Archive for the ‘Stuart Lindsay’s Lab’ Category

Project 2 Team Members

Tuesday, March 5th, 2013

Project Lead: Stuart Lindsay

  • Stuart Lindsay is Edward and Nadine Carson Professor of Physics and Chemistry and Director of the Center for Single Molecule Biophysics in the Biodesign Institute. His research focuses on biology at the nanoscale. He was a co-founder of Molecular Imaging Corporation. His textbook “Introduction to Nanoscience” has just been published by Oxford University Press. More Info

  • Team Members

  • Parminder kaur Is a final year PhD grad student. She has been working on the DNA methylation and chromatin project. She has a physics background and would further like to put her combined knowledge of physics, biology and chemistry in the pharmaceutical world. She has worked as an intern in Bristol Myers Squibb.

  • Trent Bowen is a senior studying physics and applying to medical
    school. Trent pursued his interest in applying a physical science background in furthering the understanding of biological molecules by studying centromere dynamics at the National Cancer Institute this
    past summer.

  • Ian Vicino is an undergraduate student majoring in Biochemistry. He hopes to learn as much as he can about this world through the research he does, whether it be within his undergraduate degree or when he eventually gets his PhD.

  • Peter Costa is an undergrad studying physics and he has been helping tremendously in the lab with the DNA methylation and chromatin project.

Stuart Lindsay’s Publications

Monday, March 4th, 2013

Publications 2013

Application of catalyst-free click reactions in attaching affinity molecules to tips of atomic force microscopy for detection of protein biomarkers.Langmuir. November 2013
Application of catalyst-free click reactions in attaching affinity molecules to tips of atomic force microscopy for detection of protein biomarkers.

Long Lifetime of Hydrogen-bonded DNA Basepairs by Force Spectroscopy. Biophys J. May 2012
Long lifetime of hydrogen-bonded DNA basepairs by force spectroscopy.

Publications 2012

Optical and electrical detection of single-molecule translocation through carbon nanotubes

Optical and Electrical Detection of Single-Molecule Transolcation

Hydrophobicity of methylated DNA as a possible mechanism for gene silencing

Hydrophobicity of Methylated DNA

DNA translocating through a carbon nanotube can increase ionic current

DNA Translocating through a carbon nonotube

Identifying Single Bases in a DNA Oligomer with Electron Tunneling. Nat Nanotechnol. December 2010.
Identifying single bases in a DNA oligomer with electron tunneling.

Mass transport through vertically aligned large diameter MWCNTs embedded in parylene

Mass Transport through vertically aligned large diameter MWCNTS

Palladium electrodes for molecular tunnel junctions

Palladium electrodes for Molecular Tunnel Junctions

1,8-Naphthyridine-2,7-diamine: A potential universal reader of Watson-Crick base pairs for DNA sequencing by electron tunneling

Base pairs for DNA sequencin by electron tunneling

Solution Synthesis of Ultrathin Single-Crystalline SnS Nanoribbons for Photodetectors via Phase Transition and Surface Processing

Solution Synthesis

Chemical Recognition and Binding Kinetics in a functionalized tunnel junction

Abstract

4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bonding sites and a short flexible linker. When tethered to a pair of electrodes, it traps target molecules in a tunnel junction. Surprisingly large recognition-tunneling signals are generated for all naturally occurring DNA bases A, C, G, T and 5-methyl-cytosine. Tunnel current spikes are stochastic and broadly distributed, but characteristic enough so that individual bases can be identified as a tunneling probe is scanned over DNA oligomers. Each base yields a recognizable burst of signal, the duration of which is controlled entirely by the probe speed, down to speeds of 1nms 1, implying a maximum off-rate of 3s 1 for the recognition complex. The same measurements yield a lower bound on the on-rate of 1M 1s 1. Despite the stochastic nature of the signals, an optimized multiparameter fit allows base calling from a single signal peak with an accuracy that can exceed 80% when a single type of nucleotide is present in the junction, meaning that recognition-tunneling is capable of true single-molecule analysis. The accuracy increases to 95% when multiple spikes in a signal cluster are analyzed. © 2012 IOP Publishing Ltd.

