44 results about "Stem-loop" patented technology

Provided herein are gene editing pairs comprising an engineered protein, e.g, an engineered CRISPR protein and a guide ribonucleic acid (gRNA) that may provide improved gene editing relative to reference, i.e., wild type sequences. In some embodiments, the engineered protein of the gene editing pair comprises a RuvC cleavage domain with one or more amino acid modifications relative to a reference CRISPR protein RuvC sequence. In some embodiments, a guide ribonucleic acid (gRNA) of the gene editing pair comprises at least one modification in a scaffold stem loop relative to a reference scaffold stem loop sequence.

The present application discloses partially single-stranded deoxyribonucleic acid (ssDNA) molecules comprising at least one nucleic acid sequence of interest flanked by a at least one stem loop structure, methods of making, and methods of use for delivery and expression of a transgene in host cells.

The invention relates to the technical field of biology, in particular to an engineered vesicle carrying mRNA targeting and application of the engineered vesicle to improvement of fibroblast functionality. The iRGD polypeptide carried by the engineered vesicle is used as a targeting polypeptide of fibroblasts, specifically targets human dermal fibroblasts and can be combined with rich integrin alpha v and NRP-1 on cell membranes of the fibroblasts, meanwhile, the iRGD polypeptide and exosome surface mask protein emp2b construct a fusion protein, MS2 capsid protein is fused in exosome, and the human dermal fibroblasts can be specifically targeted to the human dermal fibroblasts. According to the present invention, with the combination of the mRNA containing the MS2 stem loop, the more target mRNA can be obtained, such that the more target mRNA can enter the senescent fibroblast in the targeting manner so as to improve the activity of the fibroblast and delay the senescence of the human skin;

The invention provides a super-sensitive molecule system for biological signal amplification, a detection method and application, and belongs to the technical field of biological sensing. The super-sensitive molecular system for biological signal amplification is composed of a hairpin probe H and gold nanoparticles (TS (at) CP (at) loading a trajectory chain TS and a capture chain CP (CP), a traditional DNA walking machine walking chain is split into the hairpin probe H and the capture chain CP which are spatially isolated, and a target-dependent activation mechanism is constructed: when no target exists, the hairpin probe H realizes self-protection through a self-folding stem-loop structure; non-specific binding with the capture chain CP / trajectory chain TS is blocked; after the target is beaten on the clamping probe H, the exposed sequence of the clamping probe H is hybridized with the capture chain CP to form a complete walking chain, and a signal amplification reaction driven by cutting incision enzyme is triggered, so that the problem of false positive caused by co-localization of the walking chain and a track chain interface is fundamentally solved.

The present application discloses partially single-stranded deoxyribonucleic acid (ssDNA) molecules comprising at least one nucleic acid sequence of interest flanked by a at least one stem loop structure, methods of making, and methods of use for delivery and expression of a transgene in host cells.

The invention relates to the technical field of nucleic acid detection, and discloses a construction method of a PFLAMP-Cas12b one-tube method reaction system based on single-stranded stem loop mediation. The construction method comprises the following steps: designing an LAMP amplification primer; designing an sgRNA sequence of a single-stranded loop region in a targeted LAMP amplification product; and constructing a reaction system containing the LAMP amplification primer, the Cas12b protein, the sgRNA sequence and the fluorescence report probe. A single-chain stem-loop structure formed in an LAMP amplification product is used as a specific activation target of the Cas12b protein, PAM dependence of the Cas12b protein on double-chain DNA targeting in traditional detection is avoided, and the bottleneck that sgRNA design is limited due to lack of natural PAM sites in a highly conserved gene area is effectively overcome; and non-specific background amplification interference induced by artificially introducing a PAM sequence into the primer is eliminated, and the detection accuracy is improved.

Disclosed herein include systems, methods, compositions, and kits for molecular barcoding on the 5'-end of a nucleic acid target. After barcoding a nucleic acid target using an oligonucleotide barcode comprising a target binding region and a molecular label to generate a barcoded nucleic acid molecule, an oligonucleotide comprising a complement of the target binding region can be added to generate a barcoded nucleic acid molecule comprising the target-binding region and the complement of the target-binding region. A stem loop is formed with intra-molecular hybridization of the barcoded nucleic acid molecule, which can be extended to generate an extended barcoded nucleic acid molecule comprising the molecular label and a complement of the molecular label.

The invention provides a preparation method for preparing a scar-free circular RNA (Ribonucleic Acid), a recombinant nucleic acid molecule for preparing the scar-free circular RNA and application of the recombinant nucleic acid molecule. The recombinant nucleic acid molecule based on the IRES stem-loop structure provided by the invention can be used for preparing a circular nucleic acid molecule in which an exogenous sequence is completely eliminated, and any sequence of IRES is not changed, so that the unpredictable influence of IRES mutation on recruitment of ribosome functions is avoided.

