University of California

Logan Mulroney

Mark Albert Akeson

Manuel Ares, Jr

Jenny Vo

Hugh Edward Olsen

Miten Jain

Provided are methods of adding a polymer of non-canonical nucleotides to the 3' end of a ribonucleic acid (RNA). In certain embodiments, the methods comprise combining an RNA, a polynucleotide-3′ nucleotidyl transferase, and non-canonical nucleotides, in a reaction mixture under conditions in which the polynucleotide-3′ nucleotidyl transferase adds a polymer of the non-canonical nucleotides to the 3′ end of the RNA. Such methods may further include analyzing the RNA using a nanopore. According to some embodiments, the methods include identifying the polymer of non-canonical nucleotides added to the 3′ end of the RNA, and determining the junction between the 3′ end of the RNA and the polymer of non-canonical nucleotides to identify the 3′ end of the RNA. Kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

Methods of adding polymers to ribonucleic acids

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.

Methods of analyzing capped ribonucleic acids

Provided are methods of adding a polymer of non-canonical nucleotides to the 3′ end of a ribonucleic acid (RNA). In certain embodiments, the methods comprise combining an RNA, a polynucleotide-3′ nucleotidyl transferase, and non-canonical nucleotides, in a reaction mixture under conditions in which the polynucleotide-3′ nucleotidyl transferase adds a polymer of the non-canonical nucleotides to the 3′ end of the RNA. Such methods may further include analyzing the RNA using a nanopore. According to some embodiments, the methods include identifying the polymer of non-canonical nucleotides added to the 3′ end of the RNA, and determining the junction between the 3′ end of the RNA and the polymer of non-canonical nucleotides to identify the 3′ end of the RNA. Kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

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Santa Cruz, CA, United States of America

Logan Mulroney