Sequencing by avidity enables high accuracy with low reagent consumption.

We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer-nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.

False positive effect of sulfur sources used in growing and processing of vine (Vitis Vinifera L.) leaves on the results of dithiocarbamate analysis based on carbon disulfide measurement.

Vine leaves, which are produced fresh, brined or fermented from the leaves of Vitis Vinifera in Türkiye are an important food. Sulfur is used as a pesticide and sulfur compounds can be used as additives during the growing and processing of the vine leaves. These sulfur sources cause positive results on carbon disulfide (CS2) measurements by GC-MS. Therefore, the main objective of the present study was to investigate the effects of residues of sulfur or sulfur compounds on dithiocarbamate analysis methods based on CS2 measurement. For this, vine leaves were produced by controlled agricultural production and processed as brine under controlled conditions. The sulfur dioxide (SO2) and dithiocarbamate analysis were carried out on the vine leave obtained by applying sulfur spraying in agricultural treatments and brined vine leaves produced by adding sodium metabisulfite (SM), and control samples of each stage. SO2 was not detected in any of the samples in this study. SO2 residues did not occur in the vine leaves as a result of the sulfur spraying application and therefore did not have a false positive effect on dithiocarbamate analysis. However, approximately 0.15 mg kg-1 false positive dithiocarbamate was detected, which is thought to originate from natural sulfur in the vine leaves. The effect of SM, which was used in low concentration in the production of brined vine leaves, on dithiocarbamate results was limited. Even if SM was not used, the total false positive dithiocarbamate result in the brined vine leaves production process was approximately determined as 0.20 mg kg-1. This study showed that the dithiocarbamates analysis method based on CS2 measurement may lead to false positive results in brined vine leaves since sulfur compounds are found naturally in vine leaves.

The false positive effect of residue of sulphur sources on dithiocarbamate analysis based on CS2 measurement.

Turkey plays an important role in the international trade of apricots as it has the largest production rate in the world. Since the sulphurisation process is allowed to be used for different products, the effect of residual sulphur and its compounds (which can be found in products as pesticide residues or additive residues) on the positive detection of carbon disulphide (CS2) still creates a big challenge in international trade. Therefore, the main objective of the present study was to investigate the effects of residues of sulphur or sulphur compounds on dithiocarbamate analysis methods based on CS2 measurement. In this study, apricots were chosen since they contain sulphur residues as a result of the sulphurisation process. Sulphur dioxide and dithiocarbamate analyses were conducted on dried apricots prepared with the sulphurisation process (SA) and without the sulphurisation process (NSA); analysis was by two different accredited laboratories. No of pesticide was applied to either SA or NSA samples. Although some of the NSA samples had

Protein structure. Engineering of a superhelicase through conformational control.

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here we show that conformational control through intramolecular cross-linking of a helicase monomer with undetectable unwinding activity converts it into a superhelicase that can unwind thousands of base pairs processively, even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into the regulation of nucleic acid unwinding activity and introduces a monomeric superhelicase without nuclease activities, which may be useful for biotechnological applications.

Activated GTPase movement on an RNA scaffold drives co-translational protein targeting.

Approximately one-third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane. The proper localization of these proteins is mediated by a universally conserved protein-targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences and, through interactions with the SRP receptor, delivers them to the protein-translocation machinery on the target membrane. The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA for which precise functions have remained elusive. Here we used single-molecule fluorescence microscopy to show that the Escherichia coli SRP-SRP receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA, where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism for ensuring the productive exchange of the targeting and translocation machineries at the ribosome exit site with high spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the easy exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes.

Single-molecule nanopositioning: structural transitions of a helicase-DNA complex during ATP hydrolysis.

The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partial-duplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to single-stranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATPγS and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 Å relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation.

Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single-molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single-molecule fluorescence measurements of biomacromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions in equilibrium with the initial buffer condition. To permeabilize the barrier, Staphylococcus aureus toxin α-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single-molecule fluorescence resonance energy transfer signals from vesicle-encapsulated guanine-rich DNA that folds in a G-quadruplex motif as well as from the Rep helicase-DNA system. We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17-37 °C). Ions can efficiently pass through preformed aHL channels to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report a 35-fold improvement in the aHL channel formation efficiency, making this approach more practical for wider applications. Finally, the temperature-dependent single-molecule enzymatic study inside these nanocontainers is demonstrated by measuring the Rep helicase repetitive shuttling dynamics along a single-stranded DNA at various temperatures. The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high-throughput measurements and sensors beyond the single-molecule fluorescence measurements.

