51 results about "Pseudouridine" patented technology

The present application provides methods, compositions, and systems for decoding a pseudouridine-modified (psi-modified) codon. In some aspects, the present application provides methods for improving the translation of a target RNA in a host cell, the target RNA comprising a psi-modified codon wherein the method comprises: introducing a near homologous transport RNA (nctRNA) of the psi-modified codon or a nucleic acid encoding the nctRNA into the host cell; and wherein the psi-modified codon is decoded to an amino acid, thereby improving translation of the target RNA. In some cases, the codon of the psi modification is a premature termination codon (PTC).

A method of synthesising a 1N-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. The substrate has a structure of: Formula (I). The 1N-methyl derivative of pseudouridine has a structure of: Formula (II). R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group. R2 is H or a hydroxyl group. R3 is a methyl group or an ethyl group. The cofactor is a source of a methyl group or an ethyl group. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Enzymatic synthesis of 1N-methyl derivatives of pseudouridine may be more efficient and produce less waste than chemical syntheses. Also provided are methyltransferases and buffers useful in the method; and polynucleotides, expression vectors, and microorganisms useful for producing the methyltransferases.

The invention relates to compositions including polynucleotides encoding polypeptides which have been chemically modified by replacing the uridines with 1-methyl-pseudouridine to improve one or more of the stability and / or clearance in tissues, receptor uptake and / or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency, accessibility to circulation, protein half-life and / or modulation of a cell's status, function, and / or activity.

A method for preparing pseudouridine through whole-cell catalysis of recombinant Escherichia coli. The recombinant Escherichia coli is obtained by overexpressing pseudouridine-5-phosphoglycosidase, ribokinase, ribonucleoside hydrolase, cytosine deaminase and nucleoside permease, and not expressing pseudouracil transport protein in Escherichia coli E.coHMG1655; and whole-cell catalytic preparation of pseudouridine taking uridine or cytidine as a substrate is realized by using the recombinant Escherichia coli. A novel pseudouridine enzyme catalytic synthesis route is designed, and in optimal reaction conditions, the maximum yield of pseudouridine reaches 27.24 g / L, the conversion rate reaches 90.8%, the production efficiency is 1.135 g·h-1·L-1, and the pseudouridine yield and the production efficiency are high.

A 2-bromoacrylamide assisted cyclization sequencing (BACS) method allows quantitative profiling of pseudouridine (psi) at single base resolution. Based on bromoacrylamide cyclization chemistry, BACS induces psi-to-C mutations during reverse transcription (RT) instead of truncation or deletion of features, thus providing higher resolution and enabling more accurate quantification of psi stoichiometry compared to CMC and BS based methods. The BACS of the invention allows for precise identification of psi positions, in particular in densely modified psi regions and continuous uridine sequences, compared to known methods. Accordingly, methods, compositions, and kits for detecting psi are possible. In the case of RNA molecules, this allows psi to be detected and sequenced in such sequences.

This disclosure relates to the modified 3' region elements that confer improved therapeutic properties including, but not limited to, increased stability, increased expression, and / or a reduced innate immune response when introduced into a population of cells, even in the absence of a 5'-cap. The disclosure also provides polynucleotides (e.g., RNA molecules) containing modified 3' regions. In some embodiments of the disclosure, the modified 3' regions contain modified adenosines (e.g., 2'-0-methoxy- adenosine, 2'-fluoro-adenosine, or locked nucleic acid (LNA)-adenosine) and / or a modified thymidine (e.g., inverted deoxythymidine). In some embodiments of the disclosure, the modified 3' region may contain one or more modified internucleoside linkages (e.g., a phosphorothioate linkage) or modified nucleobase (e.g., N1-methyl-pseudouridine). The polynucleotides of the disclosure may be used to express a desired polypeptide in a subject, such as a subject having a disease or condition associated with a deficiency in the corresponding endogenous polypeptide.

The invention provides a genetically engineered bacterium for efficiently producing pseudouridine and application of the genetically engineered bacterium. The genetically engineered bacterium is based on a high-yield uridylic acid strain E. coli MB219 (the patent application number is 202410153359.6), and the high-efficiency pseudouridine engineering bacterium is constructed through systemic gene modification. The genetically engineered bacterium is characterized in that (1) gene knockout: 10 genes such as pbs1, psuK and the like are knocked out to block a metabolic pathway of byproducts; (2) metabolism regulation: replacing a J23110 strong promoter, and overexpressing zwf / gnd to enhance a phosphopentose pathway; (3) heterologous gene integration: integrating a specific dephosphorylase gene HDHD1, and introducing RjpsuG, NmygdH and thyA to construct a co-expression plasmid pRL06; the fermentation yield of the finally obtained engineering bacterium CY20 (pRL06) reaches 45.3 g / L and is increased by 6 times compared with that of a traditional method, the production efficiency is greatly improved, the product purity is high, and by-products are few.

