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1.
Nano Lett ; 18(10): 6469-6474, 2018 10 10.
Article in English | MEDLINE | ID: mdl-30187755

ABSTRACT

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.


Subject(s)
Biosensing Techniques/methods , CRISPR-Cas Systems/genetics , DNA/isolation & purification , Nanopores , Binding Sites , DNA/chemistry , DNA/genetics , Humans , RNA, Guide, Kinetoplastida/chemistry , RNA, Guide, Kinetoplastida/genetics
2.
ACS Nano ; 12(3): 2623-2633, 2018 03 27.
Article in English | MEDLINE | ID: mdl-29474060

ABSTRACT

Many theoretical studies predict that DNA sequencing should be feasible by monitoring the transverse current through a graphene nanoribbon while a DNA molecule translocates through a nanopore in that ribbon. Such a readout would benefit from the special transport properties of graphene, provide ultimate spatial resolution because of the single-atom layer thickness of graphene, and facilitate high-bandwidth measurements. Previous experimental attempts to measure such transverse inplane signals were however dominated by a trivial capacitive response. Here, we explore the feasibility of the approach using a custom-made differential current amplifier that discriminates between the capacitive current signal and the resistive response in the graphene. We fabricate well-defined short and narrow (30 nm × 30 nm) nanoribbons with a 5 nm nanopore in graphene with a high-temperature scanning transmission electron microscope to retain the crystallinity and sensitivity of the graphene. We show that, indeed, resistive modulations can be observed in the graphene current due to DNA translocation through the nanopore, thus demonstrating that DNA sensing with inplane currents in graphene nanostructures is possible. The approach is however exceedingly challenging due to low yields in device fabrication connected to the complex multistep device layout.


Subject(s)
DNA/analysis , Graphite/chemistry , Nanopores , Nanostructures/chemistry , Sequence Analysis, DNA/instrumentation , Electric Conductivity , Electrochemical Techniques/instrumentation , Equipment Design , Motion , Nanopores/ultrastructure , Nanotechnology/instrumentation
4.
Nature ; 535(7613): 566-9, 2016 07 28.
Article in English | MEDLINE | ID: mdl-27437582

ABSTRACT

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs. Here we show, using dual- and quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4-XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4-XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4-XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations.


Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Diffusion , Humans , Microscopy, Fluorescence , Movement , Optical Tweezers , Translocation, Genetic
5.
Nat Nanotechnol ; 11(2): 127-36, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26839258

ABSTRACT

Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.


Subject(s)
Graphite/chemistry , Nanotechnology/instrumentation , Sequence Analysis, DNA/instrumentation , Equipment Design
6.
ACS Nano ; 9(4): 3428-35, 2015 Apr 28.
Article in English | MEDLINE | ID: mdl-25864552

ABSTRACT

Structural defects strongly impact the electrical transport properties of graphene nanostructures. In this Perspective, we give a brief overview of different types of defects in graphene and their effect on transport properties. We discuss recent experimental progress on graphene self-repair of defects, with a focus on in situ transmission electron microscopy studies. Finally, we present the outlook for graphene self-repair and in situ experiments.


Subject(s)
Graphite/chemistry , Nanotubes, Carbon/chemistry
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