High-Sensitivity Fluorescence-Based Detection of Reverse Transcriptase Read-Through of GC-Rich Short Tandem Repeat RNA

Short tandem repeat (STR) RNAs play a pivotal role in the pathology of STR expansion-associated disorders. However, disease-related STR sequences are often GC-rich (>66% GC), which makes sample preparation and detection challenging. GC-rich STR RNAs, particularly those composed entirely of GC (100% GC), frequently cause interruptions during reverse transcription. Additionally, the GC-rich STR DNA sequences generate low-yield and heterogeneous products when amplified via polymerase chain reaction. The lack of robust processivity of polymerases for GC-only STR poses major challenges in preparing samples and detecting such sequences with physiologically relevant lengths. Herein, we report the in vitro preparation of r(CGG)29 and r(G4C2)15 RNAs, which had repeat numbers relevant to the human FMR1 and C9ORF72 genes, respectively, and achieved high yield and homogeneity of the prepared GC-only STR RNAs. Using the prepared RNAs, a fluorescence-based detection platform is developed that uses reverse transcriptases (RTases) to identify read-through cDNA products with high sensitivity, requiring minimal RNA input. Further, we demonstrate the versatile applications of this detection platform and provide structural insights into the r(CGG)29 and r(G4C2)15 RNAs during RTase processing. The findings of this study will enhance our ability to characterize and target disease-relevant STR RNAs in vitro and pave the way for future efforts in the directed evolution of RTases aimed at improving the detection of endogenous-expanded GC-rich STR RNAs.