-Sec-NCA antagonist at a dose of 2.5 mg / kg for 4 months significantly decreased the levels of aging factors P16, P21, and IL6 in mouse blood leukocytes. Figure 9C shows that treatment with the tRF-Sec-NCA antagonist at a dose of 2.5 mg / kg for 9 months significantly prolonged the length of telomeres in some organs. Figure 9D shows that treatment with the tRF-Sec-NCA antagonist at a dose of 2.5 mg / kg for 9 months significantly reduced the expression level of the aging factor P16 in some organs. Figure 9E shows that treatment with the tRF-Sec-NCA antagonist at a dose of 2.5 mg / kg for 9 months significantly reduced the expression level of the aging factor P21 in some organs. Figure 9F shows that after 9 months of treatment with tRF-Sec-NCA antagonists at a dose of 2.5 mg / kg, the expression level of the aging factor IL6 in some organs was significantly reduced.

[0190] Example 11: In vivo metabolic analysis of tRF-Sec-NCA antagonists

[0191] Wild-type Balb / c female 6-week-old mice were purchased from Zhuhai Beston Biotechnology Co., Ltd., and kept at 25°C in a pathogen-free laboratory with free access to food and water. Animal experiments were conducted according to the institution's animal care guidelines and the committee-approved protocol. Mice were injected intravenously with a high-dose solution (2.5 mg / kg) of LNP-encapsulated tRF-Sec-NCA antagonist (SEQ ID NO:2), and were euthanized by cervical dislocation after blood collection via the fundus venous plexus at specific time points (0, 5, 10, 20, 40, 60, and 120 minutes) following injection. Blood samples were centrifuged at 3,500 rpm for 10 minutes, and the supernatant was collected as plasma. Total RNA was extracted using the TRIzol method according to the manufacturer's protocol, and the prototype drug and its degradation fragments were analyzed under the ultra-high performance liquid chromatography-mass spectrometry conditions described in Example 1. Plasma concentration-time curves were plotted.

[0192] The statistical results are shown in Figure 10. The time to maximum plasma concentration (Tmax) of the tRF-Sec-NCA antagonist was 10 minutes, and its half-life in mouse plasma was relatively long, indicating that LNP may help maintain the stability of the tRF-Sec-NCA antagonist in the blood and prolong its circulation time in vivo. More importantly, 13 degradation fragments of the tRF-Sec-NCA antagonist with lengths of 4-30 nt were identified from plasma samples (Table 1), suggesting that these degradation fragments may also have anti-aging effects.

[0193] Table 1. Degradation fragments of tRF-Sec-NCA antagonists detected in mouse plasma a difference = measured value - theoretical value

Claims

1. An RNA molecule, a pharmaceutically acceptable salt, hydrate, or solvate thereof, said RNA molecule comprising a sequence having at least 80% sequence identity with the tRF-Sec-NCA antagonist sequence or a fragment thereof shown in SEQ ID NO:2, or a modified sequence of said sequence or a fragment thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, wherein... The sequence shown in SEQ ID NO.2 is as follows: 5'P-AGCCUGCACCCCAGACCACUGAGGAUCAUCCGGGC-OH 3'.

2. The RNA molecule according to claim 1, its pharmaceutically acceptable salt, hydrate, or solvate, wherein, The RNA molecule contains at least a truncated fragment at the 3' end of the tRF-Sec-NCA antagonist sequence shown in SEQ ID NO:2, with a length of 8-35 nt, and the RNA molecule has the same or substantially the same function as the tRF-Sec-NCA antagonist.

3. The RNA molecule according to claim 2, its pharmaceutically acceptable salt, hydrate, or solvate, wherein, The RNA molecule is shown in SEQ ID NO:

2.

4. The RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate according to any one of claims 1-3, wherein, The pharmaceutically acceptable salts are selected from carboxylates, alkali metal salts, ammonium salts, alkaline earth metal salts, salts formed with organic bases, and other pharmaceutically acceptable salts. Preferably, the salt is an alkali metal salt, more preferably a sodium or potassium salt; Preferably, the salt is an alkaline earth metal salt, more preferably a magnesium salt or a calcium salt; Preferably, the salt is an ammonium salt.

