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The soluble interleukin-2 receptor α (sIL-2Rα) is a broad indicator of clinical disease activity in various inflammatory diseases. Here we have developed, for the first time, a rapid, washing-free colorimetric aptasensor based on a sIL-2Rα aptamer (Kd =1.33 nm). The aptasensor was fabricated with Au nanoparticles (AuNPs) adsorbing sIL-2Rα aptamers. On addition of sIL-2Rα, the aptamers become desorbed from the AuNPs, and this in turn weakens the absorption corresponding to AuNP-catalyzed oxidation of ortho-phenylenediamine (oPD) with H2 O2 . The aptasensor was characterized by TEM imaging, ζ potential measurements, dynamic light scattering (DLS) analysis, and UV/Vis spectrometry, followed by further optimization. The fabricated sensor exhibited great analytical performance, with a linear range of 1 to 100 nm and a detection limit of 1 nm both in buffer and in spiked human serum within 25 min. Other proteins, such as bovine serum albumin (BSA), IL-17Rα, IL-5Rα, IL-13Rα2 , and CD166, showed negligible effects on the aptasensor. Thanks to the great advantages of the aptamers and AuNPs, this aptasensor provides a rapid, simple, and inexpensive process that might offer insights into various diagnostic applications of sIL-2Rα.

Well-ordered bioreceptors on atomically flat Au surfaces can be a high-performance biosensor. Cardiac troponin I proteins (cTnIs) have been regarded as a specific biomarker for acute myocardial infarction (AMI). Here, we report the accurate detection of cTnIs using an aptamer-immobilized Au nanoplate platform. The single-crystalline and atomically flat Au nanoplate was characterized by atomic force microscopy. For the precise detection of cTnI, we immobilized an aptamer that can strongly bind to cTnI onto an atomically flat Au nanoplate. Using the aptamer-immobilized Au nanoplate, cTnIs were successfully detected at a concentration of 100 aM (2.4 fg/mL) in buffer solution. Furthermore, cTnIs in serum could be identified at a concentration of 100 fM (2.4 pg/mL). The total assay time was ~7 h. Importantly, the aptamer-immobilized Au nanoplate enabled us to diagnose AMI patients accurately, suggesting the potential of the present method in the diagnosis of AMI.

Propionibacterium acnes is a lipophilic commensal bacterium mainly found on the skin and in the gastrointestinal tract. Pathophysiological effects of P. acnes have recently been reported not only in acne progression but in various diseases. As an emerging mode of bacterial communication, extracellular vesicles (EVs) have been demonstrated to conduct critical pathophysiological functions. To provide information on P. acnes lipid composition for the first time, we conducted a comparative lipidomic analysis of P. acnes and P. acnes EVs and identified 214 lipids with high confidence using triplicated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. P. acnes EVs contained substantially more PCs, DGs, PAs, PEs, LPAs, LPCs, and MGs than P. acnes, and contained fewer PSs, SO1Ps, SA1Ps, LPGs, LPIs, and LPSs. Distinctively, P. acnes EVs possessed a markedly reduced amount of TG. These findings will provide useful clues for understanding the biological and pathophysiological mechanisms of P. acnes and for clinical applications such as vaccine development, diagnostics and therapeutics.

Patients with pancreatic and colorectal cancers have elevated levels of activated leukocyte cell adhesion molecule (ALCAM) in their blood compared to healthy individuals. To this end, sensitive detection of soluble fraction of ALCAM (sALCAM), also called CD166, was performed using enzyme-linked antibody aptamer (ELAA) assay. For the assay, aptamer specific for sALCAM was used as a capturing probe and polyclonal antibody modified with an enzyme was used as a detecting probe for colorimetric visualization. The ELAA assay was able to detect sALCAM as low as 0.31 ng/mL. In addition, the assay enabled the differentiation of sALCAM from other proteins in buffer as well as in human serum. The ELAA assay provides cost-effective, highly sensitive, and reproducible detection of sALCAM.

