Showing papers by "Jens H. Gundlach published in 2012"

Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Abstract: Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.

Torsion-balance tests of the weak equivalence principle

Abstract: We briefly summarize motivations for testing the weak equivalence principle and then review recent torsion-balance results that compare the differential accelerations of beryllium–aluminum and beryllium–titanium test-body pairs with precisions at the part in 1013 level. We discuss some implications of these results for the gravitational properties of antimatter and dark matter and speculate about the prospects for further improvements in experimental sensitivity.

Molecular Dynamics Study of MspA Arginine Mutants Predicts Slow DNA Translocations and Ion Current Blockades Indicative of DNA Sequence

Abstract: The protein nanopore Mycobacteria smegmatis porin A (MspA), can be used to sense individual nucleotides within DNA, potentially enabling a technique known as nanopore sequencing. In this technique, single-stranded DNA electrophoretically moves through the nanopore and results in an ionic current that is nucleotide-specific. However, with a high transport velocity of the DNA within the nanopore, the ionic current cannot be used to distinguish signals within noise. Through extensive (∼100 μs in total) all-atom molecular dynamics simulations, we examine the effect of positively charged residues on DNA translocation rate and the ionic current blockades in MspA. Simulation of several arginine mutations show a ∼10–30 fold reduction of DNA translocation speed without eliminating the nucleotide induced current blockages. Comparison of our results with similar engineering efforts on a different nanopore (α-hemolysin) reveals a nontrivial effect of nanopore geometry on the ionic current blockades in mutant nanopores.

Torsion-balance tests of the weak equivalence principle

Abstract: We briefly summarize motivations for testing the weak equivalence principle and then review recent torsion-balance results that compare the differential accelerations of beryllium-aluminum and beryllium-titanium test body pairs with precisions at the part in $10^{13}$ level. We discuss some implications of these results for the gravitational properties of antimatter and dark matter, and speculate about the prospects for further improvements in experimental sensitivity.

MspA Nanopores from Subunit Dimers

Abstract: Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.