High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments

Single molecule biophysics experiments have enabled the observation of biomolecules with a great deal of precision in space and time, e.g. nucleic acids mechanical properties and protein–nucleic acids interactions using force and torque spectroscopy techniques. The success of these experiments strongly depends on the capacity of the researcher to design and fabricate complex nucleic acid structures, as the outcome and the yield of the experiment also strongly depend on the high quality and purity of the final construct. Though the molecular biology techniques involved are well known, the fabrication of nucleic acid constructs for single molecule experiments still remains a difficult task. Here, we present new protocols to generate high quality coilable double-stranded DNA and RNA, as well as DNA and RNA hairpins with ∼500–1000 bp long stems. Importantly, we present a new approach based on single-stranded DNA (ssDNA) annealing and we use magnetic tweezers to show that this approach simplifies the fabrication of complex DNA constructs, such as hairpins, and converts more efficiently the input DNA into construct than the standard PCR-digestion-ligation approach. The protocols we describe here enable the design of a large range of nucleic acid construct for single molecule biophysics experiments.

Single-Molecule Analysis with Solid-State Nanopores

Solid-state nanopores and nanopipettes are an exciting class of single-molecule sensors that has grown enormously over the last two decades. They offer a platform for testing fundamental concepts of stochasticity and transport at the nanoscale, for studying single-molecule biophysics and, increasingly, also for new analytical applications and in biomedical sensing. This review covers some fundamental aspects underpinning sensor operation and transport and, at the same time, it aims to put these into context as an analytical technique. It highlights new and recent developments and discusses some of the challenges lying ahead.