Nanopores map the acid-base properties of a single site in a single DNA molecule.

Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas.

Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase (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 double-stranded DNA. Our data reveal nsp13's single-nucleotide steps, translocating at ∼1000 nt/s or unwinding at ∼100 bp/s. Nanopore tweezers' high spatiotemporal resolution enables 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. We construct a detailed picture of inhibition in which ATPγS has multiple mechanisms of inhibition. The dominant mechanism of inhibition depends on the application of assisting force. This lays the groundwork for future single-molecule inhibition studies with viral helicases.

Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores.

Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such "artificially expanded genetic information systems" are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter "hachimoji" expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatis porin A) nanopore. We find that hachimoji DNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and that hachimoji single-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules along hachimoji DNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible with hachimoji DNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.

Inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

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.