titration, we created an abasic mapping, in 2M and 1M KCl, that details a-he- Download full-text
molysin’s sensitivity with respect to electrical conductance as a homopolymer
with a single abasic traverses through the pore. From our map, we are able to
reveal the smallest (minimal) voltages that can reveal DNA translocation prog-
ress through pore, e.g., during enzyme-catalysis on the pore. These results are
part of preliminary studies that aim to measure hydrolysis of DNA by the Exo-
nuclease I (ExoI) of E. coli on the nanopore. ExoI is catalytically active in both
1 and 2 M KCl. In this research, we present voltages that make it possible to
observe ExoI-catalyzed DNA hydrolysis on the nanopore in 1 and 2 M KCl,
Mechanical Model of Cell Membrane Penetration by Vertical Nanowires
Xi Xie, Nicholas A. Melosh.
Stanford University, Stanford, CA, USA.
For many therapeutic and scientific applications, direct access into a cell’s in-
terior is the key for delivering various biomolecules to alter cell behavior or for
intracellular assays. However, the lipid membrane presents a challenging bar-
rier that prevents biomolecular species from entering the cytosol.
The recent discovery of cell viability despite penetration by vertical nanowires
(NW) has opened new avenues for direct intracellular access. Cells are found to
be impaled onto small diameter nanowires without application of any external
force. Vertical NW arrays have been reported to serve as a universal and effi-
cient platform for introducing RNA, DNA, proteins and peptides into a broad
range of cell types.
However, the cell membrane penetration mechanism by vertical nanowires is
still unknown. Several recent experiments have shown that the penetration ef-
ficiency is greatly reduced with increasing NW diameter. Understanding the
penetration mechanism is the key to optimizing the design of nanowires and
developing advanced devices based on this technique.
In this work a mechanical model is developed to predict cell membrane pene-
tration by vertical nanowires. The tension and strain on cell membranes due to
indentation by nanowire are calculated using a model of axisymmetric defor-
mation of elastic membrane indented by a probe with a hemispherical tip.
The critical membrane rupture conditions are determined under different fail-
ure criteria, either tension or strain. The effects of NW radius, NW aspect ratio
and cell membrane stiffness on penetration are investigated based on the me-
chanical model. Our results provide a practical guide to designing nanowires
for applications in cell membrane penetration.
Detection of Methylated DNA by Modified GP10 Nanopore
Elizabeth Wurtzler1, Murali Venkatesan2, Rashid Bashir2, David Wendell1.
1University of Cincinnati, Cincinnati, OH, USA,2University of Illinois at
Urbana-Champaign, Urbana, IL, USA.
We report the electrophysiology of a biological sensor for the detection of
methylation state changes of DNA characteristic of carcinogenesis. This sensor
is an engineered nanopore composed of a freestanding lipid bilayer containing
the capsid portal protein GP10 and several mutants in a freestanding lipid bi-
layer. The measured conductance is on par with some of the largest biological
nanopores, like those of the mechanosensitive channels and porins found in
many prokaryotes. The variable region and the C-terminal crown both appear
to play a role in restricting conductance. These two areas are known to interact
with the viral DNA but remain unresolved in the crystal structure. We have
used the C-terminal interaction as a basis for distinguishing dsDNA methyla-
tion state by engineering a methylated DNA binding domain onto the crown
of GP10. Of the two methylated DNA binding domains tested, MBD2 and
MeCP2, MeCP2 imparts greater stability to the nanopore possibly due to its in-
creased ability to interact with DNA or other proteins. The engineered pore has
the ability to electrically distinguish methylated and hydroxymethylated DNA
from the unmodified form. We envision this sensor as a future tool for detecting
the alterations in DNA methylation state commonly associated with
Probing the Transport of Ionic Liquids in Aqueous Solution through
Kozhinjampara R. Mahendran, Pratik Raj Singh, Niraj Modi, Robert Schulz,
Ulrich Kleinekatho ¨fer, Mathias Winterhalter.
