Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/micros translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.
Voltage biased solid-state nanopores are used to detect and characterize individual single stranded DNA molecules of fixed micrometer length by operating a nanopore detector at pH values greater than approximately 11.6. The distribution of observed molecular event durations and blockade currents shows that a significant fraction of the events obey a rule of constant event charge deficit (ecd) indicating that they correspond to molecules translocating through the nanopore in a distribution of folded and unfolded configurations. A surprisingly large component is unfolded. The result is an important milestone in developing solid-state nanopores for single molecule sequencing applications.
The authors measured ionic current blockages caused by protein translocation through voltage-biased silicon nitride nanopores in ionic solution. By calculating the mean amplitude, time duration, and the integral of current blockages, they estimated the relative charge and size of protein molecules at a single molecule level. The authors measured the change in protein charge of bovine serum albumin (BSA) protein induced by pH variation. They also confirmed that BSA molecules indeed traverse nanopores using an improved chemiluminescent analysis. They demonstrated that a larger protein fibrinogen could be distinguished from BSA by a solid-state nanopore measurement.
A prior genetic study indicated that activity of Sir silencing proteins at a hypothetical AGE locus is essential for long life span. In this model, the SIR4-42 mutation would direct the Sir protein complex to the AGE locus, giving rise to a long life span. We show by indirect immunofluorescence that Sir3p and Sir4p are redirected to the nucleolus in the SIR4-42 mutant. Furthermore, this relocalization is dependent on both UTH4 a novel yeast gene that extends life span, and its homologue YGL023. Strikingly, the Sir complex is relocalized from telomeres to the nucleolus in old wild-type cells. We propose that the rDNA is the AGE locus and that nucleolar function is compromised in old yeast cells in a way that may be mitigated by targeting of Sir proteins to the nucleolus.
The CCAAT-binding factor is a conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites (UASs) within the promoters of numerous eukaryotic genes. The The transcriptional activation of gene expression is mediated by the binding of distinct regulatory factors to specific cis-acting DNA sequence elements, referred to as enhancers or upstream activation sites (UASs), located within the promoters of eukaryotic genes. Many of these transcription factors are composed of multiple polypeptides that interact in a specific manner to form an oligomeric complex that is capable of recognizing specific cisacting DNA sequence elements. Several families of hetero-oligomeric transcription factors have been identified in eukaryotes (McKnight et al. 1987;Olesen et al. 1987;
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