Methods For Adding Polymers Of Modified Nucleotides To Natural RNAs

Tech ID: 33040 / UC Case 2019-503-0

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

The addition of a short polymer of non-canonical (or modified) nucleotides (i.e. not found in natural RNA or DNA) results in a strong and unique signal in a nanopore sequencer that clearly indicates the junction between the added modified nucleotides and the natural nucleotides.

 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

Country Type Number Dated Case
United States Of America Published Application 20200377875 12/03/2020 2019-503
 

Additional Patent Pending

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Inventors

  • Akeson, Mark A.
  • Ares Jr, Manny
  • Mulroney, Logan
  • Vo, Jenny

Other Information

Keywords

Nanopore sequencing, RNAseq, Full length RNA sequencing, Determining poly-A length, RNA sequencing

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