5. The RNA molecule according to any one of claims 1-4, its pharmaceutically acceptable salt, hydrate, or solvate, wherein, The RNA molecule contains at least one modified nucleotide; Preferably, the modified nucleotides include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, 2'-O-methylpseudouridine, β,D-galactosonucleotide, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylinosine, 1-methylguanosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 5-methyluridine, and 3-methylcytidine. N4-methylcytosine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, β,D-mannose-Q nucleoside, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxyaminomethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-thiomethyl-N6-isopentenyl Adenosine, N-((9-β-D-furanosyl-2-thiomethylpurine-6-yl)carbamoyl)threonine, N-((9-β-D-furanosylpurine-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid-methyl ester, uridine-5-oxyacetic acid, wybutoxosine, pseudouridine, Q nucleoside, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine Urate, 5-thiouridine, N-((9-β-D-ribofuranopurine-6-yl)carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methyluridine, Wybutosine, 3-(3-amino-3-carboxy-propyl)uridine, N6-acetyladenosine, and 2-methylthio-N6-methyladenosine, one, two, or more of these; Preferably, the RNA molecule contains at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is selected from one or more of the following: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-deoxynucleotides. Preferably, the 2'-modification is selected from the following: 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluorine, 2'-O-methoxyethyl; Preferably, the RNA molecule has a 5'(E)-vinylphosphonate nucleotide at its 5' end.

6. The RNA molecule of any one of claims 1-5, its pharmaceutically acceptable salt, hydrate, or solvate, wherein, The oligonucleotide contains at least one modified internucleotide bond; Preferably, the at least one modified nucleotide inter-bond is a phosphate thioester bond; More preferably, the RNA molecule contains two phosphate thioester nucleotide bonds at its 3' end.

7. The RNA molecule of any one of claims 1-6, its pharmaceutically acceptable salt, hydrate, or solvate, wherein, The RNA molecule is a sequence having a 5' hydroxyl group and a 3' phosphate group or a sequence having a 5' hydroxyl group and a 3' hydroxyl group.

8. The RNA molecule, pharmaceutically acceptable salt, hydrate, or solvate thereof, as described in any one of claims 1-7, wherein, The RNA molecule is a double-stranded RNA molecule; Preferably, the double-stranded RNA molecule further comprises a 3' overhang; more preferably, the double-stranded RNA molecule comprises a 3' overhang of 2 nucleotides.

9. A conjugate or a pharmaceutically acceptable salt thereof, comprising: (i) an RNA molecule, a pharmaceutically acceptable salt, hydrate, or solvate thereof, as described in any one of claims 1-8, and (ii) a ligand conjugated to said RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, wherein, The RNA molecule, at least one nucleotide of its pharmaceutically acceptable salt, hydrate or solvate, is conjugated to a targeting ligand; Preferably, the ligand comprises carbohydrates, amino sugars, cholesterol, polypeptides, or lipids; Preferably, the ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion; Preferably, the ligand is L96.

10. A composition comprising an RNA molecule according to any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or a conjugate according to claim 9 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier; Preferably, the dosage form of the composition is an oral preparation, an intravenous injection, a subcutaneous injection, or an intramuscular injection.

11. Use of the RNA molecule of any one of claims 1-8, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate of claim 9 or its pharmaceutically acceptable salt, or the composition of claim 10, in the preparation of a medicament for delaying and / or improving aging or prolonging life of a subject, for treating paraquat poisoning, for preventing or treating osteoporosis in a subject, for improving cognition and / or mood in a subject, for preventing or treating sarcopenia in a subject, for improving age-related muscle function decline, for improving body composition in a subject, or for improving hair loss in a subject.

12. The use according to claim 11, wherein, The aging process is selected from one or more of cellular aging, organ aging, and physiological system aging.

13. The use according to claim 12, wherein, The cellular senescence is selected from one or more of epithelial cells, connective tissue cells, muscle cells, nerve cells, blood cells, germ cells, stem cells, immune cells, sensory cells, and glandular cells.

14. The use according to claim 12, wherein, The organ aging mentioned is selected from one or more organs such as the heart, liver, spleen, lungs, kidneys, brain, thymus, pancreas, skin, and bones.

15. The use according to claim 12, wherein, The systemic aging is selected from one or more of the following systems: musculoskeletal system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, reproductive system, immune system, skin system, and sensory system.

16. The use according to any one of claims 11-15, wherein, The subjects were selected from both mammals and non-mammals.

17. The use according to any one of claims 11-16, wherein, The subjects were mammals.

18. The use according to any one of claims 11-17, wherein, The subjects were selected from humans, cattle, dogs, monkeys, goats, sheep, dairy cows, deer, rats, mice, and other mammals.