Provided herein are RNA molecules (e.g. self-amplifying messenger ribonucleic acids (samRNA)) comprising modified nucleotides, such as N1-methyl-pseudouridine.

The present application relates to the technical field of mRNA vaccine, and specifically discloses a proinsulin mRNA vaccine for protecting the function and quantity of pancreatic beta cells. The vaccine is composed of a lipid nanoparticle (LNP) and an mRNA wrapped by the LNP; the mRNA is an mRNA sequence encoding proinsulin, and all uridines in the mRNA sequence are replaced by N1-methyl pseudouridine. The mRNA vaccine of the present application realizes the dual protection of the quantity and function of pancreatic beta cells, compared with the existing intervention means which only focus on blood glucose control or immune regulation, the present application delays the occurrence process of type 1 diabetes from the root, and provides a new technical scheme and drug selection for the prevention and early intervention of type 1 diabetes.

A solution has been discovered that provides a more effective Coronavirus vaccine. The solution is an mRNA vaccine encoding a SARS-CoV-2 nucleoprotein (N) (mRNA-N) in combination with an mRNA vaccine encoding SARS-CoV-2 spike protein(S) (mRNA-S). Chemically modified mRNA-N (pseudouridine) and / or chemically modified mRNA-S (pseudouridine) can be synthesized and packaged in lipid nanoparticles (LNP). In mouse and hamster models, it was shown that mRNA-N alone is immunogenic and can significantly diminish viral loads in the mouse lung after prime-boost intramuscular immunization. In addition, the combinatorial mRNA-N / mRNA-S vaccination induces substantially stronger protection against SARS-CoV-2 than vaccination with mRNA-S alone.

The present invention includes novel systems, methods, and compositions for the enzymatic / chemical production of pseudouridine (Ψ) and its variants, such as N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).

Provided herein are nucleoside-modified ribonucleic acid molecules encoding a chimeric influenza virus hemagglutinin (HA) polypeptide that comprises a HA globular head domain and a HA stalk domain, wherein the HA stalk domain comprises an amino acid sequence of a HA stalk domain of an influenza B, the HA globular head domain comprises an amino acid sequence of a HA globular head domain of an influenza A, or the HA globular head domain comprises an amino acid sequence of a HA globular head domain of the influenza B, wherein one or more antigenic sites have been replaced with antigenic sites of an influenza A, and the nucleoside-modified RNA molecule comprises at least one pseudouridine. Also provided herein are methods of immunizing a subject against influenza virus disease and methods of preventing influenza virus symptoms in a subject using a composition described herein.

The application provides a uridine monophosphate specific glycoside hydrolase and application thereof in biosynthetic synthesis of pseudouridine. Nmygdh The function of the gene is verified by in-vitro enzyme activity experiment Nmygdh The recombinant protein has broad-spectrum and high-efficiency catalytic activity, and can efficiently catalyze the hydrolysis of glycosidic bonds in various nucleotides, such as uridine monophosphate (UMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP) and cytidine monophosphate (CMP), and the activity of uridine monophosphate is the strongest. The application uses the protein to efficiently hydrolyze uridine monophosphate to obtain two substrates of pseudouridine, i.e. uracil and 5'-phosphoribose, and realizes the atom-economical and efficient biosynthetic synthesis of pseudouridine by combining the cascade reaction of pseudouridine glycosidase EcPsuG and dephosphorylase EcYjjG.

Provided herein is a one-pot method of synthesising a C-nucleoside-5′-monophosphate such as pseudouridine-5′-monophosphate, from an N-nucleoside using a multi-step enzymatic pathway. The method comprises reacting the N-nucleoside with a phosphate source and nucleoside phosphorylase to form a pentose-1-phosphate intermediate, reacting the pentose-1-phosphate intermediate with a phosphomutase to form pentose-5-phosphate intermediate, and reacting the pentose-5-phosphate intermediate with a nucleoside-5′-phosphate C-glycosidase to form the C-nucleoside-5′-monophosphate.