Single molecule nanocontainers made porous using a bacterial toxin.

Encapsulation of a biological molecule or a molecular complex in a vesicle provides a means of biofriendly immobilization for single molecule studies and further enables new types of analysis if the vesicles are permeable. We previously reported on using DMPC (dimyristoylphosphatidylcholine) vesicles for realizing porous bioreactors. Here, we describe a different strategy for making porous vesicles using a bacterial pore-forming toxin, alpha-hemolysin. Using RNA folding as a test case, we demonstrate that protein-based pores can allow exchange of magnesium ions through the vesicle wall while keeping the RNA molecule inside. Flow measurements indicate that the encapsulated RNA molecules rapidly respond to the change in the outside buffer condition. The approach was further tested by coencapsulating a helicase protein and its single-stranded DNA track. The DNA translocation activity of E. coli Rep helicase inside vesicles was fueled by ATP provided outside the vesicle, and a dramatically higher number of translocation cycles could be observed due to the minuscule vesicle volume that facilitates rapid rebinding after dissociation. These pores are known to be stable over a wide range of experimental conditions, especially at various temperatures, which is not possible with the previous method using DMPC vesicles. Moreover, engineered mutants of the utilized toxin can potentially be exploited in the future applications.

CT-guided transthoracic fine needle aspiration of pulmonary lesions: accuracy and complications in 134 cases.

The aim of this study was to perform a prospective evaluation of the effectiveness of computed tomography (CT)-guided transthoracic fine needle aspiration (TFNA) in the diagnosis of pulmonary lesions and to determine the complication rate of this procedure. A prospective review was conducted of 134 patients who underwent CT-guided TFNA at our center between December 2003 and August 2005. All fine needle aspirations were performed with a 22-gauge single-pass Chiba needle under CT guidance. The biopsies were performed by one pulmonologist. Two hundred twenty two (91%) malignant lesions and 12 (9%) benign lesions were reviewed in the present study. An accurate diagnosis was made in 107 (88%) of the 122 malignant lung lesions and a specific diagnosis was obtained in 42% of the benign lesions. The sensitivity of TFNAs for the detection of malignancy was 83%, and the overall accuracy of TFNA for diagnosing malignancy was 84%. Pneumothorax occurred in 22 of the 134 patients (16%). Pneumothorax was more frequently observed in centrally located lesions (p= 0.001). Our results suggest that CT-guided TFNA has a high diagnostic accuracy and an acceptable rate of complications. Moreover, we suggest that the most important factor increasing the risk of pneumothorax is an increase in the depth of aerated lung traversed for sampling.

Serum arylesterase activity is negatively correlated with inflammatory markers in patients with acute coronary syndromes.

OBJECTIVES: To examined whether serum paraoxonase (PON1) and arylesterase (ARE) activities are correlated with inflammatory biomarkers (procalcitonin and high sensitivity C-reactive protein (hs-CRP) in patients with acute coronary syndrome (ACS). METHODS: This cross-sectional study was conducted at the Departments of Cardiology and Biochemistry, Uludag University School of Medicine, Bursa, Turkey, from April 2007 to December 2007. Seventy-eight consecutive patients with ACS and 39 healthy controls were investigated. Acute coronary syndrome patients were divided into 3 groups according to their clinical presentation: unstable angina pectoris (UAP) (Braunwald III-B, n=25), non-ST elevation myocardial infarction (NSTEMI) (n=18), and ST-elevation myocardial infarction (STEMI) (n=35). Serum PON1/ARE activities were measured spectrophotometrically. Levels of procalcitonin and hs-CRP were measured by immunoassay. RESULTS: Paraoxonase/ARE activities were significantly lower in all patient groups compared to controls. No correlation between PON1/ARE activities and high-density-cholesterol levels was seen. Among ACS patients, serum ARE activity correlated inversely with baseline and 48-hour procalcitonin (r=-0.577, p=0.009, and r=-0.642, p=0.019) and hs-CRP levels (r=-0.614, p=0.03, and r=-0.719, p=0.044). CONCLUSION: Serum ARE activity is reduced in ACS patients and inversely correlated with inflammatory markers.

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids.

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5' ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.