High-SensitivityFluorescence-Based Detection of ReverseTranscriptase 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 <i>in vitro</i> preparation of r(CGG)₂₉ and r(G₄C₂)₁₅ RNAs, which had repeat numbers relevant to the human <i>FMR1</i> and <i>C9ORF72</i> 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 <i>in vitro</i> 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.