Electrochemical biosensors using five anticancer drug and lipid molecules attached on the conducting polymer layer to obtain the orientation of drug molecules toward cancer cells, were evaluated as sensing materials and their performances were compared. Conjugation of the drug molecules with a lipid, phosphatidylcholine (PC) has enhanced the sensitivity towards leukemia cells and differentiates cancer cells from normal cells. The composition of each layer of sensor probe was confirmed by electrochemical and surface characterization experiments. Both impedance spectroscopy and voltammetry show the enhanced interaction of leukemia cells using the drug/lipid modified sensor probe. As the number of leukemia cells increased, the charge transfer resistance (R ct ) in impedance spectra increased and the amine oxidation peak current of drug molecules in voltammograms decreased at around 0.7–1.0 V. Of test drug molecules, raltitrexed (Rtx) showed the best performance for the cancer cells detection. Cancer and normal cell lines from different origins were examined to evaluate the degree of expression of folate receptors (FR) on cells surface, where cervical HeLa cell line was found to be shown the highest expression of the receptor. Impedance and chronoamperometric experiments for leukemia cell line (Jurkat E6-1) showed linear dynamic ranges of 1.0×10 3 –2.5×10 5 cells/mL and 1.0×10 3 –8.0×10 3 cells/mL with detection limits of 68±5 cells/mL and 21±3 cells/mL, respectively.

The emergence of a plant vascular system was a prerequisite for the colonization of land; however, it is unclear how the photosynthate transporting system was established during plant evolution. Here, we identify a novel translational regulatory module for phloem development involving the zinc-finger protein JULGI (JUL) and its targets, the 5' untranslated regions (UTRs) of the SUPPRESSOR OF MAX2 1-LIKE4/5 (SMXL4/5) mRNAs, which is exclusively conserved in vascular plants. JUL directly binds and induces an RNA G-quadruplex in the 5' UTR of SMXL4/5, which are key promoters of phloem differentiation. We show that RNA G-quadruplex formation suppresses SMXL4/5 translation and restricts phloem differentiation. In turn, JUL deficiency promotes phloem formation and strikingly increases sink strength per seed. We propose that the translational regulation by the JUL/5' UTR G-quadruplex module is a major determinant of phloem establishment, thereby determining carbon allocation to sink tissues, and that this mechanism was a key invention during the emergence of vascular plants.

Structural elements are key elements for understanding single-stranded nucleic acid folding. Although various RNA structural elements have been documented, structural elements of single-stranded DNA (ssDNA) have rarely been reported. Herein, we determined a crystal structure of PvLDH in complex with a DNA aptamer called pL1. This aptamer folds into a hairpin-bulge contact by adopting three novel structural elements, viz, DNA T-loop-like motif, base–phosphate zipper, and DNA G·G metal ion zipper. Moreover, the pL1:PvLDH complex shows unique properties compared with other protein:nucleic acid complexes. Generally, extensive intermolecular hydrogen bonds occur between unpaired nucleotides and proteins for specific recognitions. Although most protein-interacting nucleotides of pL1 are unpaired nucleotides, pL1 recognizes PvLDH by predominant shape complementarity with many bridging water molecules owing to the combination of three novel structural elements making protein-binding unpaired nucleotides stable. Moreover, the additional set of Plasmodium LDH residues which were shown to form extensive hydrogen bonds with unpaired nucleotides of 2008s does not participate in the recognition of pL1. Superimposition of the pL1:PvLDH complex with hLDH reveals steric clashes between pL1 and hLDH in contrast with no steric clashes between 2008s and hLDH. Therefore, specific protein recognition mode of pL1 is totally different from that of 2008s.

SummaryThe 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. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR.

Finding a highly sensitive diagnostic technique for malaria has challenged scientists for the last century. In the present study, we identified versatile single-strand DNA aptamers for Plasmodium lactate dehydrogenase (pLDH), a biomarker for malaria, via the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The pLDH aptamers selectively bound to the target proteins with high sensitivity (K(d)=16.8-49.6 nM). The selected aptamers were characterized using an electrophoretic mobility shift assay, a quartz crystal microbalance, a fluorescence assay, and circular dichroism spectroscopy. We also designed a simple aptasensor using electrochemical impedance spectroscopy; both Plasmodium vivax LDH and Plasmodium falciparum LDH were selectively detected with a detection limit of 1 pM. Furthermore, the pLDH aptasensor clearly distinguished between malaria-positive blood samples of two major species (P. vivax and P. falciparum) and a negative control, indicating that it may be a useful tool for the diagnosis, monitoring, and surveillance of malaria.