19. The use according to any one of claims 11-18, wherein, The subjects were human.

20. The use according to any one of claims 11-19, wherein, The subjects were at least approximately 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old.

21. The use according to any one of claims 11-20, wherein, The subjects were between approximately 16 and 95 years old, or between approximately 18 and 95 years old.

22. The use according to any one of claims 11-21, wherein, The subjects were between approximately 50 and 65 years old, or between approximately 60 and 75 years old, or between approximately 70 and 85 years old, or between approximately 80 and 95 years old.

23. The use according to any one of claims 11-22, wherein, The subjects were adults aged 25-60.

24. The use according to any one of claims 11-23, wherein, The subjects were young people aged 18 to 25.

25. The use according to any one of claims 11-24, wherein, The subjects were elderly people aged 60 and above.

26. The use according to any one of claims 11-25, wherein, The subjects were elderly people aged 65 and above.

27. The use according to any one of claims 11-26, wherein, The drug comprises at least about 1 μg of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition.

28. The use according to claim 27, wherein, The drug comprises at least about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 9 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 100 μg, 150 μg, 250 μg, 500 μg, 750 μg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 6 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 150 mg, 250 mg, or 500 mg of the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition.

29. The use according to any one of claims 11-28, wherein, The composition may be administered at a dose of at least about 0.2 μg / kg of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvation, or the conjugate or its pharmaceutically acceptable salt, or at a dose in which the active ingredient comprises at least about 0.2 μg / kg of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvation, or the conjugate or its pharmaceutically acceptable salt.

30. The use according to any one of claims 11-29, wherein, The composition may be administered at a dose of at least about 50 μg / kg to 10 mg / kg, or at a dose in which the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt is contained, or at a dose in which the active ingredient comprises at least about 50 μg / kg to 10 mg / kg of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt.

31. The use according to any one of claims 9-20, wherein, At least approximately 50μg / kg, 55μg / kg, 60μg / kg, 65μg / kg, 70μg / kg, 75μg / kg, 80μg / kg, 85μg / kg, 90μg / kg, 95μg / kg, 100μg / kg, 105μg / kg, 110 μg / kg, 115μg / kg, 120μg / kg, 125μg / kg, 130μg / kg, 135μg / kg, 140μg / kg, 145μg / kg, 150μg / kg, 180μg / kg, 200μg / kg, 250μg / kg, 30 The RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate thereof, or its pharmaceutically acceptable salt, or as an active ingredient, shall be administered at doses of 0 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, and 10 mg / kg. Contains at least approximately 50μg / kg, 55μg / kg, 60μg / kg, 65μg / kg, 70μg / kg, 75μg / kg, 80μg / kg, 85μg / kg, 90μg / kg, 95μg / kg, 100μg / kg, 105μg / kg, 11 0μg / kg, 115μg / kg, 120μg / kg, 125μg / kg, 130μg / kg, 135μg / kg, 140μg / kg, 145μg / kg, 150μg / kg, 180μg / kg, 200μg / kg, 250μg / kg, 3 The composition is administered at doses of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate thereof, at doses of 00 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg.

32. The use according to any one of claims 11-31, wherein, The RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition may be applied via enteral route, such as oral route, for example, in tablet or capsule form, or via parenteral route, such as in injectable solution or suspension form, or via topical route, such as in lotion, gel, ointment or cream form, or via nasal or suppository form.

33. The use according to any one of claims 11-32, wherein, The RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition, is formulated for oral administration.

34. The use according to any one of claims 11-33, wherein, The RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition, is a food, beverage, feed composition or nutritional supplement.

35. The use according to any one of claims 11-34, wherein, The RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition, is a liquid, syrup, tablet, lozenge, gummy, capsule, powder, gel or film.

36. The use according to any one of claims 11-35, wherein, The use of the RNA molecule, its pharmaceutically acceptable salt, hydrate or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition in combination with a second therapeutic agent in the preparation of a medicament for delaying and / or improving aging of a subject or prolonging the lifespan of a subject.

37. The use according to any one of claims 11-36, wherein, The second therapeutic agent is selected from one or more of the following dietary supplements or drugs with anti-aging effects: rapamycin, β-nicotinamide mononucleotide (NMN), metformin, urolithiasis A, vitamin C, taurine, spermidine, quercetin, resveratrol, α-ketoglutarate, ergothioneine, etc.

