Observation of DNA Knots Using Solid-State Nanopores

Abstract

Long DNA molecules can self-entangle into knots. Experimental techniques to observe such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kbp in length. Here we show that solid-state nanopores can be used to directly observe individual DNA knots in both linear and circular single molecules of arbitrary length. DNA knots are observed as short spikes in the nanopore current traces of traversing DNA molecules. The observation of knots is dependent on sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of DNA molecules with knots for different DNA molecules, up to 166 kbp in length. We find that the knotting probability rises strongly with length, and compare our experimental data to simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with small sizes below 100 nm. In the case of linear molecules, we observe that knots are able to slide out upon applying high driving forces (voltage). Our results demonstrate that the solid-state nanopore technique can provide a wealth of information about the position and the size of knots, including the number of DNA strands inside DNA knots. 831-Pos Board B611Enhanced Electrostatic Force Microscopy Imaging Reveals Mechanism of TRF2 Mediated DNA Compaction Parminder kaur. Physics, North Carolina State University, Raleigh, NC, USA. T-loop formation at telomeres is proposed to play an important role in telomere protection through the sequestration of the 3' single-stranded overhang. Previous studies indicated that shelterin protein TRF2 modulates telomere structure by promoting DNA compaction and T-loop formation. To further understand the mechanism underlying TRF2-mediated DNA compaction, we applied Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM), a recently developed technique capable of high-resolution imaging of weak electrostatic potentials. DREEM images of nucleosomes clearly reveal DNA strands wrapped around histone proteins in nucleosomal arrays. In contrast, DREEM imaging shows DNA compacted inside TRF2 complexes through a 3-dimensional stacking of TRF2 dimers mediated by collective actions of multiple copies of TRF2 proteins. TRF2-mediated DNA compaction leads to electric potential gradients across the complexes. Surprisingly, while DNA wrapped around histones displays similar electrostatic potential signals compared to bare DNA, TRF2 DNA compaction leads to significant differences in the electrical potential signals inside multioligomeric TRF2 complexes compared to protein or DNA alone. These results clearly demonstrate the electrostatic changes in TRF2-DNA complexes upon oligomerizaton of proteins and DNA compaction, and underscore the importance of developing new electrostatic force microscopy imaging techniques for studying biological systems. 832-Pos Board B612Grab & Watch: Correlative Optical Tweezers-Flu...

Periodic forces trigger knot untying during translocation of knotted proteins

“…Another possibility is to use modern artificial nanopore technology. Engineered protein nanopores have been successfully used for the detection of DNA chains 67 68 , including the knotted ones 69 . Recently, it was demonstrated that they can also be used for the detection of proteins 70 71 72 .…”

Detection of Individual Proteins Bound along DNA Using Solid-State Nanopores

“…This approach differentiates the high amplitude spikes produced by the antibodies from smalleramplitude folds and knots that are always observed in highbandwidth measurements on long DNA in large pores. 16 Statistics on the percentage of events with spikes reveal a clear correlation between the addition of antibodies and the appearance of the spikes.…”

“…We chose to use a spike detection threshold of 3.5I 1 as it differentiates quite well between spikes caused by antibodies and spikes observed in DNA-only experiments, which are due to DNA knots and folds. 16 The typical spike amplitudes are sufficiently large that the vast majority of spikes are captured at the 3.5I 1 threshold used (Figure S4, Supporting Information). Figure 2, panel b shows the fraction of events with spikes of amplitude larger than 3.5I 1 at voltages ranging from 100−400 mV for experiments with both DNA and antibodies as well as control experiments containing only DNA.…”

Steady-State and Transient Behavior of Knotted Chains in Extensional Fields

“…It is unsurprising to physicists that molecular topology plays a vital role in influencing the dynamics of polymers. − One such example is a knot on an open chain . These architectures have been found naturally in DNA , and proteins, and they have been discovered in nanotechnology applications that perform single-molecule sequencing − and genetic mapping of DNA. The removal of self-entanglements is important for the latter technologies as these structures give rise to incorrect readings of base-pair sequences. , Since these issues become inevitable as chain sizes become longer, , there has been a recent push to understand the physics of knotted polymers in more detail.…”