Recently, aptamers have attracted the attention of many scientists, because they not only have all of the advantages of antibodies, but also have unique merits, such as thermal stability, low cost, and unlimited applications. In this review, we present the reasons why aptamers are known as alternatives to antibodies. Furthermore, several types of in vitro selection processes, including nitrocellulose membrane filtration, affinity chromatography, magnetic bead, and capillary electrophoresis-based selection methods, are explained in detail. We also introduce various applications of aptamers for the diagnosis of diseases and detection of small molecules. Numerous analytical techniques, such as electrochemical, colorimetric, optical, and mass-sensitive methods, can be utilized to detect targets, due to convenient modifications and the stability of aptamers. Finally, several medical and analytical applications of aptamers are presented. In summary, aptamers are promising materials for diverse areas, not just as alternatives to antibodies, but as the core components of medical and analytical equipment.

The angiotensin II type I receptor (AGTR1) has been implicated in diverse aspects of human disease, from the regulation of blood pressure and cardiovascular homeostasis to cancer progression. We sought to investigate the role of AGTR1 in cell proliferation, epithelial-mesenchymal transition (EMT), migration, invasion, angiogenesis and tumor growth in the breast cancer cell line MCF7. Stable overexpression of AGTR1 was associated with accelerated cell proliferation, concomitant with increased expression of survival factors including poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP), as well as extracellular signal-regulated kinase (ERK) activation. AGTR1-overexpressing MCF7 cells were more aggressive than their parent line, with significantly increased activity in migration and invasion assays. These observations were associated with changes in EMT markers, including reduced E-cadherin expression and increased p-Smad3, Smad4 and Snail levels. Treatment with the AGTR1 antagonist losartan attenuated these effects. AGTR1 overexpression also accelerated tumor growth and increased Ki-67 expression in a xenograft model. This was associated with increased tumor angiogenesis, as evidenced by a significant increase in microvessels in the intratumoral and peritumoral areas, and enhanced tumor invasion, with the latter response associated with increased EMT marker expression and matrix metallopeptidase 9 (MMP-9) upregulation. In vivo administration of losartan significantly reduced both tumor growth and angiogenesis. Our findings suggest that AGTR1 plays a significant role in tumor aggressiveness, and its inhibition may have therapeutic implications.

SUMMARY Recent studies have shown that stromal cell derived factor-1 (SDF-1), first known as a cytokine involved in recruiting stem cells into injured organs, confers myocardial protection in myocardial infarction, which is not dependent on stem cell recruitment but related with modulation of ischemia-reperfusion (I/R) injury. However, the effect of SDF has been studied only in a preischemic exposure model, which is not clinically relevant if SDF is to be used as a therapeutic agent. Our study was aimed at evaluating whether or not SDF-1 confers cardioprotection during the reperfusion period. Hearts from SD rats were isolated and perfused with the Langendorff system. Proximal left coronary artery ligation, reperfusion, and SDF perfusion in KH buffer was done according to study protocol. Area of necrosis (AN) relative to area at risk (AR) was the primary endpoint of the study. Significant reduction of AN/AR by SDF in an almost dose-dependent manner was noted during both the preischemic exposure and reperfusion periods. In particular, infusion of a high concentration of SDF (25 nM/L) resulted in a dramatic reduction of infarct size, which was greater than that achieved with ischemic pre- or postconditioning. SDF perfusion during reperfusion was associated with a similar significant reduction of infarct size as preischemic SDF exposure. Further studies are warranted to assess the potential of SDF as a therapeutic agent for reducing I/R injury in clinical practice.

Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ssDNA excision tracts.

The ability of MutS to recognize mismatched DNA is required to initiate a mismatch repair (MMR) system. ATP binding and hydrolysis are essential in this process, but their role in MMR is still not fully understood. In this study, steady-state ATPase activities of MutS from Escherichia coli were investigated using the spectrophotometric method with a double end-blocked heteroduplex containing gapped bases. The ATPase activities of MutS increased as the number of gapped bases increased in a double end-blocked heteroduplex with 2-8 gapped bases in the chain, indicating that MutS dissociates from DNA when it reaches a scission during movement along the DNA. Since movement of MutS along the chain does not require extensive ATP hydrolysis and the ATPase activity is only enhanced when MutS dissociates from a heteroduplex, these results support the sliding clamp model in which ATP binding by MutS induces the formation of a hydrolysis-independent sliding clamp.

