StraDiVari-RT for Improved Transcriptome Analysis in Fixed Samples

Background & Unmet Need

• Recent advancements in spatial and single-cell transcriptomics enable transcriptome profiling in degraded RNA samples using paired oligonucleotide probes that hybridize to targeted transcripts
• Current probe-based methods lose original RNA sequence information including mutations during ligation and sequencing, limiting genetic variant detection in fixed samples
• Existing approaches using non-contiguous probes with gaps are limited by reverse transcriptases (RT) that possess high strand displacement activity
• Standard RT enzymes continue synthesizing past the second probe during gap-filling, displacing downstream probes and preventing proper capture of original RNA sequences
• Unmet Need: A method that enables accurate capture of original RNA sequences and mutations in spatial and single-cell transcriptomic analysis of formalin-fixed samples

Technology Overview

• The Technology: StraDiVari-RT (Strand Displacement Variant Reverse Transcriptase) is an engineered enzyme that enables incorporation of original RNA sequences into locus-specific probe sets
• The enzyme contains engineered mutations that abolish strand displacement activity and eliminate RNase H activity to preserve target integrity
• PoC Data: StraDiVari-RT successfully resolves admixtures of wild-type and mutant cells at single-cell resolution in both fixed cell lines and primary samples from AML patients 
• Compatibility with existing single-cell RNA-seq workflows was demonstrated through a cell line mixing experiment
• Probes targeting GAPDH and CTCF, with a 9-nucleotide space between probes, efficiently filled and ligated, enabling precise genotyping of these sequences in single cells

Technology Applications

• Research tool to detect mutations in tumor samples and understand their effects on cellular behavior
• Develop more precise diagnostic methods that account for genetic variations at the single-cell level
• Identifying cellular responses to drug treatments at a granular level, facilitating targeted therapy development

Technology Advantages

• Seamless integration with commercially available probe-based single-cell and spatial transcriptomics products
• Precise identification of mutations within RNA sequences, including small deletions or duplications
• Compatible with fixed samples