Measuring hybridization kinetics in live cells using a single-molecule imaging and tracking method.

Abstract

The performance of many biosensors relies on effective hybridization of the nucleic acid probe with its complementary target, but the binding kinetics of probe-target pairs are often difficult to predict or measure in live cells. Current methods for studying annealing/melting kinetics of nucleic acids (NAs) mostly rely on in-vitro models that do not reflect the molecular constituents of native cellular environment. Here by using a transmembrane receptor labeled with SNAPf-tag and a DNA probe dually conjugated with benzylguanine (BG) and fluorophore Atto633 at the two ends, we measured the annealing/melting kinetics of a probe-target model system in live cells on a microscope. Upon electroporation, the BG-DNA-Atto633 probes were covalently linked to the SNAPf tags on receptors, thus anchored on the intracellular surfaces of plasma membrane. With Cy3-labeled complementary strands also in the cytoplasm, transient nucleic acid hybridization and melting between the two strands were observed through colocalization analysis at the single-molecule level. kon, koff and Kd were derived from the fluorescence time traces of Atto633 and Cy3 using the hidden Markov model (HMM). Although the observation depth (within 200 nm from the cell membrane) is much limited compared to that of the 3D tracking techniques (30 μm), hundreds of molecules can be tracked simultaneously and the resulting track durations are extended from a few hundreds of milliseconds to a few seconds, thus leading to more precise kinetics measurements. Our method provides a simple, direct and precise way to evaluate nucleic acid binding kinetics in cytoplasm. The acquired kinetics information is important for designing small nucleic acid probes or oligonucleotide drugs that bind with their specific targets effectively in live cells.