A fluorescent method was developed for the detection of unpaired and mismatched DNAs using a MutS-fluorophore conjugate. The fluorophore, 2-(4'-(iodoacetoamido)anilino) naphthalene-6-sulfonic acid (IAANS), was site-specifically attached to the 469 position of Thermus aquaticus (Taq.) MutS mutant (C42A/T469C). The fluorophore labeled residue located at the dimer interface of the protein undergoes a drastic conformational change upon binding with mismatched DNA. The close proximity of the two identical fluorescent molecules presumably causes the self-quenching of the fluorophore, since fluorescence emission of the biosensor decreases with increasing concentrations of mismatched DNA. The order of binding affinity for each unpaired and mismatched DNA obtained by this method was DeltaT (Kd=52 nM)>GT (62 nM)>DeltaC (130 nM)>CT (160 nM)>DeltaG (170 nM)>DeltaA (250 nM)>CC (720 nM)>AT (950 nM). This order is comparable to the previous results of the gel mobility shift assay. Thus, this method can be a simple, useful tool for elucidating the mechanism of DNA mismatch repair as well as a novel probe for detecting of genetic mutation.

Polymyxin B, a cationic antimicrobial peptide has been isolated from Paenibacillus polymyxa. The cationic peptide has polycationic, amphipathic and cyclic unique features. These characteristics of the cationic antimicrobial peptide are thought to contribute to the destruction of gram-negative bacteria by through an initial interaction with the negatively charged surface molecule lipopolysaccharide (LPS), which leads to self promoted uptake across the outer membrane into the negatively charged cytoplasmic membrane of the bacterium. This leads to membrane perturbation and probably translocation of the peptide across the membrane. Some gram-negative bacteria resistant to polymyxin B possess mechanism that modify the LPS through neutralization of its negative charge, which decreases the binding affinity of polymyxin B. Several of the genes regulated by PhoP/PhoQ and PmrA/ PmrB encode enzymes that modify either the acyl chain region or the phosphate group of LPS. In Salmonella, polymyxin B resistance is mainly controlled by the PmrA/PmrB two-component system (Figure 1). PmrA-activated genes encode periplasmic and cause the modification of the LPS. Transcription of PmrA-activated genes is not only promoted by high levels of extra-cytoplasmic Fe, which is sensed by the PmrB protein, also it is activated by the PhoP/PhoQ system in response to low levels of extra-cytoplasmic Mg. For activation of PmrA at the low level Mg condition not only requires the noncognate sensor PhoQ protein but also its cognate regulator PhoP, and the PhoP activated PmrD gene. The PmrD protein appears to control the PmrA/PmrB system at a post-transcriptional level. The PmrD protein activates the PmrA/PmrB system via a protection mechanism through dephosphorylation of PmrA. The protective activity of PmrD protein is suggestive of the behavior displayed by certain members of the 14-3-3 protein family in eukaryotes to prevent the phosphorylated state of CDC25. It has recently been reported that the differential regulation of homologous genes is the mechanism responsible for the divergence of Salmonella enterica and E. coli in their ability to make LPS modifications and mediate resistance to polymyxin B. In contrast to Salmonella, E. coli K-12 induces PmrA-activated gene transcription and polymyxin B resistance in response to Fe, but it makes no response to low levels of Mg. This discrepancy suggests that the structure of E.coli PmrD differs from Salmonella PmrD. In particular, the N-terminal region of PmrD, which differs between the E.coli and Salmonella proteins, is in part responsible for the distinct behaviors of the PmrD proteins (Figure 2). In the present report, we describe the crystallization and preliminary X-ray analysis of E. coli PmrD.

