Aspects of the invention include methods for preparing sequencing libraries comprising a plurality of RNA molecules. In some embodiments, the subject methods comprise sequencing at least a portion of a sequencing library to obtain sequencing data or sequence reads from a test sample (e.g., a biological sample from a subject).

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 present invention provides, among other things, circular guide RNA (cgRNA) compositions and methods for making same. For example, in some aspects, a method of making egRN A is provided comprising enzymatically ligating two ends of a linear guide RNA, creating a cgRNA. In some aspects, a. method is provided for making cgRNA comprising contacting a linear guide RNA with a ligating enzyme, wherein the contacting brings together a first end and a second end of the guide RN A. and wherein the ligating enzyme ligates the first and the second end of the guide RNA thus creating a cgRNA. In some aspects, circularization is carried out by self-splicing introns. In some aspects, provided are circular RNA compositions, for example, circular messenger RNA, and methods for making same.

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 present disclosure provides engineered RNA ligases, recombinant polynucleotides encoding the engineered RNA ligases, and compositions of the engineered RNA ligases. The present disclosure further provides uses of the engineered RNA ligases for ligation of polynucleotide substrates.

The present disclosure provides a complex for synthesizing one or more nucleotide strands of interest and a method for synthesizing one or more nucleotide strands of interest. The complex for synthesizing one or more nucleotide strands of interest includes nucleotide fragments and a double-stranded oligonucleotide ligase, wherein the nucleotide fragments are hybridized to form a double-stranded oligonucleotide; the double-stranded oligonucleotide ligase is a ligase capable of sealing nicks in the double-stranded oligonucleotide; and at least one strand of the double-stranded oligonucleotide is a nucleotide strand of interest; and the nucleotide fragments include a first fragment and a second fragment, the first fragment contains a 5-phosphate group, and the first fragment is synthesized in a 3- to 5-direction; and the second fragment contains a 3-hydroxyl group, and the second fragment is synthesized in a 5- to 3-direction.

The present disclosure relates to engineered RNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA ligase polypeptides. The present disclosure also provides methods of using the engineered RNA ligase polypeptides or compositions thereof for molecular biological, diagnostic, and other purposes.

The present disclosure provides a complex for synthesizing one or more nucleotide strands of interest and a method for synthesizing one or more nucleotide strands of interest. The complex for synthesizing one or more nucleotide strands of interest includes nucleotide fragments and a double-stranded oligonucleotide ligase, wherein the nucleotide fragments are hybridized to form a double-stranded oligonucleotide; the double-stranded oligonucleotide ligase is a ligase capable of sealing nicks in the double-stranded oligonucleotide; and at least one strand of the double-stranded oligonucleotide is a nucleotide strand of interest; and the nucleotide fragments include a first fragment and a second fragment, the first fragment contains a 5-phosphate group, and the first fragment is synthesized in a 3- to 5-direction; and the second fragment contains a 3-hydroxyl group, and the second fragment is synthesized in a 5- to 3-direction.

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.

Disclosed herein, inter alia, are compositions and methods of use thereof for amplifying polynucleotides within cells and tissues.