The present invention includes novel systems, methods, and compositions for the enzymatic / chemical production of pseudouridine (Ψ) and its variants, such as N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).

The invention relates to the technical field of biological medicine, in particular to placenta-derived tRF and application of the placenta-derived tRF in prevention and treatment of bronchial lung dysplasia, the placenta-derived tRF is a derivative fragment generated after glycine tRNA is modified by pseudouridine, and the nucleotide sequence of the placenta-derived tRF is shown as SEQ ID No.1. The invention innovatively finds that the placenta-derived tRNAGly derivative fragment tRF-0009 is an important active metabolite for mediating placenta and alveolar development, can inhibit mitochondrial integration stress reaction, negatively regulate AECII proliferation and differentiation, and perform deep function and mechanism exploration on tRF-0009, and can provide a potential strategy and a new target for prevention and treatment of BPD.

The present invention comprises novel systems, methods and compositions for the enzymatic / chemical production of pseudouridine (Ψ) and its variants, such as N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP).

The invention relates to a biomarker for diagnosis, disease course analysis and prognosis of paraquat poisoning and application of the biomarker. The biomarker is a urine metabolite Pseudouridine. Urine non-target metabonomics analysis shows that the relative amount of Pseudouridine is relatively low in urine of early-stage poisoning patients, is gradually reduced along with aggravation of illness, is increased in follow-up visit of five years, and is recovered to the level of a healthy control group; according to the invention, the prediction efficiency is judged through the area under the line (AUC) of a subject's working characteristic (ROC) curve, the Pseudouridine has good efficiency (AUC = 0.732) in the aspect of diagnosing whether paraquat is poisoned or not, has good efficiency (AUC = 0.855) in the aspect of judging the progression of paraquat poisoning, has good efficiency (AUC = 0.807) in the aspect of judging long-term prognosis in the early stage of poisoning (1-4 days), and has excellent efficiency (AUC = 0.979) in the aspect of judging long-term prognosis in the progression stage of poisoning (7-14 days). And the prediction efficiency of the Pseudouridine is better than the blood routine examination index reported in the literature. In conclusion, the Pseudouridine, as a biomarker for paraquat poisoning diagnosis, disease course analysis and prognosis, shows a good clinical application prospect.

The invention discloses construction and application of an engineering strain for efficiently producing pseudouridine, and belongs to the technical field of microorganisms. According to the invention, Escherichia coli BL21 (DE3) is used as a starting strain, and key enzyme genes psuG, YjjG, rihA and rbsK are over-expressed to optimize a synthesis route of pseudouridine in E.coli; thrA, argF and pepA genes are knocked out to increase uracil accumulation; the psuT gene is knocked out to prevent pseudouridine from being transferred back to cells; uridine degradation pathways are blocked by knocking out udk, udp and ppnp genes, and precursor substance supply is increased. In a 5L fermentation tank, high-efficiency production of the pseudouridine is realized through a fed-batch fermentation method, so that the yield of the pseudouridine of the recombinant escherichia coli reaches 102.2 g / L. The preparation method has the characteristics of low cost, high efficiency, simplicity in operation, wide prospect and the like.

The invention discloses a method for preparing high-purity houridine (formula 1). According to the method, a formula 44 is used as a raw material, and high-purity pseudouridine (formula 1) is prepared through condensation, reduction, cyclic etherification and deprotection in sequence. The method has the excellent effects of high yield, high purity and the like. ,

The invention belongs to the technical field of mRNA vaccines, and particularly provides mRNA of an FASN gene and application of the mRNA. The sequence of the FASN gene is shown as SEQ ID No.1, a 3-O-Me-GAG cap structure is added to the 5'end of mRNA of the optimized FASN gene, a polyadenylic acid polyA tail structure is added to the 3 'end of mRNA of the optimized FASN gene, and all U basic groups in the mRNA sequence are replaced with pseudouracil psi-UTP. After codon optimization is carried out on mRNA of the FASN gene, in-vitro modification and synthesis are carried out, the in-vitro synthesized mRNA which is high in synthesis efficiency, high in translation efficiency, accurate in translation and good in stability is obtained, the in-vitro synthesized mRNA can be applied to modeling, the modeling period is shortened, the modeling cost is reduced, a new tool is provided for research of metabolism-related diseases, and the in-vitro synthesized mRNA has good application prospects. And the biomedical research with low cost and high safety is realized.

