Using PNA and LNA as Handles for Tethering Single DNA Molecules

Abstract

Single-molecule manipulation (SMM) instruments, such as the optical trap or magnetic tweezer, require a means to immobilize the studied biomolecule on a solid substrate. In the case of DNA, this is accomplished by adding moieties, e.g. biotin, using enzymatic labeling; however, this strategy introduces extra steps in the processing of the sample, and makes direct application of SMM to DNA/protein interactions and chromatin structure difficult. Here, we develop a novel tethering strategy based on the properties of the nucleic acid analogs (NAAs) peptide nucleic acid (PNA) and locked nucleic acid (LNA). PNA and LNA are known to stably bind to double-stranded DNA in a sequence-specific manner, either through triplex formation or strand-invasion. Using a magnetic tweezer, we explore the ability of biotinylated NAAs to immobilize a DNA molecule in a sequence-specific fashion, and to remain bound under applied force. Our results indicate that both LNA and PNA can tether a DNA molecule and withstand mechanical force, but that PNA suffers from non-specific binding, particularly to DNA extremities. We discuss rules for the optimal design of NAA probes for single-molecule experiments.