Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores

Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concentration. For our proof-of-principle experiments we use two standard binding systems, biotin-streptavidin and digoxigenin-antidigoxigenin, that allow for measurements of the concentration down to the low nanomolar range. The results demonstrate the potential for a novel quantitative and specific protein detection scheme using the DNA carrier method.The authors thank Howarth Lab, Oxford for providing the monovalent streptavidin sample, Yizhou Tan for the help with Labview programs, and Dr. Janet Kumita for the help with FP measurement. J. Kong acknowledges funding from Chinese Scholarship Council and Cambridge Trust. N.A.W.B. acknowledges funding from an EPSRC doctoral prize award and an ERC starting grant (Passmembrane 261101); U.F.K. acknowledges support from an ERC starting grant (Passmembrane 261101)

Free-standing graphene membranes on glass nanopores for ionic current measurements

A method is established to reliably suspend graphene monolayers across glass nanopores as a simple, low cost platform to study ionic transport through graphene membranes. We systematically show that the graphene seals glass nanopore openings with areas ranging from 180 nm2 to 20 μm2, allowing detailed measurements of ionic current and transport through graphene. In combination with in situ Raman spectroscopy, we characterise the defects formed in ozone treated graphene, confirming an increase in ionic current flow with defect density. This highlights the potential of our method for studying single molecule sensing and filtration.The authors would like to thank S. Purushothaman and K. Göpfrich for careful reading of the manuscript and V. Thacker for useful discussions. This work was supported by the EPSRC Cambridge NanoDTC, EP/G037221/1, and EPSRC grant GRAPHTED, EP/K016636/1. R.S.W. acknowledges a Research Fellowship from St. John's College, Cambridge. N.A.W.B. acknowledges an EPSRC doctoral prize award.This is the accepted manuscript. Copyright 2015 American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The final version is available in Applied Physics Letters 106, 023119 (2015); doi: 10.1063/1.490623

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show - through the use of biotinylated DNA and streptavidin - that this selective adsorption extends to DNA-protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+. Our findings suggest a mechanism for PARP1 in the organization of chromatin structure

Single-molecule observation of the intermediates in a catalytic cycle

The development of catalysts benefits from knowledge of the intermediate steps that accelerate the transformations of substrates into products. However, key transient species are often hidden in ensemble measurements. Here, we show that a protein nanoreactor can sample the intermediate steps in a catalytic cycle by the continuous single-molecule observation of a stoichiometric reaction in solution. By monitoring changes in the flow of ionic current through an α-hemolysin protein pore, we observed three intermediate metal–ligand complexes in a gold(I)-catalyzed reaction that converts an acetylenic acid to an enol lactone, revealing a transitional coordination complex that had been previously unobserved. A kinetic isotope effect helped assign the various metal–ligand species. Measurements of the lifetimes of the intermediates allowed a full kinetic analysis of the metal-catalyzed reaction cycle

Free-standing graphene membranes on glass nanopores for ionic current measurements

A method is established to reliably suspend graphene monolayers across glass nanopores as a simple, low cost platform to study ionic transport through graphene membranes. We systematically show that the graphene seals glass nanopore openings with areas ranging from 180 nm2 to 20 μm2, allowing detailed measurements of ionic current and transport through graphene. In combination with in situ Raman spectroscopy, we characterise the defects formed in ozone treated graphene, confirming an increase in ionic current flow with defect density. This highlights the potential of our method for studying single molecule sensing and filtration

Dynamics of driven polymer transport through a nanopore

The transport of polymers across nanoscale pores underpins many biological processes, such as the ejection of bacteriophage DNA into a host cell and the transfer of genes between bacteria. The movement of polymers into and out of confinement is also the basis for a wide range of sensing technologies used for single-molecule detection and sequencing. Acquiring an accurate understanding of the translocation dynamics is an essential step in the quantitative analysis of polymer structure, including the localization of binding sites or sequences. Here, we use synthetic nanopores and nanostructured DNA molecules to directly measure the velocity profile of driven polymer translocation through synthetic nanopores. Our results reveal a two-stage behaviour in which the translocation initially slows with time before accelerating close to the end of the process. We also find distinct local velocity correlations as the DNA polymer chain passes through the nanopore. Brownian dynamics simulations show that the two-stage behaviour is associated with tension propagation, with correlations arising from the random-walk conformation in which the DNA begins

Nanotubes complexed with DNA and proteins for resistive-pulse sensing

We use a resistive-pulse technique to analyze molecular hybrids of single-wall carbon nanotubes (SWNTs) wrapped in either single-stranded DNA or protein. Electric fields confined in a glass capillary nanopore allow us to probe the physical size and surface properties of molecular hybrids at the single-molecule level. We find that the translocation duration of a macromolecular hybrid is determined by its hydrodynamic size and solution mobility. The event current reveals the effects of ion exclusion by the rod-shaped hybrids and possible effects due to temporary polarization of the SWNT core. Our results pave the way to direct sensing of small DNA or protein molecules in a large unmodified solid-state nanopore by using nanofilaments as carriers. © 2013 American Chemical Society