Trna Handles

Tech ID: 33259 / UC Case 2014-725-0

Background

tRNA is notoriously difficult to manipulate . Sequencing of tRNA presents various problems because of complex and tightly bound secondary structures and associated proteins. Current methods of tRNA analysis include RNA sequencing, microarray analysis and mass spectrometry. Each has limitations, however. RNA sequencing requires extensive library preparation and PCR amplification followed by reverse transcription. This results in the loss of the original RNA strand and its secondary structure. Reverse transcription is also impeded by the structural and nucleotide modifications that commonly occur in tRNA's. These so-called RTstops result in truncated cDNAs that do not reflect natural tRNA's.

tRNA can be sequenced with nanopore sequencers, so long as they can be unfolded and electrically attracted to the nanopore. So a mechanism to capture tRNA molecules, unfold them, and initiate threading them into a nanopore is needed.

Technology Description

The invention includes attaching DNA or RNA "handles" to a tRNA molecule. These handles allow manipulation of the tRNA molecule, including unfolding its structure and acting as targets for attaching other molecules to the tRNA.

One example is a double stranded oligonucleotide adapter that can be ligated to a tRNA from a biological sample. Such an adepter can be a Y shaped double stranded DNA-RNA adapter with a 3' RNA overhang complementary to the CCA tail present in tRNA. The adapter can also include a cholesterol tag within its 3' end.

tRNA Handle 

 

Examples of sequences used in oligonucleotide adaptors include:

CTCACCTATCCTTCCACXCATACTATCATTATCTXTCAGATCTCACTAUCUGGU

GATXGTGAGATCTGATTTTTTTTTTTTTTTZ

GATAGTGAGATCTGATTTTTTTTTTTTTTTZ

CTCACCTATCCTTCCACTCATACTATCATTATCTCTCAGATCTCACTAUCUGGU

GATXGTGAGATCTGATTTTTTTTTTTTTTTZ

CTCACCTATCCTTCCACTCATACTATCATTATCTCTCAGATCTCACTAUCUGGU

X indicates an abasic 1'2' dideoxyribose; Z indicates a triethylene glycol cholesterol

Applications

  • Identification of tRNA 
  • Sequencing of tRNA 
  • Nanopore sequencing of tRNA 

 

Advantages

  • Molecular adaptor that facilitates tRNA seqeuncing by nanopore
  • Rapid identification of tRNA species in a biological sample

Intellectual Property Information

Country Type Number Dated Case
United States Of America Issued Patent 10,131,944 11/20/2018 2014-725
Patent Cooperation Treaty Published Application WO 2015/148567 10/01/2015 2014-725
 

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Inventors

  • Bernick, David L.
  • Smith, Andrew

Other Information

Keywords

tRNA sequencing, sequencing adapter, tRNA, nanopore sequencing, RNA, RNAseq, long read sequencing

Categorized As

Related cases

2014-725-0

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