354 results about "Circular RNA" patented technology

The present invention relates to a single-chain circular RNA having a sustained or slow-releasing RNA interference effect, characterized in that the single-chain circular RNA comprises a sense strand sequence, an antisense strand sequence complementary to the sense strand sequence, identical or different two loop sequences between the sense strand and the antisense strand, connecting both strands, wherein the sense strand and the antisense strand are paired to form a stem.

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other.

The invention provides a DNA (deoxyribonucleic acid) sequence used for circular RNA (ribonucleic acid) expression and a vector containing the DNA sequence used for circular RNA expression. The vector is inserted in a eukaryotic expression vector, a lentiviral vector, an adenovirus vector or a retroviral vector to form a series of special expression vectors for circRNA. The DNA sequence and the corresponding expression vector have the best effects, are simple and convenient to operate and are stable in results and efficient in expression (the circRNA expression level is increased by more than a hundredfold). The sequence and the corresponding vector can be widely applied to expression of various circRNA, provide a powerful research tool for the functions and mechanisms of circRNA and provide theoretical support for research and development of further determination of circRNA molecules as novel markers and disease treatment targets.

The present invention relates to inhibition of microRNAs (miRNA), other types of RNA and RNA-binding proteins. In particular the present invention relates to nucleic acid sequences that are circular and allow inhibition of the interactions between microRNAs and their RNA target and being resistant to degradation by exonucleases. Also, the present invention relates to nucleic acid sequence that are circular and acts as an RNA blocker or protein decoy.

The invention relates to a recombinant nucleic acid molecule for transcribing circular RNA and application of the recombinant nucleic acid molecule in protein expression. Specifically, the invention relates to the recombinant nucleic acid molecule for transcribing circular RNA, a recombinant expression vector, linear RNA, circular RNA, a recombinant host cell, a pharmaceutical composition and a method for preparing protein. The recombinant nucleic acid molecule is transcribed to form circular RNA containing a specific IRES element, the IRES element can improve the protein expression level of the circular RNA in eukaryotic cells, efficient and durable expression of protein is achieved, and the recombinant nucleic acid molecule has important application values for preparing mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines and mRNA-based dendritic cell tumor vaccines, and in the fields of gene therapy based on mRNA, chimeric antigen receptor T cell therapy based on mRNA,protein supplement therapy and the like.

The invention discloses an establishing method of a circular RNA high-throughput sequencing library and a kit thereof. The establishing method sequentially comprises the following steps: (S1) extracting total RNA from a sample; (S2) removing DNA in the sample; (S3) detecting and evaluating the quality of the total RNA in the sample, and determining that the RNA quality meets a requirement; (S4) removing rRNA; (S5) removing linear RNA; (S6) constructing the circular RNA library for high-throughput sequencing. The establishing method of the circular RNA high-throughput sequencing library, provided by the invention, is high in efficiency, stable, low in residual ratio of the rRNA, high in circular RNA detection efficiency, good in data reproducibility, and high in sequencing result verifying success rate, and is especially applicable to an FFPE tissue and other samples with poor RNA quality.

The invention discloses a circular RNA-MET gene in liver cancer as well as an expression method and a fluorescent quantitative PCR (polymerase chain reaction) detection method of the circular RNA-MET gene. The cDNA nucleotide sequence of the circular RNA-MET gene in the liver cancer is shown by SEQ ID NO:1. By designing a specific primer capable of amplifying the circular RNA, the circular RNA of the MET gene is amplified out by PCR, and an accurate cyclization site of circular RNA-MET is measured by a method for sequencing a PCR product namely cloned DNA; and the MET gene is determined to objectively contain the circular RNA consisting of 1214 nucleotides. By using the fluorescent quantitative PCR detection method of the circular RNA-MET gene in the liver cancer, the existence and expressive differences of the circular RNA-MET in samples of liver cancer cells and liver cancer clinical tissues can be specifically detected. The invention can provide an ideal gene detection method for early clinical diagnosis of liver cancer.

The invention discloses a method for screening specific circular RNA (ribonucleic acid) of triple-negative breast cancer. The method includes comparing circular gene expression of triple-negative breast cancer plasma samples and normal comparison groups by the aid of 70 parts of specific circular RNA of breast cancer; screening the specific circular RNA to obtain the circular RNA with functions of specifically obviously expressing the triple-negative breast cancer. The specific circular RNA is used as primers. The method has the advantage that results obtained by the aid of the method have important significance on triple-negative breast cancer diagnosis and treatment.

A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation.