38. The use according to any one of claims 11-37, wherein, Compared to a control group not treated with the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition, the lifespan of the subject treated with the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition was extended by at least 10 days, 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, 20 years, 30, 40 years, 50 years, 60 years, 70 years, or 80 years.

39. The use according to any one of claims 11-38, wherein, Compared to a control group not treated with the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition, the lifespan of the subject treated with the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition was extended by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, or 90% or longer.

40. The use according to any one of claims 11-39, wherein, Compared to a control group not treated with the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition, the subjects treated with the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvate, or the conjugate or its pharmaceutically acceptable salt, or the composition experienced a delay or improvement in aging symptoms of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 100%, 200%, or higher.

41. The use according to claim 30, wherein, The improvement is defined as being at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher than the control level.

42. A method for delaying and / or improving aging of a subject or prolonging the lifespan of a subject, comprising administering to the subject an effective amount of an RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or a conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or a composition of claim 10.

43. The method according to claim 42, wherein, The composition may be administered daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or annually at a dose of at least about 0.2 μg / kg, or at a dose of at least about 0.2 μg / kg of the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvation, or the conjugate or its pharmaceutically acceptable salt.

44. The method according to claim 43, wherein, The composition may be administered once, twice, or three times daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or annually at a dose of at least about 0.2 μg / kg, consisting of the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvation, or the conjugate thereof, or at a dose containing at least about 0.2 μg / kg of the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvation, or the conjugate thereof, or at a dose of at least about 0.2 μg / kg, consisting of the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvation, or the conjugate thereof, or at a dose of at least about 0.2 μg / kg, consisting of the RNA molecule, its pharmaceutically acceptable salt, hydrate, or solvation, or at a dose of at least about 0.2 μg / kg, consisting of the RNA molecule, or the conjugate thereof ...

45. A method for treating paraquat poisoning, comprising administering to a subject an effective amount of the RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or the conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or the composition of claim 10.

46. ​​A method for preventing or treating osteoporosis in a subject, comprising administering to the subject an effective amount of the RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or the conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or the composition of claim 10; Preferably, the osteoporosis is senile osteoporosis.

47. A method for improving a subject’s cognition and / or mood, comprising administering to the subject an effective amount of an RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or a conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or a composition of claim 10.

48. A method for preventing or treating sarcopenia in a subject, comprising administering to the subject an effective amount of an RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or a conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or a composition of claim 10.

49. A method for improving age-related muscle function decline, comprising administering to a subject an effective amount of the RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or the conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or the composition of claim 10.

50. A method for improving the body composition of a subject, comprising administering to the subject an effective amount of the RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, or the conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or the composition of claim 10; Preferably, the improvement of the subject's body composition includes reducing the subject's fat percentage and / or increasing the lean meat percentage.

51. A method for improving hair loss in a subject, comprising administering to the subject an effective amount of the RNA molecule of any one of claims 1-8, a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, or the conjugate of claim 9 or a pharmaceutically acceptable salt thereof, or the composition of claim 10; Preferably, the hair loss is age-related hair loss.

52. A small non-coding RNA with anti-aging function, comprising a sequence having at least 80% sequence identity with the sequence or a fragment thereof of tRF-Sec-NCA shown in SEQ ID NO:1, or a modified sequence of said sequence or fragment thereof, preferably having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, wherein... The sequence shown in SEQ ID NO.1 is as follows: 5'P-GCCCGGAUGAUCCUCAGUGGUCUGGGGUGCAGGCU-OH 3'.

53. A diagnostic kit for aging, the kit being used to detect the content of the small non-coding RNA of claim 45 in a sample; the kit comprising: The reverse transcription primers and detection primers for the small non-coding RNAs mentioned above.

54. The use of the small non-coding RNA of claim 35 in the preparation of a diagnostic reagent for aging, wherein the reagent determines the risk level of aging in a subject by detecting the content of the small non-coding RNA in a subject's biological sample.

55. A detection method for predicting or diagnosing the risk of aging, comprising: Blood leukocytes were collected from the subject, and the small non-coding RNA as described in claim 45 was measured. When the mass spectrometry response / RNA mass (ng) was greater than 800, the subject was at risk of aging.

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