Jens H. Gundlach

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

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 pores 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.

Tests of the Gravitational Inverse-Square Law below the Dark-Energy Length Scale

We conducted three torsion-balance experiments to test the gravitational inverse-square law at separations between 9.53 mm and 55 microm, probing distances less than the dark-energy length scale lambda(d)=[4 -root](variant Plancks over 2pic/rho(d) approximately 85 microm. We find with 95% confidence that the inverse-square law holds (|alpha|<or=1) down to a length scale lambda=56 microm and that an extra dimension must have a size R<or=44 microm.

Nanopore DNA sequencing with MspA

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

Single-molecule DNA detection with an engineered MspA protein nanopore

Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of ≈20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule ≈100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.

Submillimeter Test of the Gravitational Inverse-Square Law: A Search for “Large” Extra Dimensions

Motivated by higher-dimensional theories that predict new effects, we tested the gravitational 1/r(2) law at separations ranging down to 218 microm using a 10-fold symmetric torsion pendulum and a rotating 10-fold symmetric attractor. We improved previous short-range constraints by up to a factor of 1000 and find no deviations from Newtonian physics.

Test of the equivalence principle using a rotating torsion balance.

We used a continuously rotating torsion balance instrument to measure the acceleration difference of beryllium and titanium test bodies towards sources at a variety of distances. Our result Deltaa(N),(Be-Ti)=(0.6+/-3.1)x10(-15) m/s2 improves limits on equivalence-principle violations with ranges from 1 m to infinity by an order of magnitude. The Eötvös parameter is eta(Earth,Be-Ti)=(0.3+/-1.8)x10(-13). By analyzing our data for accelerations towards the center of the Milky Way we find equal attractions of Be and Ti towards galactic dark matter, yielding eta(DM,Be-Ti)=(-4+/-7)x10(-5). Space-fixed differential accelerations in any direction are limited to less than 8.8x10(-15) m/s2 with 95% confidence.

Sub-millimeter tests of the gravitational inverse-square law

Motivated by a variety of theories that predict new effects, we tested the gravitational

Decoding long nanopore sequencing reads of natural DNA

{1/r}^{2}

Measurement of Newton's constant using a torsion balance with angular acceleration feedback.

law at separations between 10.77 mm and

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

137\ensuremath{\mu}\mathrm{m}