The invention discloses a method for sequencing high-flux circular RNA (ribonucleic acid), belonging to the field of molecular biology. The method comprises the following steps: artificially synthesizing exogenous linear ERCC-0004-RNA and exogenous circular ERCC-0013-RNA; performing quality control on the exogenous circular ERCC-0013-RNA; mixing the exogenous linear ERCC-0004-RNA and the exogenous circular ERCC-0013-RNA at an equal mole ratio to obtain a mixed exogenous RNA; adding the mixed exogenous RNA into total RNA of a to-be-sequenced sample to obtain a first mixture; removing ribosome RNA in the first mixture to obtain a second mixture; performing 3' tail end biotin marking on the second mixture, and removing lariat RNA and linear RNA marked with biotin to obtain a third mixture; removing linear RNA in the third mixture to obtain a fourth mixture; and performing standard high-flux transcriptome sequencing on the fourth mixture and performing biological analysis on sequencing data. By adopting the method disclosed by the invention, the expression of genes in an organism under a specific condition can be accurately reflected, and the cost is reduced.

The invention discloses an application of circular RNA WHSC1 in preparation of clinical diagnosis markers in colorectal cancer tumorigenesis and hepatic metastases diseases. The invention further discloses an application of the circular RNA WHSC1 serving as a therapeutic target of the colorectal cancer drug. The invention further discloses a preparation for treating colorectal cancer. The preparation comprises a sequence for specifically interfering the circular RNA WHSC1, wherein the sequence is used for inhibiting the level of the circular RNA WHSC1. Research results show clinical significances of the circ-WHSC1 in diagnosis of colorectal cancer and metastasis and an application in preparation of drugs for treating the colorectal cancer metastasis.

The invention discloses a goat circular RNA as well as an identification method and functional application thereof. Nucleotide sequences of primers used for detecting the goat circular RNA circ_ZCCHC24 are respectively shown in the SEQ ID NO: 2 and the SEQ ID NO: 3. An over-expression vector constructed by an over-expression vector plasmid construction method of the goat circular RNA circ_ZCCHC24contains a circular RNA expression vector pCD2.1-ciR as well as full-length sequence information of the goat circular RNA circ_ZCCHC24, and is capable of promoting proliferation of goat follicular granulosa cells; and thus, new ideas for further understanding genetic mechanisms of goat follicular granulosa cell development are provided by the goat circular RNA circ_ZCCHC24. Therefore, the goat circular RNA circ_ZCCHC24 is of great significance for studying the genetic essence of goat reproductive traits and breeding high-breed-rate goat breeds.

The invention relates to intron-source circular RNA molecules and an application of cyclization key nucleotide sequences of intron-source circular RNA molecules. The invention specifically discloses sequences of circular RNA novel molecules and genome mapping and the cyclization key nucleotide sequences of the circular RNA novel molecules and further discloses a set of plasmids which contain the cyclization key nucleotide sequences for generating the circular RNA molecules comprising any nucleotide sequences. The invention discloses the intron-source circular RNA molecules and the application of cyclization key nucleotide sequences of intron-source circular RNA molecules for the first time, thereby providing a brand-new concept and method for generating the circular RNA molecules comprising any nucleotide sequences.

A method of amplifying DNA from RNA in a sample by using circular RNA is provided.

The invention provides a human circular RNA overexpression vector framework and overexpression vector and preparation methods of the overexpression vector framework and overexpression vector. The human circular RNA overexpression vector framework and vector can be used as a universal circular RNA overexpression vector, and a breakthrough in the field of circular RNA overexpression is obtained; meanwhile, the circular RNA overexpression vector solves the problem of low RNA cyclization efficiency in the early stage of circRNA in vivo, the cyclization rate is increased to 95%, the defect that a circRNA precursor is not cyclized and mainly exists in a linear form is overcome, and the false positive problem of the circular circRNA function due to a linear circRNA precursor is solved; overexpression efficiency of circRNA is greatly improved, and overexpression efficiency is improved to 500-2,000 times.

The invention establishes a circRNA expression profile for a patient with craniosynostosis, provides, provides serum circRNA marker hsa_circ_0001788 for the patient with craniosynostosis, discloses diagnostic value of hsa_circ_0001788 of serum for craniosynostosis, applies the marker herein to prepare a diagnostic kit for the patient with craniosynostosis, and provides supporting for clinical early discovery and treatment of the patient with craniosynostosis. Serum circRNA has good stability, degradation difficulty and the other advantage, has high value in auxiliary diagnosis, and is easy toclinically popularize and use. A circRNA chip is used herein to carry out detecting to obtain disease-specific and abnormally-expressed serum circRNA expression profiles; verifying is performed by means of qRT-PCR; a strict design and evaluation system is provided. It is discovered for the first time that the marker hsa_circ_0001788 in serum is an important biological detection index which causesminimal invasion and has low cost and which is applicable to clinical detection; the marker hsa_circ_0001788 is important to the clinical early diagnosis and differential diagnosis of craniosynostosis; a new idea is provided for the diagnosis of craniosynostosis in molecular level, and it is expected that early diagnostic rate of craniosynostosis is increased.

