Meni Wanunu

DNA Translocation through Graphene Nanopores

We report on DNA translocations through nanopores created in graphene membranes. Devices consist of 1-5 nm thick graphene membranes with electron-beam sculpted nanopores from 5 to 10 nm in diameter. Due to the thin nature of the graphene membranes, we observe larger blocked currents than for traditional solid-state nanopores. However, ionic current noise levels are several orders of magnitude larger than those for silicon nitride nanopores. These fluctuations are reduced with the atomic-layer deposition of 5 nm of titanium dioxide over the device. Unlike traditional solid-state nanopore materials that are insulating, graphene is an excellent electrical conductor. Use of graphene as a membrane material opens the door to a new class of nanopore devices in which electronic sensing and control are performed directly at the pore.

Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient.

Solid-state nanopores are sensors capable of analyzing individual unlabelled DNA molecules in solution. While the critical information obtained from nanopores (e.g., DNA sequence) is the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Here we study the process of DNA capture into nanofabricated silicon nitride pores of molecular dimensions. For fixed analyte concentrations we find an increase in capture rate as the DNA length increases from 800 to 8,000 basepairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. In addition, we show that application of a 20-fold salt gradient enables detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analyzing unamplified DNA samples using nanopores.

Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors

Small RNA molecules have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these molecules without the use of time-consuming radioactive labelling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane containing the nanopore to 6 nm leads to increased signal amplitudes from biomolecules, and reducing the diameter of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their physical dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.

Nanopores: A journey towards DNA sequencing

Much more than ever, nucleic acids are recognized as key building blocks in many of lifes processes, and the science of studying these molecular wonders at the single-molecule level is thriving. A new method of doing so has been introduced in the mid 1990s. This method is exceedingly simple: a nanoscale pore that spans across an impermeable thin membrane is placed between two chambers that contain an electrolyte, and voltage is applied across the membrane using two electrodes. These conditions lead to a steady stream of ion flow across the pore. Nucleic acid molecules in solution can be driven through the pore, and structural features of the biomolecules are observed as measurable changes in the trans-membrane ion current. In essence, a nanopore is a high-throughput ion microscope and a single-molecule force apparatus. Nanopores are taking center stage as a tool that promises to read a DNA sequence, and this promise has resulted in overwhelming academic, industrial, and national interest. Regardless of the fate of future nanopore applications, in the process of this 16-year-long exploration, many studies have validated the indispensability of nanopores in the toolkit of single-molecule biophysics. This review surveys past and current studies related to nucleic acid biophysics, and will hopefully provoke a discussion of immediate and future prospects for the field.

DNA Translocation Governed by Interactions with Solid-State Nanopores

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

Integrated nanopore sensing platform with sub-microsecond temporal resolution.

Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1 MHz, which to our knowledge is the highest bandwidth nanopore recording to date. We demonstrate transient signals as brief as 1 μs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.

Discrimination of methylcytosine from hydroxymethylcytosine in DNA molecules

Modified DNA bases are widespread in biology. 5-Methylcytosine (mC) is a predominant epigenetic marker in higher eukaryotes involved in gene regulation, development, aging, cancer, and disease. Recently, 5-hydroxymethylcytosine (hmC) was identified in mammalian brain tissue and stem cells. However, most of the currently available assays cannot distinguish mC from hmC in DNA fragments. We investigate here the physical properties of DNA with modified cytosines, in efforts to develop a physical tool that distinguishes mC from hmC in DNA fragments. Molecular dynamics simulations reveal that polar cytosine modifications affect internal base pair dynamics, while experimental evidence suggest a correlation between the modified cytosines polarity, DNA flexibility, and duplex stability. On the basis of these physical differences, solid-state nanopores can rapidly discriminate among DNA fragments with mC or hmC modification by sampling a few hundred molecules in the solution. Further, the relative proportion of hmC in the sample can be determined from the electronic signature of the intact DNA fragment.

Nanopore based sequence specific detection of duplex DNA for genomic profiling.

We demonstrate a purely electrical method for the single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes in order to achieve high affinity and sequence specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for fighting infectious diseases.

High-Bandwidth Protein Analysis Using Solid-State Nanopores

High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ~50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.

Slow DNA Transport through Nanopores in Hafnium Oxide Membranes

We present a study of double- and single-stranded DNA transport through nanopores fabricated in ultrathin (2-7 nm thick) freestanding hafnium oxide (HfO2) membranes. The high chemical stability of ultrathin HfO2 enables long-lived experiments with <2 nm diameter pores that last several hours, in which we observe >50 000 DNA translocations with no detectable pore expansion. Mean DNA velocities are slower than velocities through comparable silicon nitride pores, providing evidence that HfO2 nanopores have favorable physicochemical interactions with nucleic acids that can be leveraged to slow down DNA in a nanopore.