3' Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3'OH ends in DNA or RNA.

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various genomic tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to insert 5' phosphorylated blunt-end double-stranded DNA to DNA breaks at 3'-recessive ends, gaps, or nicks to form a Y-shaped 3'-branch structure. Therefore, this base pairing-independent ligation is termed 3'-branch ligation (3'BL). In an extensive study of optimal ligation conditions, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency to more than 80%. Ligation efficiency was slightly varied between different donor and acceptor sequences. More interestingly, we discovered that T4 DNA ligase efficiently ligated DNA to the 3'-recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, suggesting a ternary complex formation preference of T4 DNA ligase. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important genomic applications, such as 3'-end labelling by adding a universal sequence; directional tagmentation for NGS library construction that achieve theoretical 100% template usage; and targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling.

Site saturation mutagenesis (SSM) is a powerful mutagenesis strategy for protein engineering and directed evolution experiments. However, limiting factors using this method are either biased representation of variants, or limiting library size. To overcome these hurdles, we generated large scale targeted synthetic SSM libraries using massively parallel oligonucleotide synthesis and benchmarked this against an error-prone (epPCR) library. The yeast glucose activated GPCR-Gpr1 was chosen as a prototype to evolve novel glucose sensors. We demonstrate superior variant representation and several unique hits in the synthetic library compared to the PCR generated library. Application of this mutational approach further builds the possibilities of synthetic biology in tuning of a response to known ligands and in generating biosensors for novel ligands.

Considering the future direction of children's and young people's nursing.

Sonya Clark, Senior Lecturer in Children's Nursing at Queen's University Belfast, Emeritus Professor Alan Glasper, from the University of Southampton and Jim Richardson, Senior Lecturer in Children's Nursing at Kingston University and St George's, University of London, discuss the concerns of the academic community.

Results of ulnar shortening osteotomy with a new plate compression system.

PURPOSE: The gold standard for treatment of ulnar impaction has become ulnar shortening osteotomy. Previous reports in the literature have shown not only good results with relief of ulnar-sided wrist pain but also significant nonunion rates and painful hardware necessitating further surgery and potentially, metal removal. The purpose of this paper is to review the success rate of ulnar shortening osteotomy utilizing a low profile compression plate designed specifically for ulnar shortening osteotomy. METHODS: Ninety-three patients with ulnar abutment syndrome underwent ulnar shortening osteotomy with the low profile osteotomy plate. There were 47 males and 46 females. The Acumed's ulnar shortening system was utilized in all cases. The patients were evaluated for pain, range of motion, grip strength, return to work, time to union, and hardware removal. The patients' results were validated using the Mayo Wrist Score. RESULTS: There was a 100 % union rate in the 93 patients. There were no nonunions or delayed unions, or any hardware removal. All patients noted an improvement in their ulnar-sided wrist pain. Utilizing the Mayo wrist classification, the average postoperative score was 84.5. The average preoperative Mayo score was 49.4, for an average increase of 35.1 points. CONCLUSION: The Acumed's low-contact plate designed specifically for ulnar shortening osteotomy demonstrated 100 % union rate and no implant removal in our series. This is the largest study to our knowledge of a series of ulnar shortening osteotomies and successful healing without the removal of any implants. Furthermore, the specifically designed ulnar shortening osteotomy plate significantly simplifies the procedure for the surgeon and improves patient outcomes with relief of ulnar-sided wrist pain.

Real-time DNA sequencing from single polymerase molecules.

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides.

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.

Determination of ligand-protein dissociation constants by electrospray mass spectrometry-based diffusion measurements.

A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N' '-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MS-based methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.

Screening for noncovalent ligand-receptor interactions by electrospray ionization mass spectrometry-based diffusion measurements.

The application of a novel method for the identification of low-molecular-weight noncovalent ligands to a macromolecular target is reported. This technique is based on the measurement of analyte diffusion coefficients by electrospray mass spectrometry (ESI-MS) (Clark et al., Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462). Potential ligands have large diffusion coefficients as long as they are free in solution. Binding to a macromolecular target, however, drastically reduces the diffusional mobility of any ligand species. Mixtures containing six different saccharides [ribose, rhamnose, glucose, maltose, maltotriose, and N,N',N''-triacetylchitotriose (NAG(3))] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG(3) is known to bind to the protein. In "direct" binding tests, NAG(3) shows a significantly reduced diffusion coefficient in the presence of the protein. No changes were observed for any of the other saccharides. In a second set of experiments, the use of a "competition" screening method was explored in which mixtures of candidate saccharides were tested for their ability to displace a reference ligand from the target. The addition of NAG(3)-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG(4) (N,N',N'',N'''-tetraacetylchitotetrose), whereas mixtures that did not contain NAG(3) had no effect. These data clearly indicate the potential of ESI-MS-based diffusion measurements as a novel tool to screen compound libraries for binding to proteins and other macromolecular targets. In contrast to conventional ESI-MS-based ligand-receptor binding studies, this method does not rely on the preservation of noncovalent interactions in the gas phase.

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions.

This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gas- phase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation.

Taylor dispersion monitored by electrospray mass spectrometry: a novel approach for studying diffusion in solution.

This work describes a novel approach for monitoring analyte diffusion in solution that is based on electrospray ionization mass spectrometry (ESI-MS). A mass spectrometer at the end of a laminar flow tube is used to measure the Taylor dispersion of an initially sharp boundary between two solutions of different analyte concentration. This boundary is dispersed by the laminar flow profile in the tube. However, this effect is diminished by analyte diffusion that continuously changes the radial position, and hence the flow velocity of individual analyte molecules. The steepness of the resulting dispersion profile therefore increases with increasing diffusion coefficient of the analyte. A theoretical framework is developed to adapt the equations governing the dispersion process to the case of mass spectrometric detection. This novel technique is applied to determine the diffusion coefficients of choline and cytochrome c. The measured diffusion coefficients, (11.9 +/- 1.0) x 10(-10) m(2) s(-1) and (1.35 +/- 0.08) x 10(-10) m(2) s(-1), respectively, are in agreement with the results of control experiments where the Taylor dispersion of these two analytes was monitored optically. Due to the inherent selectivity and sensitivity of ESI-MS, it appears that the approach described in this work could become a valuable alternative to existing methods for studying diffusion processes, especially for experiments on multicomponent systems.