The invention relates to a method for detecting a circular RNA for bladder cancer screening and an application thereof; in particular, with use of a circular RNA chip, through difference analysis, the circRNA significantly highly expressed in a human bladder cancer tissue is screened and is named as cRNA-ZFR. Compared with a normal tissue, the cRNA-ZFR is significantly highly expressed in the human bladder cancer tissue, and the cRNA-ZFR is further confirmed to have the expression quantity in the human bladder cancer tissue significantly higher than that of the normal tissue in large-sample fluorescent quantitative PCR experiments. The novel cRNA-ZFR provides a new idea for further study of pathogenesis of bladder cancer, and also provides a new target for early screening and prognosis monitoring of bladder cancer.

The invention relates to a general expression framework of artificial circular RNA and application thereof. The general expression framework of artificial circular RNA has multi-microRNA combination inhibition ability. The artificial circular RNA overexpression framework consists of three parts: an upstream sequence, an intermediate sequence and a downstream sequence. The artificial circular RNA overexpression framework designed by the invention can achieve effective overexpression and efficient cyclization of the intermediate sequence in cells to produce the artificial circular RNA with multi-microRNA combination inhibition ability. The invention also relates to application of the general expression framework of the artificial circular RNA and eukaryotic expression plasmids, lentiviruses,adenoviruses, adeno-associated viruses and retroviruses carrying the framework in development and preparation of gene therapy drugs.

The invention discloses a high-flux chip data processing and analysis process control method for circular RNA. The method comprises the steps of generating a self-defined parameter configuration file by a system first and then generating a batch processing executable file corresponding to a data process according to a self-defined parameter file after parameter setting by a user and a high-flux chip data processing process module; and executing the batch processing executable file by the system, implementing data process automation, and finally generating a result report file. Therefore, bio-information analyzers can be efficiently assisted to finish a set of standardized high-flux data analysis process, and even scientific research personnel who do not understand high-flux data analysis can be enabled to finish the high-flux data analysis. In addition, the purposes of optimizing working efficiency of the scientific research personnel and reducing scientific research cost can be achieved. Reliable and diversified circular RNA analysis methods are proposed; and the control method also can be used for high-flux data analysis processes of long-chain non-coding RNA and the like, and is universal in different fields, simple to implement and wide in application range.

The present invention pertains to extracellular vesicle (EV) therapeutics, wherein the EVs comprise nucleic acid (NA)-based therapeutics such as m RNAs, circular RNAs, mi RNAs, sh RNAs, circular RNA and / or DNA molecules. The NA therapeutics are loaded into EVs using inventive engineering protein and NA engineering strategies to enhance loading into EVs and to facilitate release of the NA cargo molecules inside target cells.

The invention belongs to the technical field of biological detection, and particularly relates to a biomarker for detecting mild cognitive impairment (MCI) and / or Alzheimer disease (AD). The biomarkerfor detecting mild cognitive impairment and / or Alzheimer disease is circular RNA, and the circular RNA is ciRS-7. The invention further relates to application of the circular RNA ciRS-7 in detectingmild cognitive impairment and / or Alzheimer disease. By detecting the content of the ciRS-7 in peripheral blood, it is found that the content of the ciRS-7 in the peripheral blood of an MCI or AD patient is higher than that of a normal person, and therefore the ciRS-7 can be used as the biomarker of MCI or AD early diagnosis.

The invention discloses a prediction method for a correlation between circular RNA and a disease based on a gradient enhancement decision-making tree. A circular RNA-disease relationship network is converted into an undirected graph, the circular RNA base sequence similarity, the function annotation semantic similarity and the expression similarity are calculated, and the disease function similarity and the semantic similarity are calculated; a multi-network integration algorithm is adopted for integrating a plurality of kinds of circular RNA similarity networks and conducting weighted averageintegration on a disease similarity network, statistical characteristics of the integrated circular RNA and disease similarity network and the circular RNA-disease relationship network are extracted,and the integrated circular RNA and disease similarity network is converted into an unweighted graph-related characteristic, a circular RNA base sequence characteristic and a circular RNA-disease relationship network implicit vector characteristic; a gradient enhancement decision-making tree learning machine is trained, and a potential circular RNA-disease relationship is predicted. By means of the method, the potential circular RNA-disease relationship can be accurately predicted; and the prediction accuracy of the circular RNA-disease relationship is improved.

