A novel class of polymeric fluorescent dyes assembled using a DNA synthesizer.

In the pursuit of a novel class of fluorescent dyes we have developed a programmable polymer system that enables the rational design and control of macromolecular constructs through simple control of polymer primary sequence. These polymers are assembled using standard phosphoramidite chemistry on a DNA synthesizer which allows for extremely rapid prototyping and enables many permutations due to the large selection of phosphoramidite monomers presently available on the market. This programmability to some extent allows us to control the interactions/spacing of payload molecules distributed along the designed polymeric backbone. Control of molecular architecture using this technology has allowed us to address the long-standing technical issue of contact quenching between fluorescent dyes offering new possibilities in the life sciences arena. Much like peptidic sequences coding for enzymes, cofactors, and receptors (all needing control of tertiary structure for proper function via primary sequence) our programmable system approaches a similar endpoint using a phosphate based polymeric backbone assembled in a completely automated fashion. Using this novel technology, we have efficiently synthesized several types of fluorescent dyes and demonstrated the programmability in molecule design, including the increases in brightness of the fluorescence emission.

iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.

The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.

A 4.8-µVrms-Noise CMOS-Microelectrode Array With Density-Scalable Active Readout Pixels via Disaggregated Differential Amplifier Implementation.

We demonstrate a 4.8-µVrms noise microelectrode array (MEA) based on the complementary-metal-oxide-semiconductor active-pixel-sensors readout technique with disaggregated differential amplifier implementation. The circuit elements of the differential amplifier are divided into a readout pixel, a reference pixel, and a column circuit. This disaggregation contributes to the small area of the readout pixel, which is less than 81 µm2. We observed neuron signals around 100 µV with 432 electrodes in a fabricated prototype chip. The implementation has technological feasibility of up to 12-µm-pitch electrode density and 6,912 readout channels for high-spatial resolution mapping of neuron network activity.

Image-based evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: Correlation and complementarity with extracellular electrophysiology.

In this study, we used high-speed video microscopy with motion vector analysis to investigate the contractile characteristics of hiPS-CM monolayer, in addition to further characterizing the motion with extracellular field potential (FP), traction force and the Ca(2+) transient. Results of our traction force microscopy demonstrated that the force development of hiPS-CMs correlated well with the cellular deformation detected by the video microscopy with motion vector analysis. In the presence of verapamil and isoproterenol, contractile motion of hiPS-CMs showed alteration in accordance with the changes in fluorescence peak of the Ca(2+) transient, i.e., upstroke, decay, amplitude and full-width at half-maximum. Simultaneously recorded hiPS-CM motion and FP showed that there was a linear correlation between changes in the motion and field potential duration in response to verapamil (30-150nM), isoproterenol (0.1-10µM) and E-4031 (10-50nM). In addition, tetrodotoxin (3-30µM)-induced delay of sodium current was corresponded with the delay of the contraction onset of hiPS-CMs. These results indicate that the electrophysiological and functional behaviors of hiPS-CMs are quantitatively reflected in the contractile motion detected by this image-based technique. In the presence of 100nM E-4031, the occurrence of early after-depolarization-like negative deflection in FP was also detected in the hiPS-CM motion as a characteristic two-step relaxation pattern. These findings offer insights into the interpretation of the motion kinetics of the hiPS-CMs, and are relevant for understanding electrical and mechanical relationship in hiPS-CMs.

Serial intermediates with a 1 nt 3'-flap and 5' variable-length flaps are formed by cooperative functioning of Pyrococcus horikoshii FEN-1 with either B or D DNA polymerases.

Flap endonuclease-1 (FEN-1) plays important roles with DNA polymerases in DNA replication, repair and recombination. FEN-1 activity is elevated by the presence of a 1 nucleotide expansion at the 3' end in the upstream primer of substrates called "structures with a 1 nt 3'-flap", which appear to be the most preferable substrates for FEN-1; however, it is unclear how such substrates are generated in vivo. Here, we show that substrate production occurred by the cooperative function of FEN-1(phFEN-1) and Pyrococcus horikoshii DNA polymerase B (phPol B) or D (phPol D). Using various substrates, the activities of several phFEN-1 F79 mutants were compared with those of the wild type. Analysis of the activity profiles of these mutants led us to discriminate "structures with a 1 nt 3'-flap" from substrates with a 3' -projection longer than 2 nt or from those without a 3'-projection. When phFEN-1 processed a gap substrate with phPol B or phPol D, "structures with a 1 nt 3'-flap" were assumed the reaction intermediates. Furthermore, the phFEN-1 cleavage products with phPol B or D were from 1mer to 7mer, corresponding to the sizes of the strand-displacement products of these polymerases. This suggests that a series of 1 nt 3'-flap with 5'-variable length-flap configurations were generated as transient intermediates, in which the length of the 5'-flaps depended on the displacement distance of the downstream strand by phPol B or D. Therefore, phFEN-1 might act successively on displaced 5'-variable flaps.

Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin.

Domain structures and inter-domain interactions defining the holoenzyme architecture of archaeal d-family DNA polymerase.

Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.

Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis.

