One-step Discrimination Of BCR/ABL^p210 Transcript Isoform With Fusion-triggered Rolling Circle Amplification

BCR/ABL~p210210 fusion gene,the characteristic biomarker of chronic myelogenous leukemia(CML),contains two different transcription isoforms,e13a2 and e14a2,which lead to differences in the pathological features and response to targeted drug.At present,there is short of simple and fast technology to distinguish these two transcript isoforms.In this paper,RNA fusion-triggered rolling circle amplification(RF-RCA)strategy was developed to distinguish BCR/ABL~p210210 transcripts directly from RNA extraction in one step.The simultaneous binding of dumbbell template and corresponding primer with target fused RNA can induce their proximal hybridization and trigger the RCA to produce lots of tandem repeat G-quadruplexes sequences for real time fluorescence readout with the interaction of Thioflavin T and G-quadruplex.The proposed strategy can detect as low as 0.1 aM target and discriminate e13a2(0.01%)and e14a2(0.1%)transcript isoforms directly from complex genomic RNA extraction,proving high sensitivity and specificity.Furthermore,the RF-RCA was successfully applied to analyze BCR/ABL ~p210210 isoforms from clinica samples for accurately molecular subtyping and monitoring the response of imatinib treatment.The developed RF-RCA strategy presented an ultrasensitive,accurate and pragmatic toolbox to simple and rapid discriminate BCR/ABL~p210210 fusion isoforms for promoting clinical research and personalized treatment of CML.