The present disclosure relates to methods, compositions and kits for synthesizing moderate length RNAs (mlRNAs, including gRNAs) by splint-mediated ligation of RNA fragments. The synthesis of moderate length RNAs can be followed by DNase treatment. In some embodiments, splint DNA oligonucleotides that are no longer than 32 nucleotides are used.

Ligase mutants of the following (1), (2), or (3): (1) a ligase mutant comprising an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1, and having a nucleic acid-linking activity; (2) a ligase mutant comprising an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and having a nucleic acid-linking activity; or (3) a ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3, and having a nucleic acid-linking activity, have excellent properties.

Provided are a method for large-scale synthesis of a long-chain RNA and a method for site-specific modification of the long-chain RNA. The method for large-scale synthesis of a long-chain RNA comprises: designing short RNA fragments and splint DNA fragments; ligating; capping; and removing the splint DNA fragments and other steps. A large number of short RNA fragments and different splint DNA fragments are chemically synthesized, and then the different short RNA fragments are ligated by a biological method so as to form a target long-chain RNA. The product long-chain RNA has a low mutation rate, a plurality of the short RNA fragments can be assembled in a single reaction, and the long-chain RNA can be synthesized at a high throughput so as to fulfill the large-scale production of the long-chain RNA. In addition, by chemical modification of the short RNA fragments, the site-specific modification of the long-chain RNA can be realized.

The present invention provides, among other things, a method of producing a synthetic RNA using a self-templating approach. For example, in some embodiments a synthetic gRNA is produced comprising: contacting a first RNA with a second RNA, wherein the first RNA and the second RNA comprise at least five RNA nucleotides that are complementary, and wherein the contacting forms a stem structure or a stem loop structure, and ligating the first RNA and the second RNA with a ligating enzyme (i) within the stem structure, or (ii) at an end of the stem structure, thereby forming a loop at the end of the stem structure.

The present invention provides a method for producing a single-stranded RNA, the method comprising:

(I) a step of reacting a first single-stranded RNA having a phosphate group at the 5′-terminal and a second single-stranded RNA having a hydroxy group at the 3′-terminal with an RNA ligase classified into EC 6.5.1.3 determined by International Union of Biochemistry as an Enzyme Commission number and having a nick repair activity in a double-strand to link said first single-stranded RNA to said second single-stranded RNA; and
(II) a step of purifying the reaction product comprising the single-stranded RNA produced in the step (I) by reverse-phase column chromatography using a mobile phase comprising at least one ammonium salt(s) selected from the group consisting of monoalkylammonium salts and dialkylammonium salts.

Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

Provided are methods of analyzing capped ribonucleic acids (RNAs). The methods include translocating an adapted RNA through a nanopore of a nanopore device. The adapted RNA includes an RNA region, a 5′ cap, and an adapter polynucleotide attached to the 5′ cap. The methods include monitoring ionic current through the nanopore during the translocating, translocating the 5′ cap through the nanopore, and identifying one or more ionic current features characteristic of the 5′ cap (e.g., a triphosphate linkage between the 5′ cap and nucleotide N1 of the RNA region, a 5′ to 5′ orientation of the 5′ cap and nucleotide N1 of the RNA region, and/or the like), translocating through the nanopore. Also provided are computer-readable media, computer devices, and systems that find use, e.g., in practicing the methods of the present disclosure.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.