Methods For Adding Polymers Of Modified Nucleotides To Natural RNAs

Background

When this invention was originally conceived, there were no systems available for sequencing the entire length of an RNA transcript. Next Generation Sequencing (exemplified by the Illumina® sequencing platform) only allows sequencing of short (70-150 base reads) and only cDNA templates.

 Direct, long read RNA sequencing, (exemplified by nanopore sequences produced by Oxford Nanopore Technologies) can sequence an entire polyadenylated transcript (when a poly-T adapter is included), but not a non-polyadenylated transcript - it misses the last nucleic acids. It is also unable to accurately determine the length of the poly-A region of the polyadenylated transcript. 

Researchers at UC Santa Cruz developed a system that solves this problem. The entire length of a natural RNA can now be sequenced, unlocking new insights into RNA expression and variability.  

Technology Description

 In one application, modified nucleotides can be added to the end of the poly-A tail of a eukaryotic mRNA and the length of the poly-A tail readily determined. 

Sequences prior to the junction indicate the natural end of the original RNA. This further enables quantification of  the numbers of RNAs in a sample with a given 3' end structure.

Non-canonical nucleotides are added by a polynucleotide-3' nucleotidyl transferase and can be any non-canonical nucleotide that elicits a strong signal in nanopore sequencing. 

Applications

Sequencing the natural 3' ends of RNAs by nanopore sequencing

Determining the length of an RNA poly-A tail by nanopore sequencing

Measuring specific RNA concentration in a sample

Advantages

Only method that results in accurately identifying the natural sequence of the entire 3' end of an RNA - including the length of the poly-A tail in a eukaryotic mRNA. 

Non-canonical nucleic acids clearly signal of the end of the natural sequence.  

Intellectual Property Information

Related Materials

• Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: application to direct RNA sequencing on nanopores - 08/26/2021