Large Instrument- and Detergent-Free Assay for Ultrasensitive Nucleic Acids Isolation via Binary Nanomaterial.

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.

Improved Reversible Cross-Linking-Based Solid-Phase RNA Extraction for Pathogen Diagnostics.

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications.

Crosslinking constraints and computational models as complementary tools in modeling the extracellular domain of the glycine receptor.

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel superfamily, is the major inhibitory neurotransmitter-gated receptor in the spinal cord and brainstem. In these receptors, the extracellular domain binds agonists, antagonists and various other modulatory ligands that act allosterically to modulate receptor function. The structures of homologous receptors and binding proteins provide templates for modeling of the ligand-binding domain of GlyR, but limitations in sequence homology and structure resolution impact on modeling studies. The determination of distance constraints via chemical crosslinking studies coupled with mass spectrometry can provide additional structural information to aid in model refinement, however it is critical to be able to distinguish between intra- and inter-subunit constraints. In this report we model the structure of GlyBP, a structural and functional homolog of the extracellular domain of human homomeric α1 GlyR. We then show that intra- and intersubunit Lys-Lys crosslinks in trypsinized samples of purified monomeric and oligomeric protein bands from SDS-polyacrylamide gels may be identified and differentiated by MALDI-TOF MS studies of limited resolution. Thus, broadly available MS platforms are capable of providing distance constraints that may be utilized in characterizing large complexes that may be less amenable to NMR and crystallographic studies. Systematic studies of state-dependent chemical crosslinking and mass spectrometric identification of crosslinked sites has the potential to complement computational modeling efforts by providing constraints that can validate and refine allosteric models.

Imidate-based cross-linkers for structural proteomics: increased charge of protein and peptide ions and CID and ECD fragmentation studies.

Chemical cross-linking is an attractive low-resolution technique for structural studies of protein complexes. Distance constraints obtained from cross-linked peptides identified by mass spectrometry (MS) are used to construct and validate protein models. Amidinating cross-linkers such as diethyl suberthioimidate (DEST) have been used successfully in chemical cross-linking experiments. In this work, the application of a commercial diimidate cross-linking reagent, dimethyl suberimidate (DMS), was evaluated with model peptides and proteins. The peptides were designed with acetylated N-termini followed by random sequences containing two Lys residues separated by an Arg residue. After cross-linking reactions, intra- and intermolecular cross-linked species were submitted to CID and ECD dissociations to study their fragmentation features in the gas phase. Fragmentation of intramolecular peptides by collision induced dissociation (CID) demonstrates a unique two-step fragmentation pathway involving formation of a ketimine as intermediate. Electron capture and electron transfer dissociation (ECD and ETD) experiments demonstrated that the cyclic moiety is not dissociated. Intermolecular species demonstrated previously described fragmentation behavior in both CID and ECD experiments. The charge state distributions (CSD) obtained after reaction with DMS were compared with those obtained with disuccinimidyl suberate (DSS). CSDs for peptides and proteins were increased after their reaction with DMS, owing to the higher basicity of DMS modified species. These features were also observed in LC-MS experiments with bovine carbonic anhydrase II (BCA) after cross-linking with DMS and tryptic proteolysis. Cross-linked peptides derived from this protein were identified at high confidence and those species were in agreement with the crystal structure of BCA.

HMGB1-facilitated p53 DNA binding occurs via HMG-Box/p53 transactivation domain interaction, regulated by the acidic tail.

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic.

[Inhibition of functional activity of macrophages in vitro by dimer RNase Bacillus intermedius].

The influence of cross-linked by dimethylsuberimidate dimeric RNAse from Bacillus intermedius on peritoneal rat macrophages has been investigated in vitro. It has been shown that dimeric RNase with concentrations of 0.5-40.0 mg/ml decreases the functional activities of macrophages. This is manifested in the inhibition of the phagocyte function of macrophages and suppression of the fusion of phagosomes with lysosomes. The change in the cytoplasmatic membrane surface structure induced by the dimers, which is stronger than that induced by monomers, has been demonstrated using atomic force microscopy. The role of membrane properties modification in the inhibition effect of RNase dimers on the functional activities of macrophages is discussed.