The invention relates to compositions including polynucleotides encoding polypeptides which have been chemically modified by replacing the uridines with 1-methyl-pseudouridine to improve one or more of the stability and / or clearance in tissues, receptor uptake and / or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency, accessibility to circulation, protein half-life and / or modulation of a cell's status, function, and / or activity.

This invention provides RNA, oligonucleotide and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising them, methods of synthesizing them and methods for gene replacement, gene therapy, gene transcription suppression and delivery of therapeutic proteins to tissue in vivo comprising the molecules. The present invention also provides methods of reducing the immunogenicity of RNA, oligonucleotide and polyribonucleotide molecules.

The invention provides a mutant tRNA (transfer ribonucleic acid) capable of specifically decoding a pseudouracil modified codon in a transcriptome and a translation group. In addition, the invention also provides a translation system containing the mutant tRNA, and a method for realizing site-specific amino acid insertion by responding to the pseudouracil modification codon based on the mutant tRNA. Furthermore, the invention also provides an RNA modification assisted codon expansion (RCE) technology based on the mutant tRNA.

A method of synthesising a IN-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the I N-alkylderivative of pseudourid

The invention discloses a method for preparing high-purity houridine (formula 1) by adopting tert-butyl diphenyl silyl protection. According to the method, a formula 42 is used as a raw material, and the high-purity pseudouridine (formula 1) is prepared through hydroxyl protection, condensation, reduction, cyclic etherification and deprotection in sequence. The method has the advantages of high yield, high purity and the like.

Disclosed is a construct for preventing antibody-dependent enhancement of dengue virus infection, comprising: (a) a ribonucleic acid encoding a non-structural protein of a dengue virus wherein each of the uridine residues of the ribonucleic acid encoding a non-structural protein of a dengue virus is replaced with pseudouridine; (b) a 7-methylguanylate cap; (c) a 5' untranscribed region preferably comprising a kozak consensus ribonucleic acid sequence; (d) a 3' untranscribed region; and (e) a poly A tail and a method for using the construct to prevent antibody-dependent enhancement of dengue virus infection

The invention relates to a method for separating and purifying pseudouridine, which comprises the following steps: (1) sequentially dissolving and filtering a pseudouridine crude product to obtain a pseudouridine solution; and (2) loading the pseudouridine solution obtained in the step (1) to a chromatographic column filled with polymer microspheres for chromatography, eluting the pseudouridine solution by using an acidic aqueous solution as a flowing phase, collecting the pseudouridine solution with a target peak value in sections, and collecting component solutions meeting requirements. According to the separation and purification method provided by the invention, a high-purity and high-yield pseudouridine product can be obtained, the effect is stable, the operation is simple, the used stationary phase can be repeatedly utilized, the use amount of the mobile phase is greatly reduced, and the separation and purification method has good economic benefits and environmental benefits.

The present application relates to the fields of biotechnology and RNA editing. The invention relates to an RNA single base editing system and method based on pseudouridine modification. In particular, the present application relates to a method for inhibiting an early termination codon (PTC) in a target RNA in a host cell. The present application also relates to an engineered guide small nucleolus RNA (gsnoRNA), an isolated nucleic acid molecule comprising the same, a composition, and a host cell. The present application also relates to their use in the preparation of medicaments. According to the method provided by the invention, the efficiency of pseudouridine modification can be remarkably improved, so that the coding information of mRNA is recovered, the PTC site is decoded, and the read-through efficiency of PTC is remarkably improved.

The invention provides a preparation method of 2 '-OMe modified pseudouridine and a phosphoramidite monomer of N1-methyl pseudouridine, and belongs to the field of medicine synthesis, and the preparation method comprises the following steps: methylating 2'-OH of pseudouridine / N1-methyl pseudouridine by using trimethylsilyl diazomethane; reacting the methylation product with 4, 4 '-dimethoxytriphenylmethyl chloride to obtain an intermediate with a DMTr protecting group at the 5'position; and reacting the obtained intermediate with bis (diisopropylamino) (2-cyano ethyoxyl) phosphine to obtain a target product. According to the invention, pseudouridine / N1-methyl pseudouridine is taken as a raw material, the 2-OMe modified pseudouridine / N1-methyl pseudouridine phosphoramidite monomer can be prepared by only three steps, the synthesis route is greatly shortened, the total yield is more than 40%, the purity of the target product is more than 99%, and the method is very suitable for large-scale industrial synthesis.