Henry Brinkerhoff

Decoding long nanopore sequencing reads of natural DNA

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.

Significance Cells attach a methyl group (—CH3) to certain cytosines in DNA to control gene expression. These methylation patterns change over time and can be related to cell differentiation and diseases such as cancer. Existing methylation detection techniques are not ideal for clinical use. We pulled single-stranded DNA molecules through the biological pore MspA and found that ion currents passing through the pore reveal the methylation sites with high confidence. Hydroxymethylation, which differs from methylation by only one oxygen atom, also produces distinct signals. This technique can be developed into a research tool and may ultimately lead to clinical tests. Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.

Subangstrom single-molecule measurements of motor proteins using a nanopore

Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.

Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore

Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and Hel308 helicase are capable of controlling the motion of DNA containing dNaM and d5SICS through the pore and that single reads are sufficient to detect the presence and location of dNaM and d5SICS within single molecules.

Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers

Significance DNA helicases are enzymes that use energy from ATP hydrolysis to move along nucleic acid tracks and unwind double-stranded DNA. Helicases are involved in every aspect of DNA metabolism and are therefore vital to maintaining genomic integrity. Using the single-molecule technique single-molecule picometer-resolution nanopore tweezers (SPRNT), which measures the position of DNA through the biological membrane protein MspA as an enzyme moves along the DNA, we monitored the kinetics of the helicase Hel308 at 1,000 times better temporal resolution than was previously possible. We derived a detailed mechanism for how ATP hydrolysis coordinates the motion of Hel308 along single-stranded DNA that can likely be applied to other structurally similar helicases and showed that the DNA sequence in Hel308 affects its kinetics. Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.

Direct Single Molecule Measurement of ATP Hydrolysis Substates in Hel308 DNA Helicase using Nanopore Tweezers

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single molecule tool for studying enzymes that move on nucleic acids (NA). SPRNT measures NA position relative to the enzyme at sub-Angstrom spatial resolution on millisecond timescales, while simultaneously providing the NA sequence near the enzyme. We use this method to resolve two substates of the ATP hydrolysis cycle of a Hel308 DNA Helicase. By examining the dwell times of each state, we derive a kinetic model of Hel308s translocation along single-stranded DNA, and find that the models rate constants depend on the DNA sequence within the enzyme. This is, to our knowledge, the first evidence of sequence-dependent translocase behavior in a helicase system.

Continuously Scanning DNA with Nanopore MspA

Nanopore sequencing is a promising next-generation technology that is being enabled by the biological nanopore MspA. In this sequencing technique, current is driven through the ∼1 nm constriction of MspA. Single stranded DNA molecules are first drawn into the pore by an applied voltage. Next, a polymerase or helicase moves the DNA by discrete steps as it interacts with the pore. As the DNA moves through the pore, different nucleobases modulate the pores conductance to varying extents, allowing for extraction of sequence information from the current trace. In this experiment, we gain more complete information by using a variable applied voltage to stretch the DNA within the pore. Changing the voltage continuously repositions the DNA in the constriction. Coupling this voltage-induced movement with the enzyme-induced motion we reconstruct a profile characteristic of the DNA as it is pulled continuously through the constriction. This profile provides a tool for improving the de novo sequencing accuracy of the nanopore technique.