University of California

Manuel Ares, Jr

Mark Albert Akeson

Logan Mulroney

Jenny Vo

Ethan E Ford

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 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.

A prototype RNA cyclase ribozyme that allows efficient production of circular RNA. Methods for modifying the prototype to produce a wide variety of custom circular RNA are detailed. The method utilizes a new plasmid which enables production of a wide variety of imaginable RNA sequences in a covalent, circular form free from intron sequences in vitro. At a particular site in the plasmid, a sequence coding for the desired circular RNA is inserted to create a new RNA cyclase ribozyme gene. RNA transcribed from RNA cyclase ribozyme genes autocatalytically converts the desired RNA sequence it contains into circular form. RNA cyclase genes may be placed into appropriate expression vectors for synthesis of circular RNA in vivo as part of ribozyme or antisense gene regulation approaches to genetic engineering.

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Santa Cruz, CA, United States of America

Manuel Ares, Jr