Chemical Recognition and Binding kinetics in a functionalized tunnel junction

Long Lifetime of Hydrogen-Bonded DNA Basepairs by Force Spectroscopy

Abstract

Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ∼2 s for two-hydrogen bonds (A-T) and ∼4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements. © 2012 by the Biophysical Society.

Long Lifetime of Hydrogen-Bonded DNA Basepairs by Force Spectroscopy

Synthesis, Phsiochemical Properties and Hydrogen Bonding of 4(5)-Substituted 1-H-Imidazole-2-Carboxamide, a Potential Universal Reader for DNA Sequencing by Recognition Tunneling

Abstract

We have developed a chemical reagent that recognizes all naturally occurring DNA bases, a so called universal reader, for DNA sequencing by recognition tunneling in nanopores.1 The primary requirements for this type of molecules are the ability to form non-covalent complexes with individual DNA bases and to generate recognizable electronic signatures under an electrical bias. 1-H-imidazole-2-carboxamide was designed as such a recognition moiety to interact with the DNA bases through hydrogen bonding. In the present study, we first furnished a synthetic route to 1-H-imidazole-2-carboxamide containing a short ω-functionalized alkyl chain at its 4(5) position for its attachment to metal and carbon electrodes. The acid dissociation constants of the imidazole-2-carboxamide were then determined by UV spectroscopy. The data show that the 1-H-imidazole-2-carboxamide exists in a neutral form between pH 6-10. Density functional theory (DFT) and NMR studies indicate that the imidazole ring exists in prototropic tautomers. We propose an intramolecular mechanism for tautomerization of 1-H-imidazole-2-carboxamide. In addition, the imidazole-2-carboxamide can self-associate to form hydrogen bonded dimers. NMR titration found that naturally occurring nucleosides interacted with 1-H-imidazole-2-carboxamide through hydrogen bonding in a tendency of dG>dC≤laquo;dT>dA. These studies are indispensable to assisting us in understanding the molecular recognition that takes place in the nanopore where routinely used analytical tools such as NMR and FTIR cannot be conveniently applied. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis,Physiochemical Properties and Hyrdogen Bonding

Biochemistry and Semiconductor Electronics- the next big hit for silicon?

Abstract

Two recent developments portend a new era for silicon electronics in biomedical applications. Firstly, highly specific chemical recognition and massively parallel sample preparation techniques are being combined with VLSI to make new kinds of analytical chips. Secondly, critical dimensions are beginning to approach the size of biomolecules, opening new pathways for physical interactions between molecules and semiconductor structures. Future generations of hybrid chemicalCMOS devices could revolutionize diagnosis and make personalized medicine cheap enough to become widespread. © 2012 IOP Publishing Ltd.

Biochemistry and Semiconductor Electronics – the next big hit for Silicon?

Insulated Gold Scanning tunneling microscopy probes for recognition tunneling in an aqueous environment

Abstract

Chemically functionalized probes are required for tunneling measurements made via chemical contacts (Recognition Tunneling). Here, we describe the etching of gold STM probes suitable for chemical functionalization with moieties bearing thiol groups. Insulated with high density polyethylene, these probes may be used in aqueous electrolytes with sub pA leakage currents. The area of the exposed probe surface was characterized via the saturation current in an electroactive solution (0.1 M K 3Fe(CN) 6). Twenty five percent of the probes had an exposed region of 10 nm radius or less. © 2012 American Institute of Physics.Insulated Gold Scanning tunnelling microscopy probes for recognition tunneling in an aqueous environment

Robert Ros’ Publications

Wednesday, March 2nd, 2011

Publications 2013

A Physical Sciences Network Characterization of Non-tumorigenic and Metastatic Cells. Sci Rep. 2013
A physical sciences network characterization of non-tumorigenic and metastatic cells.

Publications 2012

Long Lifetime of Hydrogen-bonded DNA Basepairs by Force Spectroscopy. Biophys J. May 2012
Long lifetime of hydrogen-bonded DNA basepairs by force spectroscopy.

Identifying single bases in a DNA oligomer with electron tunnelling, published in Nature Nanotechnology, ONLINE: 14 NOVEMBER 2010 (http://www.nature.com/nnano/journal/v5/n12/full/nnano.2010.213.html)

Publications 2011

Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging, published in the Biophysical Journal, Volume 100, January 2011. 243–250.

