While most single-molecule DNA sequencing methods rely upon optical detection of fluorescent substrates, for nearly 20 years a subset of approaches have strived to infer sequence using electrical current. The best-known of these efforts involve nanopore sequencing, in which a DNA strand travels through a nanosized opening across which a voltage has been applied. The current passing through the pore varies in a characteristic way depending on the identity of the nucleotide in transit. This technology remains in development, and it is not clear that the approach will be able to deliver the long read lengths and low error rates needed to out-compete highly parallelized sequencing-by-synthesis approaches. At the same time, nanopore techniques have some new competition. Observing that electrical conductivity is useful for probing single-molecule dynamics, Chen et al., writing in Nature Nanotechnology, introduce a sequencing method based on changes in the electrical conductance of DNA polymerase as it incorporates nucleotides into a growing DNA strand. To build polymerase into an electrical circuit, the authors prepared a chip containing a 10 nm gap between 2 electrodes, then placed a nanoparticle adjacent to each electrode. The nanoparticles, which were exposed to a liquid channel, were then functionalized with Φ29 polymerase. For sequencing, an annealed primer and template were introduced, cross-electrode current was monitored, and, after current fluctuations subsided, nucleotides were added. Polymerization was visible as current peaks, which were revealed to be of four different kinds, each differing slightly in their component spikes and plateaus. The four peak types corresponded to the four nucleotides, as confirmed by experiments in which one nucleotide was added at a time. Because homopolymeric sequences are a sequencing bugbear, the authors tested a template with a string of 20 Ts, but this protein transistor-based method had no difficulty managing an accurate read. Φ29 is known to have a low error rate, and the authors sequenced over 50,000 nucleotides without picking up any errors. Since Φ29 is highly processive, long sequence reads may also be possible. However, the longest template tested was only about 120 nucleotides, and the authors caution that a long product might change conductance patterns and increase noise. Nevertheless, these promising results suggest current-based detection of single-molecule sequencing reactions may be exactly the jolt the field needs.
Y.S. Chen et al. 2013. DNA sequencing using electrical conductance measurements of a DNA polymerase.Nat Nanotechnol. 8:452-8.Making His-tory
Poor histidine. Phosphorylation of this amino acid was first recognized in 1962, but since then, phospho-serine, threonine, and tyrosine have gotten the lion's share of attention. In part, an “analytical blind spot” is to blame—there are no good tools to probe phosphohistidine (pHis) globally. Whereas anti-phosphotyrosine antibodies were developed in the 1980s, the high-energy nitrogen-phosphorus bond of pHis makes it prone to dephosphorylation under acidic conditions, dooming efforts to raise antibodies. Some groups have developed structural mimetics of pHis-containing peptides, using such analogues to prepare sequence-specific anti-pHis antibodies. Because these antibodies do not address the need for global assessment of histidine phosphorylation, a paper in Nature Chemical Biology from Kee et al. that describes the first pan-specific anti-pHis antibody marks a significant boost to the field. To generate a more stable version of pHis, the authors synthesized phosphoryl-triazolylethylamine (pTze), which, among other changes, replaces the nitrogen-phosphorus bond with a carbon-phosphorus bond. After conjugating pTze to keyhole limpet hemocyanin, Kee et al. immunized rabbits and enriched anti-pHis antibodies by affinity purification using chemically phosphorylated BSA. The resulting polyclonal antibody was tested bu both ELISA and Western blotting of recombinant pHis-containing proteins; as expected, treatment with acid or hydroxylamine, which dephosphorylate pHis, gave background levels of signal. These tests were performed using proteins with different pHis sequence contexts, suggesting the pan-specificity of the new polyclonal antibody. While cross-reactivity with phosphoserine or phosphothreonine was ruled out, the new antibody did have a low but detectable tendency to bind to phosphotyrosine. However, because commercially available antibodies for phosphotyrosine do not meaningfully react with pHis, phosphotyrosine-containing proteins can be immunodepleted, isolating the pHis-specific signal. In tests using E. coli lysates, Kee et al. verified that the antibody could detect endogenous proteins containing the pHis modification. Of note, having an antibody that can immunoprecipitate the pHis proteome enables the mass spectrometry-based analyses that have been so successful in identifying and assigning function to other phosphoamino acids. While future efforts to prepare a monoclonal antibody, preferably one without phosphotyrosine cross-reactivity, will be crucial for the wider implementation of pan-specific anti-pHis antibodies, this work heralds an alternative to radiolabel-dependent in vitro assays, and may finally help uncover the extent and significance of the pHis modification in eukaryotes.
J.M. Kee et al. A pan-specific antibody for direct detection of protein histidine phosphorylation.Nat Chem Biol.[Epub ahead of print, May 26, 2013; doi:10.1038/nchembio.1259].