The application discloses a modified crRNA, a light-controlled nucleic acid detection system, a kit and application, and belongs to the cross field of biotechnology, intelligent sensing and molecular diagnosis. In view of the technical defects of strong target sequence dependence and high ultraviolet irradiation requirement of the existing light-controlled CRISPR technology, the application innovatively introduces a photosensitive protection group 6-nitropiperidin oxymethyl (NPOM) at a specific key node of a stem loop skeleton of crRNA maintaining conformation. In the constant temperature amplification stage, preferred double-site cooperative modification can transiently inhibit RNP complex assembly to realize target non-interference enrichment; subsequently, only 10 mW / cm 2 of extremely low intensity ultraviolet light irradiation for 30 seconds can restore the crRNA conformation and activate the trans cleavage. The preferred technical scheme of the application can eliminate the target sequence limitation, realizes a detection limit of as low as 2 copies in a single reaction tube, and the result can be obtained within 15 minutes. The system is widely applicable to rapid diagnosis of infectious agents, high-specificity typing of single nucleotide polymorphism and portable intelligent molecular diagnosis terminal.

Provided herein are double-stranded nucleic acid inhibitor molecules having a shortened sense strand with a stem loop structure and an antisense strand. Also provided are methods and compositions for reducing target gene expression and methods and compositions for treating a disease of interest.

The present invention provides, among other things, linear mRNA compositions comprising a poly A tail and a stem loop structure downstream to the poly A tail. Also provided herein are methods of producing mRNA by ligating a stem loop exonuclease blocker using a double-stranded RNA ligase.

The present invention relates to an oligonucleotide comprising: a) a nucleotide sequence of a broom-kojin-ricin loop (SRL) of a eukaryotic / mammalian 60S ribosomal subunit wherein the SRL nucleotide sequence comprises at least one adenine; and b) at least one cut-dependent marker; wherein the oligonucleotide is a single strand. Preferably, the oligonucleotides form at least one loop structure or stem-loop structure. In another aspect, the invention relates to a method of detecting active shiga toxin in a sample, the method comprising the steps of: a) providing at least one oligonucleotide according to the invention; b) providing a sample of shiga toxin to be detected; c) incubating the sample with at least one single-stranded oligonucleotide; and d) detecting a labeled signal, wherein the signal indicates the presence of Shiga toxin in the sample. The invention additionally comprises a kit comprising at least one oligonucleotide according to the invention.

The invention discloses a group of nucleic acid aptamers specifically combined with CD8 molecules and application of the nucleic acid aptamers. The invention provides a nucleic acid aptamer of a targeted CD8 molecule. The nucleic acid aptamer comprises a core structure consisting of two stem loops, a multi-branch loop and a main stem; the multi-branch ring is positioned between the two stem rings; in the two stem loops, the stem loop close to the 5'end is composed of a random base sequence, the stem loop close to the 3 'end is composed of GC paired bases, and the loop structure is formed by a fixed sequence AGCTTGAAAT; the multi-branch ring is provided with a fixed base GTGA; the main stem is formed by pairing random basic groups. According to the method, a machine learning clustering method is adopted to identify a core region in an aptamer family, and secondary structure analysis is performed, so that a common structure capable of being specifically combined with CD8 protein is successfully found, the truncation and optimization process of an aptamer candidate library is simplified, the design of a novel aptamer is also promoted, and the method is suitable for large-scale popularization and application. These aptamers are capable of binding to CD8 proteins with high specificity and high affinity.

Provided is a prime editing guide RNA (pegRNA), aprime editing (PE) system, and use thereof, pertaining to the technical field of prime editing. The prime editing guide RNA provided herein positions the reverse transcriptase template and primer binding site (RTT-PBS) at the stem loop 2 region of the sgRNA scaffold based on the canonical pegRNA design. This configuration not only effectively mitigates degradation of the RTT-PBS but also increases its binding capability to Cas9. Consequently, this design preserves the efficiency of prime editing without interference.

The present disclosure provides compositions and methods for dynamic control of transgene expression by inclusion of optimized 3' UTR stem loops that are bound by Regnase-1 (Reg1) and / or Roquin-1 (Roq1).