AFM Stiffness Nanotomography of Normal, Metaplastic and Dysplastic Human Esophageal Cells, published in Phys. Biol. 8 (2011) 015007.

Single-Molecule Force Spectroscopy: a Method for Quantitative Analysis of Ligand-Receptor Interactions, published in Nanomedicine (2010) 5(4).

Publications 2010

Identifying Single Bases in a DNA Oligomer with Electron Tunneling. Nat Nanotechnol. December 2010.
Identifying single bases in a DNA oligomer with electron tunneling.

Self-Assembled Water-Soluble Nucleic Acid Probe Tiles for Label-Free RNA Hybridization Assays (pdf)

Saturday, February 27th, 2010

Self-Assembled Water-Soluble Nucleic Acid Probe Tiles for Label-Free RNA Hybridization Assays

The DNA origami method, in which long, single-stranded DNA segments are folded into shapes by short staple segments, was used to create nucleic acid probe tiles that are molecular analogs of macroscopic DNA chips. One hundred trillion probe tiles were fabricated in one step and bear pairs of 20-nucleotide-long single-stranded DNA segments that act as probe sequences. These tiles can hybridize to their targets in solution and, after adsorption onto mica surfaces, can be examined by atomic force microscopy in order to quantify binding events, because the probe segments greatly increase in stiffness upon hybridization. The nucleic acid probe tiles have been used to study position-dependent hybridization on the nanoscale and have also been used for label-free detection of RNA.

The potential and challenges of nanopore sequencing (pdf)

Saturday, February 27th, 2010

The potential and challenges of nanopore sequencing

ABSTRACT

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.

Characterization of an antibody scFv that recognizes fibrillar insulin and β-amyloid using atomic force microscopy (pdf)

Saturday, February 27th, 2010

Characterization of an antibody scFv that recognizes fibrillar insulin and β-amyloid using atomic force microscopy

ABSTRACT

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer’s and Parkinson’s diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes β-amyloid fibrils, a hallmark feature of Alzheimer’s disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif—probably the cross-β-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.

Evolution of a Histone H4-K16 Acetyl-Specific DNA Aptamer (pdf)

Saturday, February 27th, 2010

Evolution of a Histone H4-K16 Acetyl-Specific DNA Aptamer

Imaging Glycosylation (pdf)

Saturday, February 27th, 2010

Imaging Glycosylation

Epigenetics & Chromatin (Single-epitope recognition imaging of native chromatin) (pdf)

Saturday, February 27th, 2010

Epigenetics & Chromatin (Single-epitope recognition imaging of native chromatin)

ABSTRACT

Background: Direct visualization of chromatin has the potential to provide important insights into epigenetic processes. In particular, atomic force microscopy (AFM) can visualize single nucleosomes under physiological ionic conditions. However, AFM has mostly been applied to chromatin that has been reconstituted in vitro, and its potential as a tool for the dissection of native nucleosomes has not been explored. Recently we applied AFM to native Drosophila chromatin containing the centromere-specific histone 3 (CenH3), showing that it is greatly enriched in smaller particles. Taken together with biochemical analyses of CenH3 nucleosomes, we propose that centromeric nucleosomes are hemisomes, with one turn of DNA wrapped around a particle
consisting of one molecule each of centromere-specific CenH3, H4, H2A and H2B.

Results: Here we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3- containing hemisomes.

Conclusion: Efficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.

Translocation of Single-Stranded DNA Through Single-Walled Carbon Nanotubes (pdf)

Saturday, February 27th, 2010

Translocation of Single-Stranded DNA Through Single-Walled Carbon Nanotubes

ABSTRACT

We report the fabrication of devices in which one single-walled carbon nanotube spans a barrier between two fluid reservoirs, enabling direct electrical measurement of ion transport through the tube. A fraction of the tubes pass anomalously high ionic currents. Electrophoretic transport of small single-stranded DNA oligomers through these tubes is marked by large transient increases in ion current and was confirmed by polymerase chain reaction analysis. Each current pulse contains about 107 charges, an enormous amplification of the translocated charge. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurements, and may open avenues for control of DNA translocation.