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.

High-efficiency solid-phase capture using glass beads bonded to microcentrifuge tubes: immunoprecipitation of proteins from cell extracts and assessment of ras activation.

We have bonded glass microbeads (425-600 microm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A- or G-agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G-agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20-30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A- or G-agarose beads.

Evidence for a dimeric structure of rat liver serine dehydratase.

Rat liver serine dehydratase (SDH) is known to be involved in gluconeogenesis. It has long been believed to be a dimeric protein with the subunit molecular weight (M(r)) of 34,000. Recently, sheep liver SDH was reported to be a monomer with a M(r) of 38,000. The native M(r) of rat SDH was only determined by the ultracentrifugation method more than three decades ago, and that of sheep SDH was done by the method of gel chromatography. The primary to quaternary structures of a given enzyme in a specific mammalian organ are usually conserved among various species. The aim of the present investigation is to clarify the structural differences between rat and sheep SDHs. First, we found that the amino acid composition reported for sheep SDH was statistically similar to that of rat SDH. Second, immunoblot analysis using anti-rat SDH IgG as the probe showed the size of sheep SDH to be a M(r) of 30,500, whereas that of SDH was about M(r) of 35,000. On the other hand, the native size of rat SDH was assessed by two methods: (1) the laser light scattering method demonstrated that rat SDH had a M(r) of 66,800, consistent with the previous value (M(r)=64,000); (2) cross-linking experiments of the purified rat SDH with dimethyl suberimidate revealed the existence of a dimeric form by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present results clearly confirm that rat SDH is a dimer, and suggest that sheep SDH is similar to rat SDH immunologically, but with a molecular weight 7500 smaller than reported previously.

Dimethyl suberimidate cross-linking of oligo(dT) to DNA-binding proteins.

Dimethyl suberimidate is a bifunctional reagent that is used for cross-linking the protein components of oligomeric macromolecules. In this report, dimethyl suberimidate is shown to specifically cross-link oligo(dT) of varying lengths to the DNA-binding subunits of a multimeric helicase-primase encoded by herpes simplex virus type 1. This result indicates that dimethyl suberimidate and other imidoester cross-linking reagents may be useful for characterizing the interaction of oligo(dT) with proteins that bind single-stranded DNA.

Changes in interfacial behaviour, emulsifying and foaming properties of faba bean legumin after modification with dimethylsuberimidate.

The effect of a rising rigidity and surface hydrophobicity of the 11S storage protein from faba beans--legumin--induced by chemical modification with dimethylsuberimidate (DMS) on some surface functional properties was studied. Short-time adsorption kinetics using a droplet-volume tensiometer, pressure transformation and desorption behaviour of monolayer using a film balance, and emulsifying and foaming properties were determined to characterize surface activity and interfacial film forming behaviour. Tensio-active properties at the air-water interface, i.e. decay in surface tension and pressure transformation in monolayer, were improved by modification. However, a decrease in emulsifying activity, foam capacity and foam expansion after modification of the legumin points to an overall deterioration of energy-induced film forming behaviour. The results support the view that surface activity is generally governed more by molecular flexibility than by surface hydrophobicity.

[Polydispersity of Serratia marcescens nuclease at optimal pH values]. / Polidispersnost' nukleazy Serratia marcescens pri optimal'nom znachenii pH.

Treatment with dimethyl suberimidate, a cross-linking bifunctional agent, showed that Sm1 and Sm2 nucleases of Serratia marcescens B10M1 are polydisperse in solution and consist of monomers and dimers at the level of pH optimal for the enzyme activity. The data suggest that nucleases from the strain B10M1 and any other strain are polydisperse at pH optimum if their amino acid sequences are identical.