An object of the present invention is to provide RNA aptamers capable of binding to ASP7967 or analogs thereof. The RNA aptamer of the present invention is an RNA aptamer that binds to ASP7967 or an analog thereof, and has the sequence: -X1-L1-X2-L2-X3- (wherein X1 has the sequence Y1GY2GY3Y4Y5; L1 is a first stem-loop nucleotide sequence comprising a first stem region, a first loop region, and a second stem region, wherein the first stem region and the second stem region are two or more base pairs in length and are substantially complementary to each other; X2 is A, G, C, or U; L2 is a second stem-loop nucleotide sequence comprising a third stem region, a second loop region, and a fourth stem region, wherein the third stem region and the fourth stem region are two or more base pairs in length and are substantially complementary to each other; the first base in the third stem region is G and the last base in the fourth stem region is C; and X3 has the sequence UY6). , Y1, Y2, Y3, Y4, Y5, and Y6 are each independently A, G, C, or U), or a third stem loop region comprising the sequence: -S1-X2-L2-X3-L3-X1-S2- (wherein S1 and S2 are each independently A, G, C, or U, and S1 and S2 can form base pairs or wobble base pairs with each other, and L3 comprises a fifth stem region, a third loop region, and a sixth stem region). the fifth stem region and the sixth stem region are one or more base pairs in length and are substantially complementary to each other, and X1, X2, X3, and L2 are as defined above), or the sequence: -S3-X3-L3-X1-L1-X2-S4- (wherein S3 is C, S4 is G, and X1, X2, X3, L1, and L3 are as defined above).

The application belongs to the technical field of biological medicine, and discloses a gene leading editing system, which comprises a carrier, the carrier contains a gene coding pegRNA and a gene coding fusion protein, the pegRNA comprises sgRNA, reverse transcription template RTT, primary binding site PBS and RNA stem loop structure, and the fusion protein is a fusion protein of Cas9 nickase or a variant thereof and reverse transcriptase. The application develops sPEs by adding an RNA neck ring structure to the 3' end of the pegRNA, develops tPEs by allowing the 3' end of the pegRNA to bind to Cas9, so as to improve the stability of the Cas9 / pegRNA complex, and further improve the editing efficiency of different genomic sites in different cells.

Provided is a 5'-ligation-based single-stranded DNA-specific high-throughput sequencing method, relating to the technical field of biology. The present invention specifically comprises a method for preparing a 5'-end-ligation-based single-stranded DNA sequencing library. The method comprises the following steps of treating a sample to be detected: step 1) using a DNA polymerase without exonuclease activity to fill in the 5' end of dsDNA; step 2) adding a tail to the 3' end; (3) ligating a 5' stem loop adapter having undergone renaturation treatment, wherein the structure of the 5' stem loop adapter is an overhang-a random base region-a first stem region-a loop region-a second stem region, and the first stem region and the second stem region form a double strand by means of renaturation; and step 4) carrying out amplification on a ligated product. The amplified product constitutes a single-stranded DNA sequencing library.

The present invention provides a recombinant plasmid which includes: (a) an adeno-associated virus (AAV) genome that includes two AAV ITR sequences and a target gene sequence flanked by the AAV ITR sequences; and (b) stuffer DNA which comprises a nucleotide sequence that does not include an inverted repeat sequence capable of forming a stem loop.

The present disclosure relates to expressing one or more reprogramming factors utilizing translational enhancing elements in the 5′ and / or 3′ untranslated region (UTR) of synthetic mRNA. The 5′ UTRs include a mini-enhancer sequence and a Kozak sequence whereas 3′ UTR includes a spacer, a stem loop structure, and optionally, a polyadenine tail. The artificial 5′ and 3′ UTRs drive reprogramming factor expression, and in certain examples, do not include modified nucleosides, microRNA sites, or immune-evading factors.

The present invention provides, inter alia, a method for producing synthetic RNA using a self-template method. For example, in some embodiments, generating synthetic gRNA comprises contacting a first RNA with a second RNA, where the first RNA and the second RNA comprise at least five complementary RNA nucleotides, and where the contacting forms a stem structure or a stem-loop structure; and ligating the first RNA and the second RNA (i) within the stem structure or (ii) at the end of the stem structure with a ligase, thereby forming a loop at the end of the stem structure.

Immunostimulatory molecules and methods of use thereof are provided. Compositions include at least one immunostimulatory RNA molecule having a stem loop containing one or more AAAA adenine rich regions and / or one or more CAA motif regions. Methods include administering said compositions to a subject in need thereof for treating viral infection and / or increasing IFN expression.

The application belongs to the technical field of live cell imaging, and particularly relates to a live cell imaging system based on CRISPR-dCas12a and fluorescent RNA aptamer and application thereof. The application discloses a CRISPR LiteColor system, which mainly comprises CRISPR-dCas12a and crRNA fused with fluorescent RNA aptamer. When dCas12a and the modified crRNA are cooperatively targeted to a target site of a genome, and a corresponding small molecule fluorescent dye is added, the visualization imaging of the target gene site can be realized. The system embeds the fluorescent RNA aptamer into the stem loop structure region of the crRNA, and only relies on a single crRNA to target and recognize the specific site of the TTTV type PAM sequence in the genome, has the characteristics of autonomous degradation and low cell transfection load, and greatly simplifies the experimental operation process on the basis of ensuring high specificity of targeted recognition.