Background and objectives Information about the role of the stromal cell-derived factor-1α (SDF-1α)/chemokine receptor type 4 (CXCR4) axis in ischemic postconditioning (IPOC) is currently limited. We hypothesized that the SDF-1α/CXCR4 signaling pathway is directly involved in the cardioprotective effect of IPOC. Methods Isolated rat hearts were divided into four groups. The control group was subjected to 30-min of regional ischemia and 2-hour of reperfusion (n=12). The IPOC group was induced with 6 cycles of 10-second reperfusion and 10-second global ischemia (n=8) in each cycle. The CXCR4 antagonist, AMD3100, was applied before reperfusion in the IPOC group (AMD+IPOC group, n=11) and control group (AMD group, n=9). Hemodynamic changes with electrocardiography were monitored and infarct size was measured. The SDF-1α, lactate dehydrogenase (LDH) and creatine kinase (CK) concentrations in perfusate were measured. We also analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation state expression. Results IPOC significantly reduced infarct size, but AMD3100 attenuated the infarct reducing effect of IPOC. IPOC significantly decreased LDH and CK, but these effects were reversed by AMD3100. ERK1/2 and Akt phosphorylation increased with IPOC and these effects were blocked by AMD3100. Conclusion Based on the results of this study, SDF-1α/CXCR4 signaling may be involved in IPOC cardioprotection and this signaling pathway couples to the ERK1/2 and Akt pathways.

It is well known that the engrailed-2 (EN2) protein, a biomarker for prostate cancer, strongly binds to a specific DNA sequence (5'-TAATTA-3') to regulate transcription. Based on this intrinsic property, DNA probes with additional flanked sequences were designed and optimized. Various measurements, such as electrophoresis mobility shift assay, surface plasmon resonance, and quantitative fluorescence assay were performed to investigate the feasibility of the DNA probes. Then, the affinities of the DNA probes to the target protein were quantitatively determined using FAM-modified DNA probes and magnetic beads, resulting in dissociation constants ranging from 61.03 to 98.84nM. To develop an early diagnosis platform for prostate cancer, an ultrasensitive electrochemical biosensor based on the electrodeposition of gold nanoparticles was designed. The EN2 protein was quantitatively detected using the electrochemical biosensor, and the calculated detection limit was found to be 5.62fM. Finally, the specificity and applicability of the biosensor were verified using several proteins and an artificial urine medium. The impedance signals increased in the cases of EN2, suggesting that the system exhibited high selectivity to only EN2.

The MutL DNA mismatch repair protein has recently been shown to be an ATPase and to belong to an emerging ATPase superfamily that includes DNA topoisomerase II and Hsp90. We report here the crystal structures of a 40 kDa ATPase fragment of E. coli MutL (LN40) complexed with a substrate analog, ADPnP, and with product ADP. More than 60 residues that are disordered in the apoprotein structure become ordered and contribute to both ADPnP binding and dimerization of LN40. Hydrolysis of ATP, signified by subsequent release of the gamma-phosphate, releases two key loops and leads to dissociation of the LN40 dimer. Dimerization of the LN40 region is required for and is the rate-limiting step in ATP hydrolysis by MutL. The ATPase activity of MutL is stimulated by DNA and likely acts as a switch to coordinate DNA mismatch repair.

MutL and its homologs are essential for DNA mismatch repair. Mutations in genes encoding human homologs of MutL cause multiorgan cancer susceptibility. We have determined the crystal structure of a 40 kDa N-terminal fragment of E. coli MutL that retains all of the conserved residues in the MutL family. The structure of MutL is homologous to that of an ATPase-containing fragment of DNA gyrase. We have demonstrated that MutL binds and hydrolyzes ATP to ADP and Pi. Mutations in the MutL family that cause deficiencies in DNA mismatch repair and a predisposition to cancer mainly occur in the putative ATP-binding site. We provide evidence that the flexible, yet conserved, loops surrounding this ATP-binding site undergo conformational changes upon ATP hydrolysis thereby modulating interactions between MutL and other components of the repair machinery.

Malaria, a major burden of disease caused by parasites of the genus Plasmodium, is widely spread in tropical and subtropical regions. Here, we have successfully developed a diagnostic technique for malaria. The proposed method is based on the interaction among the Plasmodium lactate dehydrogenase (pLDH), which is a biomarker for malaria, and pL1 aptamer against Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum lactate dehydrogenase (PfLDH). In addition, the cationic polymers, poly(diallyldimethylammonium chloride) (PDDA) and poly(allylamine hydrochloride) (PAH), aggregate gold nanoparticles (AuNPs) that should be possible to observe the change in color from red to blue, which depends on the concentration of pLDH. Using this aptasensor, pLDH proteins were successfully detected with low detection limits. Moreover, the specificity test proved that the aptasenor is very specific in targeting proteins over other interfering proteins. In addition, the pLDH from infected blood samples of the two main species of malaria were also detected. The limits of detection for P. vivax were determined as 80 parasites/μl for PDDA and 74 parasites/μl for PAH. The aptasenor has great advantages that can simply and rapidly diagnose malaria. Thus, the developed aptasensor for detection of pLDH can offer an effective and sensitive diagnosis of malaria.