The invention provides application of circular DNA (Deoxyribonucleic Acid) in detecting and imaging sense RNA (Ribonucleic Acid) and gene therapy. The circular DNA at least comprises one or more DNA probe fragments complementary to a detection target sense RNA sequence, and is formed by connecting and cyclizing 3' and 5' terminals of ssDNA (single-stranded Deoxyribonucleic Acid). The circular DNAand the detection target sense RNA form heterozygous double strands, and the heterozygous double strands are separated from a carrier, so that (1) the application in detecting and imaging through a corresponding fluorescence signal change is realized; (2) the sense RNA in the heterozygous double strands is enzymically decomposed, so that the aim of gene therapy is achieved.

The invention discloses a method for preparing an exogenous circular RNA and a method for quantitatively determining an endogenous circular RNA. The method for preparing an exogenous circular RNA comprises the following steps: cloning and multiplying an artificially synthesized DAN gene, transcribing the DAN gene into a linear RNA, modifying the tail end of the RNA, and connecting the head and the tail of the RNA to obtain the exogenous circular RNA. Exogenous circular RNA molecules are added to a total RNA according to a specific proportion to serve as a reference gene for real-time quantitative determination of the RNA, and therefore the real-time quantitative determination of the endogenous circular RNA is realized for the first time.

The invention discloses an oligonucleotide composition and a method for detecting hepatitis B virus (HBV) rcDNA (Relaxed Circular deoxyribonucleic acid) and / or cccDNA (Covalently Closed Circular DNA), a kit and application of the oligonucleotide composition. The oligonucleotide composition comprises first oligonucleotide, second oligonucleotide, or oligonucleotide complementary with the sequenceof the first oligonucleotide and oligonucleotide complementary with the sequence of the second oligonucleotide; the first oligonucleotide is specifically combined with at least one part of continuoussequence of the first sequence, and the second oligonucleotide is specifically combined with at least one part of continuous sequence of the second sequence; in addition, a distance between two continuous sequences is 30-350 nt; the first sequence is a sequence between DR (Direct Repeat) 2-DR1 of the HBV DR sequence of a cross-notch area which contains cccDNA or rcDNA, and the second sequence is asequence after the fifth basic group of the DR1. According to the oligonucleotide composition, the level of HBV rcDNA and / or cccDNA can be effectively detected without being affected by integrated HBV DNA.

The invention discloses a circular RNA recognition method based on a machine learning strategy. The method comprises the steps: inputting data, positioning each candidate circular RNA on a reference genome, and extracting Reads features nearby circular RNA regions; training a supervised machine learning model by using the extracted features; and performing true and false positive classification onthe candidate circular RNA set by using the trained model, and outputting final circular RNA. The method belongs to a machine learning filtering strategy, has the advantages of the machine learning filtering strategy, and can remarkably save the cost, time and the like in clinical practice.

The invention discloses a dye method digital quantitative PCR kit as well as application and an application method thereof. The dye method digital quantitative PCR kit comprises application of circular RNA hsa_circ_0051778 copy number as a detection index in a lung adenocarcinoma and tuberculous pleuritis identification kit according to the circular RNA hsa_circ_0051778 copy number in tuberculouspleuritis being higher than the copy number in the lung adenocarcinoma. The kit comprises a circular RNA hsa_circ_0051778 specific primer, a positive control, a negative control, a special PCR amplification premix solution for dye-method digital PCR as well as sterile deionized water; and the kit is used by virtue of steps of sampling, PCR amplification system preparation, amplification reaction and identification. By adopting the dye method digital quantitative PCR kit, the lung adenocarcinoma and the tuberculous pleuritis can be accurately and rapidly identified. The detection result provesthat the sensitivity of the detection tool can reach about 90 percent or more which is higher than the existing single method used clinically.

RNA molecules are an important member of an intracellular genetic information delivery system. Conventional RNA labeling methods cannot simultaneously have the characteristics of living cells, no invasion, high temporal and spatial resolution, single molecules, etc., so a novel method for labeling and tracking RNA need to be developed. The invention discloses a novel labeling method for messengerRNA and circular RNA based on self-ligation label bimolecular fluorescence complementation. A RNA aptamer subsystem is cooperatively used, aptamers are inserted at proper positions in the RNA, and twoparts of a self-ligation label are respectively fused with the binding proteins of the RNA aptamers. When the fusion proteins do not bind to RNA, RNA molecules do not fluoresce; when and only when the fusion proteins bind to RNA, the RNA molecules fluoresce, so the fluorescence background of an intracellular RNA marker is reduced. The novel RNA labeling method based on self-ligation label bimolecular fluorescence complementation can be used for long-term interference-free single-molecule imaging observation and tracking of messenger RNA and circular RNA of living cells and fixed cells, and has good application prospects in cell biology.