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MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Förster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.

Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26–30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

Optical and magnetic tweezers are widely employed to probe the mechanics and activity of individual biomolecular complexes. They rely on micrometre-sized particles to detect molecular conformational changes from the particle position. Real-time particle tracking with Ångström accuracy has so far been only achieved using laser detection through photodiodes. Here we demonstrate that camera-based imaging can provide a similar performance for all three dimensions. Particle imaging at kHz rates is combined, with real-time data processing being accelerated by a graphics-processing unit. For particles that are fixed in the sample cell we can detect 3-Å-sized steps that are introduced by cell translations at rates of 10 Hz, while for DNA-tethered particles 5 Å steps at 1 Hz can be resolved. Moreover, 20 particles can be tracked in parallel with comparable accuracy. Our approach provides a simple and robust way for high-resolution tweezer experiments using multiple particles at a time.