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Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an ‘active helicase,’ capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

The genome of SARS-CoV-2 encodes for a helicase called nsp13 that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds short DNA duplexes. Our data confirm that nsp13 uses the inchworm mechanism to move along the DNA in single-nucleotide steps, translocating at ~1000 nt/s or unwinding at ~100 bp/s. Nanopore tweezers’ high spatio-temporal resolution enables observation of the fundamental physical steps taken by nsp13 even as it translocates at speeds in excess of 1000 nucleotides per second enabling detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13’s motion in the presence of the ATPase inhibitor ATPγS. Our data reveals that ATPγS interferes with nsp13’s action by affecting several different kinetic processes. The dominant mechanism of inhibition differs depending on the application of assisting force. These advances demonstrate that nanopore tweezers are a powerful method for studying viral helicase mechanism and inhibition.

We used single-molecule picometer-resolution nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of superfamily 1 helicase PcrA as it translocates on, or unwinds, several kilobase-long DNA molecules. We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo , such as when the helicase encounters bound proteins or duplex DNA. We show how PcrA’s kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base pairs being unwound. Instead, the sequence of the single-stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

Helicases are essential for nearly all nucleic acid processes across the tree of life. Using Nanopore Tweezers we observed the small, fast steps taken by single RecQ helicases as they step along and unwind DNA at ultrahigh spatiotemporal resolution. By directly measuring conformational substates of RecQ we determine the coupling between helicase domain motions and chemical reactions that together produce forward motion along the DNA. Application of assisting and opposing forces shows that RecQ has a highly asymmetric energy landscape that reduces its sensitivity to opposing mechanical forces that could be encountered in vivo by molecular roadblocks such as DNA bound proteins. This energy landscape enables RecQ to maintain speed against an opposing load.

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications, as well as for the engineering of semi-synthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here, we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with non-canonical amino acids.