Abstract
The inhibition of micro RNA (miRNA) maturation by Dicer and loading matured miRNAs into the RNA‐induced silencing complex (RISC) is envisioned as a modality for treatment of cancer. Existing methods for evaluating maturation either focus on the conversion of modified precursors or detect mature miRNA. Whereas the former is not applicable to native pre‐miRNA, the latter approach underestimates maturation when both nonmatured and matured miRNA molecules are subject to cleavage. We present a set of two orthogonally labelled FIT PNA probes that distinguish between cleaved pre‐miRNA and the mature miRNA duplex. The probes allow Dicer‐mediated miR21 maturation to be monitored and Ago2‐mediated unwinding of the miR21 duplex to be assayed. A two‐channel fluorescence readout enables measurement in real‐time without the need for specialized instrumentation or further enzyme mediated amplification.
Highlights
Introduction dling stepsModern detection methods take advantage of isothermal amplification,[9] which allow measurements in homogenous phase
The design of a probe that responds to the formation of the micro-RNA 21 (miR21)-5p*miR21-3p duplex seemed more challenging given that the mature sequence is fully presented in both the pre-miR21 and the matured structures
We envisioned that opening of the double-stranded region by strand invading PNA that is complementary to the C46-C56 segment would be easier for the miR21-5p*miR21-3p duplex than for the stem region of premiR21
Summary
Modern detection methods take advantage of isothermal amplification,[9] which allow measurements in homogenous phase. The (bio)chemical steps leading to amplification prolong the assay time and it is, not feasible to monitor miRNA maturation in or near real time. An ideal assay should i) allow sensitive detection with standard equipment, ii) proceed in solution phase to enable rapid measurements near real time, iii) not require enzymes to allow – if required – the use of detergents and solvents and – if desired – enable monitoring within living cells and iv) allow simultaneous detection of both precursor and matured miRNA. A pre-miRNA was labelled with fluorescence donor and acceptor dyes.[10]. The probe showed increased donor emission upon Dicer-mediated cleavage
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