A noninvasive method for the characterization of cardiomyocyte contractile behavior is presented. Light microscopic video images of cardiomyocytes were captured with a high-speed camera, and motion vectors (which have a velocity dimension) were calculated with a high spatiotemporal resolution using a block-matching algorithm. This method could extract contraction and relaxation motions of cardiomyocytes separately and evaluate characteristics such as the beating rate, orientation of contraction, beating cooperativity/homogeneity in the monolayer, and wave propagation of impulses. Simultaneous phase-contrast imaging and calcium (Ca2+) fluorescence measurements confirmed that the timing of the maximum shortening velocity of cardiomyocytes correlated well with intracellular Ca2+ transients. Based on our analysis, gap junction inhibitors, 1-heptanol (2 mM) or 18-ß-glycyrrhetinic acid (30 µM), resulted in clear changes in beating cooperativity and the propagation pattern of impulses in the cardiomyocyte monolayer. Additionally, the time dependence of the motion vector length indicated a prolonged relaxation process in the presence of potassium (K+) channel blockers, dl-sotalol (1 µM), E-4031 (100 nM), or terfenadine (100 nM), reflecting the prolonged QT (Q wave and T wave) interval of cardiomyocytes. Effects of autonomic agents (acetylcholine or epinephrine [EPI]) or EPI and propranolol on cardiomyocytes were clearly detected by the alterations of beating rate and the motion vector length in contraction and relaxation processes. This method was noninvasive and could sensitively evaluate the contractile behavior of cardiomyocytes; therefore, it may be used to study and/or monitor cardiomyocyte tissue during prolonged culture periods and in screens for drugs that may alter the contraction of cardiomyocytes.

Novel structure of an N-terminal domain that is crucial for the dimeric assembly and DNA-binding of an archaeal DNA polymerase D large subunit from Pyrococcus horikoshii.

Archaea-specific D-family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. The N-terminal (1-300) domain structure of the large subunit was determined by X-ray crystallography, although ∼50 N-terminal residues were disordered. The determined structure consists of nine alpha helices and three beta strands. We also identified the DNA-binding ability of the domain by SPR measurement. The N-terminal (1-100) region plays crucial roles in the folding of the large subunit dimer by connecting the ∼50 N-terminal residues with their own catalytic region (792-1163).

Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii.

Archaeal/eukaryotic primases form a heterodimer consisting of a small catalytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N-terminal domain (NTD) of PriL (PriL(NTD); residues 1-222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii, at 2.9 A resolution. The PriL(NTD) structure consists of two subdomains, the helix-bundle and twisted-strand domains. The latter is structurally flexible, and is expected to contain a PriS interaction site. Pull-down and surface plasmon resonance analyses of structure-based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155-Tyr156-Ile157 region near the flexible region is the PriS-binding site, as the Y155A/Y156A/I157A mutation markedly reduces PriS binding, by 1000-fold. These findings and a structural comparison with a previously reported PriL(NTD)-PriS complex suggest that the presented alternative conformations of the twisted-strand domain facilitate the heterodimer assembly.

Molecular structure of a novel membrane protease specific for a stomatin homolog from the hyperthermophilic archaeon Pyrococcus horikoshii.

Membrane-bound proteases are involved in various regulatory functions. Our previous study indicated that the N-terminal region of an open reading frame, PH1510 (residues 16-236, designated as 1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii, is a serine protease with a catalytic Ser-Lys dyad that specifically cleaves the C-terminal hydrophobic residues of a membrane protein, the stomatin-homolog PH1511. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis, but the function of stomatin is not fully understood. Here, we report the crystal structure of 1510-N in dimeric form. Each active site of 1510-N is rich in hydrophobic residues, which accounts for the substrate-specificity. The monomer of 1510-N shows structural similarity to one monomer of Escherichia coli ClpP, an ATP-dependent tetradecameric protease. But, their oligomeric forms are different. Major contributors to dimeric interaction in 1510-N are the alpha7 helix and beta9 strand, both of which are missing from ClpP. While the long handle region of ClpP contributes to the stacking of two heptameric rings, the corresponding L2 loop of 1510-N is disordered because the region has little interaction with other residues of the same molecule. The catalytic Ser97 of 1510-N is in almost the same location as the catalytic Ser97 of E.coli ClpP, whereas another residue, Lys138, presumably forming the catalytic dyad, is located in the disordered L2 region of 1510-N. These findings suggest that the binding of the substrate to the catalytic site of 1510-N induces conformational changes in a region that includes loop L2 so that Lys138 approaches the catalytic Ser97.

Switches from pi- to sigma-bonding complexes controlled by gate voltages.

A conjugated polymer/metal ion/liquid-crystal molecular system was set between source and drain electrodes with a 100 nm gap. When gate voltage (Vg) increases, the current between source and drain electrodes increases. Infrared spectra show this system to be composed of pi and sigma complexes. At Vg = 0, the pi complex dominates the sigma complex, whereas the sigma complex becomes dominant when Vg is switched on. Calculations found that the pi complex has lower conductivity than the sigma complex.

Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii.

Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.

A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.

Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.

Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii.

DNA primases are essential components of the DNA replication apparatus in every organism. Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers. The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S). The 25KL domain has a dual function. One function is to depress the large affinity of the intrasubunit domain 20KL for the template DNA until complex (L(1)S(1) unit) formation. The other function is to tether the L subunit tightly to the S subunit, probably to promote effective interaction between the intrasubunit domain 20KL and the active center of the S subunit. The 20KL domain is a central factor to enhance the de novo DNA synthesis activity of the catalytic S subunit since the total affinity of the L(1)S(1) unit is mainly derived from the affinity of 20KL, which is elevated more than 10 times by the heterodimer formation, presumably due to the cancellation of the inhibitory activity of 25KL through tight binding to the S subunit.

Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii.

The crystal structure of flap endonuclease-1 from Pyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg(40) and Arg(42) on small loop 1, a cluster Lys(193)-Lys(195) on small loop 2, and two sites, Arg(94) and Arg(118)-Lys(119), on the large loop were identified as binding sites. Lys(87) on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg(94) and Lys(193)-Lys(195) located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg(118)-Lys(119) was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.