A rapid, homogeneous, and in-situ labelable format designed to detect single nucleotide polymorphism (SNP) is presented. Fluorescent silver nanoclusters produced following hydride-mediated reduction of Ag(+) bound to a partial double-stranded oligodeoxynucleotide were employed as probes for SNPs. The sensing mechanism is based on the fluorescence enhancement of silver nanoclusters through an irreversible cluster transfer upon hybridization. The key element of our design is modulating the "turn on" mechanism by introducing a competitor that can be displaced in response to a target DNA. In a controlled model system, the fluorescence intensity of silver nanoclusters is approximately 3-fold enhanced upon hybridization with a perfectly matched target DNA, and single-base mismatch detection is achieved within 5min, regardless of the position of mismatches. Human aldehyde dehydrogenase 2 (ALDH2), which is responsible for the oxidation of aldehydes to carboxylic acids, is then utilized as a target for SNP. Upon addition of a perfectly matched sequence, dramatically enhanced fluorescence intensity is observed (ca. 48-fold), and prompt SNP genotyping is accomplished.

The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results.

The crystal structure of the recombinant calmodulin from Paramecium tetraurelia (rPCaM, M(r) = 16 700, 148 residues) has been determined at 1.68 A resolution. X-ray intensity data were collected at 263 K using a Siemens-Nicolet area detector and Cu Kalpha radiation from a rotating-anode source. A total of 35 936 observations were processed with XENGEN1.3 and scaled to yield 16 255 unique reflections with R(symm)(I) of 4.1%. The crystals are triclinic, with unit-cell dimensions a = 29.89, b = 53.42, c = 25.35 A, alpha = 93.67, beta = 96.88, gamma = 89.24 degrees, space group P1, with one molecule in the unit cell. The atomic coordinates of the wild-type Paramecium calmodulin (PCaM) studied in our laboratory provided the starting model. Refinement of the structure by X-PLOR and refitting it into omit maps yielded an R value of 0.194 for 15 965 reflections greater than 3sigma(F) in the 6.0-1.68 A resolution range. The final model contained 1165 protein atoms for all of the 148 residues, four Ca(2+) ions, and 172 water molecules. The dumbbell structure has seven alpha-helices including a long 7.8 turn central helix connecting the two terminal domains each containing two EF-hand (helix-loop-helix motif) calcium-binding sites. The loops within each pair of EF-hand motifs in the N- and C-terminal domains are brought into juxtaposition to form a pair of hydrogen-bonded antiparallel beta-sheets which are extended at either ends by water bridges. The four calcium-binding EF-hands are superposable with r.m.s. deviations of 0.31-0.79 A. The best agreement is between site 1 and site 3 and the worst agreement is between site 1 and 4. The largest differences are in the ninth and tenth residues of the calcium-binding loops probably because of their involvement in the mini beta-sheets. The calcium coordination distances vary between 2.04 and 2.69 A, average 2.34 A. The rPCaM and wild-type PCaM have an r.m.s. deviation of 0.36 A for equivalent C(alpha) atoms. The side chains of Lys13 and Lys115 are more extended in rPCaM compared to the wild type where the post-translational modified di- and tri-methylated lysine residues are more folded. The sequence of PCaM differs from those of mammalian (MCaM) and Drosophila calmodulin (DCaM), but the overall structures are very similar, with r.m.s,. deviations of 0.44 and 1.68 A for equivalent C(alpha) atoms, respectively. However, in rPCaM, the first four N-terminal residues stretch out and make intermolecular crystal contacts, in contrast to those in recombinant Drosophila calmodulin (rDCaM), they stretch out in the opposite direction and towards the second calcium-binding site (see note below), while in MCaM and wild-type PCaM, the N-terminal residues are not visible. The central helix in rPCaM has all its backbone hydrogen bonds intact with no unusually long separation between the carbonyl and amide groups as found in MCaM and rDCaM.