Jacobs University, Bremen, Germany.
The permeation of water soluble molecules across cell membranes is controlled
by channel forming proteins and particularly the channel surface determines
the selectivity. An adequate method to study properties of these channels is
electrophysiology and in particular analyzing the ion current fluctuation in
the presence of permeating solutes provides information on possible interac-
tions with the channel surface. The temperature-dependent transport of the
ionic liquid 1-butyl-3-methyl-imidazolium chloride (BMIM-Cl) in aqueous so-
lution is studied theoretically and experimentally. Using molecular dynamics
simulations and ion-conductance measurements, the transport is examined in
bulk as well as through a biological nanopore, OmpF and its mutant D113A.
This investigation is motivated by the observation that aqueous solutions of
BMIM-Cl drastically reduce the translocation speed of DNA or antibiotics
through nanopores in electrophysiological measurements. This makes
BMIM-Cl an interesting alternative salt to improve the time resolution. In
line with previous investigations of simple salts, the size of the ions and their
orientation adds another important degree of freedom to the ion transport,
thereby slowing the transport through nanopores. An excellent agreement be-
tween theory and conductance measurements is obtained for wild type
OmpF and a reasonable agreement for the mutant. Moreover, all-atom simula-
tions allow an atomistic analysis revealing molecular details of the rate-
limiting ion interactions with the channel.
 Mahendran KR et al, J. Phys: Condens. Matter 22 (2010) 454131.
 Niraj Modi, Pratik Raj Singh et al, J. Phys. Chem. Lett. 2 (2011)
A Designed Polymer Detects the microRNAs Based on Protein Nanopore
University of Missouri, Columbia, MO, USA.
role in modulating gene expression. And it is a new candidate for some disease
diagnosis. However, it is difficult to detect because of the short length, low con-
centration and mixed with other components in cell. In our work, a synthesized
polymer is design to detect miRNA based on protein nanopore. The positively
charged polymer has high specificity to given kind of miRNA. Meanwhile, the
capture rate in a site-direct mutated nanopore is several hundred folds higher
than other kinds of probe from trans side. After binding of probe and miRNA,
the polymerprobecandetect lowconcentrationofmiRNAto tens ofpicomolar.
According the side preference of nucleic acid, the unrelated ones in trans side
will not interfere the detection. Besides, three members of miRNA family can
be distinguished using the same probe by the properties of the signatures, even
though there is only one or two bases difference in them.
SSB Enhances Detection of ssDNA Translocation through Solid-State
Deanpen Japrung1, Achim Nadzeyka2, Lloyd Peto2, Sven Bauerdick2,
Tim Albrecht1, Joshua Edel1.
1Imperial College London, London, United Kingdom,2Raith GmbH,
The small diameter and secondary structure formation are major problems in
nanopore-based analysis of hetero-sequence ssDNA/RNA. Here we report
how binding of single-stranded binding protein (SSB) can both prevent second-
ary formation and increase diameter of ssDNA. SSB is a helix-destabilizing
protein in virtue of its binding with high affinity to ssDNA and plays important
roles in DNA replication,recombinationand repair. E.coli SSB forms tetramers
and binds every 35 nucleotides (nt) under conditions used in our experiments.
We have translocated long (7.2 kb) and short SSB-coated ssDNA in the 37-100
nt range. For long SSB-coated ssDNA, current blockade levels are lower and
last significantly longer than those of the free ssDNA, which is due to straight-
ening of the globular structure. SSB-coated ssDNA molecules as short as
37-100nt translocatemuchfaster butare stilleasily detectable. Wefoundtrans-
location times of 0.9250.18 ms for 37-nt ssDNA/SSB and 1.4050.14 ms for
100-nt SSB/ssDNA. This is the first demonstration that ssDNA shorter than
ing solid-state nanopore
and applicable for future
ssDNA sizing or se-
quencing of the natural
and long ssDNA, which
forms complicated sec-
Sunday, February 26, 2012