DNA heterogeneity and phosphorylation unveiled by single-molecule electrophoresis

Abstract

Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes. Because the molecule information is quantized and digitally recorded with single-molecule resolution, the sensitivity of the system can be adjusted in real time to detect trace amounts of a particular DNA species.T he purity of a nucleic acid preparation and the chemical integrity of nucleic acid bases affect the efficiency of hybridization procedures, enzymatic reactions, and chemical modifications. These processes dictate the accuracy and reliability of routine biochemical and clinical investigations as well as the expanding field of array technologies. Although traditional techniques of electrophoresis, chromatography, and mass spectrometry can assess DNA or RNA sample purity and chemical integrity, the sensitivity of these methods is limited by the relative size and quantity of contaminating nucleic acids. More importantly, the resolution of these methods decreases with increasing DNA or RNA length. Sample evaluation is difficult for nucleic acids with Ͼ100 nt and is virtually impossible for those with Ͼ1,000 nt. In a process we call single-molecule electrophoresis, we show that a transmembrane nanopore can evaluate polynucleotides with Ͻ100 bases or Ͼ1,000 bases. The ensemble pattern produced by the individual interaction between the polymer and the nanopore reveals DNA sample properties in a manner unparalleled by other detection systems.The nanopore was formed by self-assembly of ␣-hemolysin (1), a robust channel-forming protein that has been used and engineered for stochastic sensing, characterization of small molecules, and detection and discrimination of individual DNA strands (2-7). A single ␣-hemolysin channel was embedded in a lipid membrane that partitioned a conducting solution into two chambers. A voltage bias across the membrane produced an ion current through the ␣-hemolysin and also drove negatively charged single-stranded DNA from the cis to the trans side of the channel. Translocations occurred on the microsecond timescale and were signaled by the partial blockage of ion current to 10-25% of the open pore current value (Fig.

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

“…6), increased electroosmotic flow (51,52) or a combination of the two that depends on the applied potential. The newly engineered pores or derivatives of them will be useful for enhancing the sensitivity of ␣HL as a biosensor of nucleic acids (2,(17)(18)(19)(20)(21)(22)(23)(24)(25). An additional potential application is in nanopore sequencing, where the bases in a single DNA strand are read off one by one during translocation (16,30,(32)(33)(34)(35)(36)(37).…”

“…Studies of nucleic acids have been especially intensive, following the demonstration of the transit of single strands in 1996 (16). The examination of mean transit times and current amplitudes during transit have allowed the analysis of single-strand nucleic acid length (2), base composition (17), phosphorylation state (18) and secondary structure (19). The formation (20,21) and dissociation of nucleic acid double strands (22)(23)(24) and hairpins (25,26) have also been examined.…”

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

“…Detailed insight has been gained from studies on pores of known molecular structure and the electrophoretically-driven movement of individual DNA and RNA strands. 3,11,21,23,[28][29][30] Several biophysical parameters have been determined such as threading frequency, [31][32][33][34] orientation of strands, [35][36][37][38][39] velocity of DNA transport, 40 influence of pore geometry, 41 and interactions with the pore wall. 39 In addition to nucleic acids, translocation has also been investigated for peptides, [42][43][44][45] proteins, [46][47][48] and peptide-oligonucleotide conjugates, 49 which, unlike nucleic acid strands, frequently feature inhomogeneous charge distributions and vary considerably in diameter along the polymer sequence.…”

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

“…Wang and colleagues first showed that the direction in which DNA enters the ␣HL pore (5Ј or 3Ј threading) affects the extent of current block (15), an observation supported by the data of Mathé et al (16), and recent studies have shown that the rate of capture of DNA by protein pores is enhanced when the interior surfaces bear a net positive charge (17,18). Under the high applied potentials required for threading, freely moving DNA is translocated through the wild-type (WT) ␣HL pore too quickly for bases to be identified, unless the bases are modified with bulky groups (19).…”