In Situ Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging.

Abstract

Fluorescence signal enhancement via isothermal nucleic acid amplification is an important approach for sensitive imaging of intra- or extracellular nucleic acid or protein biomarkers. Rolling circle amplification (RCA) is frequently applied for fluorescence in situ imaging but faces limitations concerning multiplexing, dynamic range, and the required multiple washing steps before imaging. Here, we show that Förster resonance energy transfer (FRET) between fluorescent dyes and between lanthanide (Ln) complexes and dyes that hybridize to β-actin-specific RCA products in HaCaT cells can afford washing-free imaging of single β-actin proteins. Proximity-dependent FRET could be monitored directly after or during (real-time monitoring) dye or Ln DNA probe incubation and could efficiently distinguish between photoluminescence from β-actin-specific RCA and DNA probes freely diffusing in solution or nonspecifically attached to cells. Moreover, time-gated FRET imaging with the Ln-dye FRET pairs efficiently suppressed sample autofluorescence and improved the signal-to-background ratio. Our results present an important proof of concept of RCA-FRET imaging with a strong potential to advance in situ RCA toward easier sample preparation, higher-order multiplexing, autofluorescence-free detection, and increased dynamic range by real-time monitoring of in situ RCA.