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A Versatile and High-throughput Assay for the Directed Evolution of Reverse Transcriptases to Profile Epigenetic Modifications With NGS

Interests: Tangible Property
Published:
Lead Inventor: Bryan Dickinson
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SUMMARY

  • Engineered reverse transcriptases (RTs) to profile epigenetic modifications have numerous advantages over traditional antibody enrichment methods including: single base pair resolution, lower sample quantities needed, and lower noise and background interference. Problematically, current RT engineering strategies are cumbersome, low throughput, and cannot be versatilely applied to multiple types of epigenetic modifications.
  • The inventors developed a fluorescence-based assay in which a mutation at a specific base pair in an RNA transcript results in the induction of fluorescence. By strategically positioning an epigenetic modification at that base pair, the assay can be used to screen for RTs that induce mutations in the presence of a specific modification.
  • The invention is a high throughput fluorescence-based assay to screen libraries of RTs for the directed evolution of enzymes that profile a desired epigenetic modification. The assay is high throughput and can be run in 384 well plates on unpurified cellular lysates. 
  • In a proof of concept experiment, 3 rounds of selection were performed on an HIV-1 RT library in 384 well plates for enzymes that identify N1-methyladenosine (m1A) epigenetic modifications. The lead enzyme was identified based off a 40-fold increase in fluorescence as compared to background. This lead had 80% read through efficiency (as compared to 40% for the wild type), and a 60% mutation rate at the desired location (as compared to 36% for the wild type).

 

FIGURE

Schematic overview of the assay with red X denoting the positioning of the epigenetic modification to be profiled. (1) The RNA probe is converted to cDNA with the given reverse transcriptase from the library. (2) PCR amplification is performed on the cDNA template. (3) The PCR product is transcribed back to RNA. If the desired mutation occurs at the site of the epigenetic modification in the original template, the RNA fluoresces. Fluorescence can be quantified to select the most promising RTs to carry over to the next round of screening.

 

 

ADVANTAGES

ADVANTAGES

  • Standardized assay for multiple epigenetic modifications
  • Does not require enzyme purification
  • High throughput (384 well plates)
  • Low background and noise 

 

APPLICATIONS

  • Enzyme engineering and directed evolution
  • NGS profiling of epigenetic modifications

 

PUBLICATIONS

 

 

 

 

 

  • Screening of HIV-1 RT library to identify mutants that detect m1A modifications
  • Validation and characterization of resulting lead RT library hit