The invention provides a detection method for absolute quantification of engineering modified exosome shRNA (short hairpin Ribonucleic Acid), and relates to a method for detecting the expression quantity of shRNA loaded by the engineering modified exosome. According to the method, aiming at the current technical situation that quantitative detection of shRNA in engineered exosomes lacks a standardized scheme, an absolute quantitative system based on combination of stem-loop reverse transcription and a standard substance is established, and the method has relatively high quantitative accuracy and repeatability. The method not only can be used for evaluating the RNA loading efficiency under different engineering loading strategies, but also can be used as a key quality attribute of an engineering exosome product, and is used for batch consistency evaluation and quality control in a large-scale preparation process. Meanwhile, the method can be used as an important reference for determining the in-vivo administration dosage and carrying out pharmacokinetics and safety evaluation.

A C9orf72 DNA repeat expansion can be detected using a CRISPR Arrayed Repeat Detection System (CARDS). Based upon the compositions and methods supporting this platform primary cell cultures and / or blood cell smears can be tested under conventional clinical diagnostic laboratory conditions to diagnose genetically-based diseases having DNA repeat expansions, including but not limited to ALS. dCas9 constructs are also contemplated as having fluorescent proteins bound to any or all stem loop sequences, wherein detection of a plurality of dCas9 constructs having different colored fluorescent proteins can simultaneously detect at least six (6) different gene target loci.

The present invention provides, among other things, linear mRNA compositions comprising a poly A tail and a stem loop structure downstream to the poly A tail. Also provided herein are methods of producing mRNA by ligating a stem loop exonuclease blocker using a double-stranded RNA ligase.

The invention discloses a hot spot self-assembly colorimetric-Raman sensing platform and an escherichia coli detection method, the hot spot self-assembly colorimetric-Raman sensing platform comprises an isothermal amplification system, a CRISPR-dCas9 system, GNPs-probe and SA-GNPs, a target bacterial gene is taken as a DNA template, RPA amplification is carried out through the isothermal amplification system, and a double-chain amplification product modified by terminal biotin is obtained; the CRISPR-dCas9 system is used for targeted recognition of a double-chain amplification product modified by terminal biotin to form a composite product; gNPs-probe is composed of gold nanoparticles, a Raman signal molecule and a DNA probe, and the GNPs-probe is combined with the stem-loop structure of the sgRNA of the composite product through the DNA probe; sA-GNPs is combined with the biotin of the composite product through streptavidin; gNPs-probe and SA-GNPs are assembled on the composite product, so that a colorimetric-Raman sensing platform for detecting target bacteria is formed. According to the present invention, the cross validation of the generated colorimetric, ultraviolet and Raman multi-mode signals is adopted to improve the result reliability, the isothermal amplification and the self-assembly hot spot are adopted to enhance the multiple sensibilization detection signal, and the specificity is enhanced based on the specific primer group and the CRISPR-dCas9 system mediated secondary recognition, such that the rapid and accurate detection of the bacteria in the actual sample is finally achieved.

The application discloses application of serum tsRNA in preparation of a non-small cell lung cancer early diagnosis or detection reagent, and is characterized in that the serum tsRNA is tsRNA-Asp-5-0013 sequence, the stem loop structure reverse transcription primer sequence is CACTCAACAGACCTGCACGAGCACGAC AGTCACAAGAGGTGCAGGTCTGT, the fluorescence quantitative PCR specific amplification upstream primer is GAGAACACTGACAGCACGAG, and the downstream primer is CCTTCCTCGTTAGTATAGT. The application also provides application of the serum tsRNA in preparation of non-small cell lung cancer and benign tumor, tumor size, non-small cell lung cancer N0 and N1-3 stage, M0 and M1 stage and TNM stage diagnosis, and has high specificity and sensitivity.

The invention relates to the technical field of biological medicine, in particular to a nucleic acid aptamer for ricin and / or abrus precatorius toxin and application of the nucleic acid aptamer. Based on a toxicity damage mechanism of ricin and abrus precatorius toxin, starting from reasonable drug design, a nucleic acid chemical modification strategy is adopted, a nucleic acid aptamer with a stable stem-loop conformation is designed, and the adopted sequence design and modification core strategy comprises but is not limited to locked nucleic acid modification of a conserved region GAGA; a stable conformation design (a plurality of CG units) of a'stem 'region on both sides of the'loop'; performing sulfo-modification on a sequence skeleton; the tail end of the sequence is modified by chemical functional groups (such as cholesterol and biotin). The nucleic acid aptamer provided by the invention has good in-vivo and in-vitro pharmacodynamic activity, and can realize remarkable animal protection rate and prolonged survival time under lethal dose.