The crystal structure of the self-complementary chimeric decamer duplex r(C)d(CGGCGCCG)r(G), with RNA base pairs at both termini, has been solved at 1.9 A resolution by the molecular replacement method and refined to an R value of 0.145 for 2,314 reflections. The C3'-endo sugar puckers of the terminal riboses apparently drive the entire chimeric duplex into an A-DNA conformation, in contrast to the B-DNA conformation adopted by the all-deoxy decamer of the same sequence. Five symmetry related duplexes encapsulate a spermine molecule which interacts with ten phosphate groups, both directly and through water molecules to form multiple ionic and hydrogen bonding interactions. The spermine interaction severely bends the duplexes by 31 degrees into the major groove at the fourth base pair G(4).C(17), jolts it and slides the 'base plate' into the minor groove. This base pair, together with the adjacent base pair in the top half and the corresponding pseudo two-fold related base pairs in the bottom half, form four minor groove base-paired multiples with the terminal base pairs of two neighboring duplexes.

We have designed multiple detection systems for the DNA strand exchange process. Thermostable Thermotoga maritima recombinase A (TmRecA), a core protein in homologous recombination, and DNAzyme, a catalytic DNA that can cleave a specific DNA sequence, are introduced in this work. In a colorimetric method, gold nanoparticles (AuNPs) modified with complementary DNAs (cDNAs) were assembled by annealing. Aggregated AuNPs were then separated irreversibly by TmRecA and DNAzyme, leading to a distinct color change in the particles from purple to red. For the case of fluorometric detection, fluorescein isothiocyanate (FITC)-labeled DNA as a fluorophore and black hole quencher 1 (BHQ1)-labeled DNA as a quencher were used; successful strand exchange was clearly detected by variations in fluorescence intensity. In addition, alterations in the impedance of a gold electrode with immobilized DNA were employed to monitor the regular exchange of DNA strands. All three methods provided sufficient evidence of efficient strand exchange reactions and have great potential for applications in the monitoring of recombination, discovery of new DNAzymes, detection of DNAzymes, and measurement of other protein activities.

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K(D) = 29 nM) while it dramatically decreases above 100 mM (K(D)>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of nonspecific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without nonspecific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

The effect of wild-type and mutant MutL on the steady-state ATPase activity of MutS from Escherichia coli has been investigated in the absence and presence of 22, 50, and 75 base pair hetero- and homoduplex DNAs with open and blocked ends. The steady-state ATPase activity of MutS has been measured at 37 degrees C using a spectrophotometric method. The presence of MutL did not affect appreciably on the ATPase activity of MutS in the absence of DNA or in the presence of blocked end homoduplex DNAs. However, the addition of MutL affected oppositely on the ATPase activity of MutS in the presence of G-T mismatched DNAs depending on their end status. We have also found that only the ATPase active forms of MutL increased the ATPase activity of MutS in the presence of G-T mismatched DNAs with blocked ends. The results suggest that MutL ATPase activity is required to catalyze dissociation of the MutS sliding clamps.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19–base-pair duplex region with 3′ overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand–mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.

MutL is required to assist the mismatch repair protein MutS during initiation of the methyl-directed mismatch repair (MMR) response in various organisms ranging from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity of MutL to aggregate has led to significant difficulties in determining its biological relationship with other MMR-related proteins. Here, we perform analysis on the thermostable MutL protein found in Thermotoga maritima MSB8 (TmL). Size exclusion chromatographic analysis indicates the lack of aggregated forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS) analysis reveals that the solution structures of the full-length TmL and its corresponding complexes with nucleotides and ssDNA undergo conformational changes. The elucidated TmL SAXS model is superimposed to the crystal structure of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal SAXS model of TmL exists as monomeric form, indicating that TmL has a structurally flexible N-terminal domain. TmL SAXS analysis can suggest a considerable possibility on a new 3D view of the previously unresolved full-length MutL molecule.

