DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging.

Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called 'DNA skybridge'. The DNA skybridge is constructed in a 3D structure with 4 μm-high thin quartz barriers in a quartz slide.

Coordinating Multi-Protein Mismatch Repair by Managing Diffusion Mechanics on the DNA.

DNA mismatch repair (MMR) corrects DNA base-pairing errors that occur during DNA replication. MMR catalyzes strand-specific DNA degradation and resynthesis by dynamic molecular coordination of sequential downstream pathways. The temporal and mechanistic order of molecular events is essential to insure interactions in MMR that occur over long distances on the DNA.

Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair.

Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10-40% of related sporadic tumours.

Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting.

Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy.

Dynamic control of strand excision during human DNA mismatch repair.

Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic.

Loading dynamics of a sliding DNA clamp.

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E.

Single-molecule views of MutS on mismatched DNA.

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides.

Aptamer-based single-molecule imaging of insulin receptors in living cells.

We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2).

ATP alters the diffusion mechanics of MutS on mismatched DNA.

The mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (∼1 s) and an unusually stable (∼600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch.

Alterations of vasoactive intestinal polypeptide receptors in allergic rhinitis.

Background: Vasoactive intestinal peptide (VIP) is one of the parasympathetic neurotransmitters involved in the regulation of airway mucus secretion. The biological functions of VIP are mediated through two receptors (vasoactive intestinal peptide receptor type 1 [VPAC1R] and type 2 [VPAC2R]). The purpose of this study was to determine the distribution of VIP receptors and to compare the level of VIP receptor expression in the nasal mucosa of patients with allergic rhinitis and normal controls.