The molecular mechanisms of the DNA mismatch repair (MMR) system have been uncovered over the last decade, especially in prokaryotes. The results obtained for prokaryotic MMR proteins have provided a framework for the study of the MMR system in eukaryotic organisms, such as yeast, mouse and human, because the functions of MMR proteins have been conserved during evolution from bacteria to humans. However, mutations in eukaryotic MMR genes result in pleiotropic phenotypes in addition to MMR defects, suggesting that eukaryotic MMR proteins have evolved to gain more diverse and specific roles in multicellular organisms. Here, we summarize recent advances in the understanding of both prokaryotic and eukaryotic MMR systems and describe various new functions of MMR proteins that have been intensively researched during the last few years, including DNA damage surveillance and diversification of antibodies.

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.

MutL stimulates the DNA duplex unwinding activity of UvrD in methyl-directed DNA mismatch repair (MMR) via their physical interactions. However, the molecular functions of MutL associated with the DNA binding and UvrD helicase have been partially understood. We present the kinetic characteristics of the single-stranded DNA (ssDNA) binding activity of MutL in the absence or the presence of UvrD helicases using the single-molecule techniques. The lengthening of the ssDNA due to the ssDNA binding of MutL allows us to observe association and dissociation of MutL from the ssDNA in real-time. In this study, we demonstrate that the nonspecific ssDNA binding of MutL can be involved in subsequent loading of UvrD helicases onto the ssDNA in a manner independent of ATP hydrolysis of MutL.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

MutS, MutL and MutH are the three essential proteins for initiation of methyl‐directed DNA mismatch repair to correct mistakes made during DNA replication in Escherichia coli . MutH cleaves a newly synthesized and unmethylated daughter strand 5′ to the sequence d(GATC) in a hemi‐methylated duplex. Activation of MutH requires the recognition of a DNA mismatch by MutS and MutL. We have crystallized MutH in two space groups and solved the structures at 1.7 and 2.3 A resolution, respectively. The active site of MutH is located at an interface between two subdomains that pivot relative to one another, as revealed by comparison of the crystal structures, and this presumably regulates the nuclease activity. The relative motion of the two subdomains in MutH correlates with the position of a protruding C‐terminal helix. This helix appears to act as a molecular lever through which MutS and MutL may communicate the detection of a DNA mismatch and activate MutH. With sequence homology to Sau 3AI and structural similarity to Pvu II endonuclease, MutH is clearly related to these enzymes by divergent evolution, and this suggests that type II restriction endonucleases evolved from a common ancestor.

A direct and label-free electrochemical biosensor for the detection of the protein-mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GTCTCC approximately AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.

Background Polymyxin B resistance protein D (PmrD) plays a key role in the polymyxin B-resistance pathway, as it is the signaling protein that can act as a specific connecter between PmrA/PmrB and PhoP/PhoQ. We conducted structural analysis to characterize Escherichia coli (E. coli) PmrD, which exhibits different features compared with PmrD in other bacteria. Results The X-ray crystal structure of E. coli PmrD was determined at a 2.00 Å resolution, revealing novel information such as the unambiguous secondary structures of the protein and the presence of a disulfide bond. Furthermore, various assays such as native gel electrophoresis, surface plasmon resonance (SPR), size-exclusion chromatography, dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS) measurements, were performed to elucidate the structural and functional role of the internal disulfide bond in E. coli PmrD. Conclusions The structural characteristics of E. coli PmrD were clearly identified via diverse techniques. The findings help explain the different protective mechanism of E. coli compared to other Gram-negative bacteria. Electronic supplementary material The online version of this article (doi:10.1186/s12900-014-0024-y) contains supplementary material, which is available to authorized users.

It has been studied that DNA repair proteins search a target via a 1-dimensional diffusion along naked DNA. Due to the absence of the target on DNA this single-molecule tracking approach lacked of understanding the catalytic function of the repair proteins and moreover the mechanism of the downstream transactions for the repair of an error on the DNA. We present the catalytic processes of MutS, mismatch repair initiation protein, on a mismatched DNA that bears an unpaired nucleotide. Our single-molecule analysis reveals that MutS undergoes two distinct conformational changes during DNA mismatch repair (MMR). MutS forms a transient clamp that scans duplex DNA for the unpaired nucleotide and a different sliding clamp that is induced by ATP binding to the mismatch-bound MutS with unusual stability on DNA. These observations suggest a mechanism of how MMR machinery can be recruited for the strand incision, which is highly controversial.