Crosslinking density and resorption of dimethyl suberimidate-treated collagen.

Collagen was purified from bovine Achilles tendon and crosslinked with dimethyl suberimidate (DMS) and glutaraldehyde (GTA). Under optimal conditions, the shrinkage temperature (Ts) was raised to 74 degrees C for collagen crosslinked with DMS and to 80 degrees C for those crosslinked with GTA. Crosslinking density measurements were done on the hydrothermally denatured collagen by the method based on the Flory-Rehner equation. GTA treatment was found to introduce more number of crosslinks than DMS. The maximum tension attained during heating (after shrinkage has occurred) was greater for GTA-treated collagen than for DMS and control. The control collagen membranes broke during heating (at 73 degrees C), while for the crosslinked membranes the tension kept on increasing up to 100 degrees C. The crosslinking density correlated well with the data determined from the in vitro and in vivo degradation studies. Uncrosslinked and DMS crosslinked collagen membranes were more susceptible to degradation by enzymes in vitro, while GTA-treated collagen was highly resistant to degradation. The biocompatibility of the collagen membranes was studied by subcutaneous implantation in rats. Uncrosslinked collagen membranes degraded within 14 days with the formation of granulation tissue. DMS crosslinked membranes degraded within 21 days and the area was replaced by numerous fibroblasts and newly formed collagen. No calcification was observed. For GTA-treated membranes, necrosis was observed after 7 days implantation and by 14 days the membrane had started to calcify.

The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking.

Transcription termination factor rho from Escherichia coli is a ring-shaped homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. Previous chemical cross-linking studies suggested that the rho hexamer might have D3 symmetry with three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F1-ATPase rather argued for C6 symmetry. To resolve this discrepancy, we have re-investigated the pattern of cross-linking of rho using various cross-linkers with different functional groups and spacer lengths. Upon reaction with dimethyl suberimidate followed by SDS-polyacrylamide gel electrophoresis, rho protein generated a series of cross-linked oligomers up to hexamers, of which dimers migrated as distinct doublet bands of approximately equal intensities. However, the lower band became much stronger than the upper one with dimethyl adipimidate and difluorodinitrobenzene, and vice versa with disuccinimidyl glutarate, disuccinimidyl suberate and disulfosuccinimidyl tartarate. Furthermore, the trimeric products also produced doublet bands, whose relative intensities were again variable with cross-linkers, but in an inverse correlation with those of the dimer bands. These results combined with theoretical considerations support a C6 symmetry model in which cross-linking is assumed to occur stochastically at one of two alternative sites within each subunit interface with variable relative frequencies depending on cross-linkers. The D3 symmetry is excluded, for the putative trimeric subspecies should always retain mutually equal intensities in that case. Detailed inspections of the cross-linking kinetics further revealed a moderate characteristic of C3 symmetry for the rho hexamer such that the collective as well as relative rates of cross-linking at the two available sites could fluctuate between alternating interfaces. The final model designated as C3/6 is also compatible with other functional and structural properties known for rho.

Physical properties of dystrophin rod domain.

We have prepared two fragments of the human dystrophin rod domain, each containing eight spectrin-like repeating units, by expression in Escherichia coli. The first corresponds to the central portion of the rod, the other to three repeats from the N-terminal end, fused to five repeats from the C-terminal end. The latter makes up the entire mutant rod, found in a patient with mild (Becker-type) muscular dystrophy. Both fragments were found to possess an ordered, stable structure, and had the form of short rod-like particles in the electron microscope. Molecular weight determinations by sedimentation equilibrium revealed that both polypeptides were monomeric in solution, suggesting that the dystrophin rod domain is incapable of forming an antiparallel homodimer. This supports the inference from sequence analyses [Winder et al., 1995: FEBS Lett. 369:27-33, 1996: Biochem. Soc. Trans. 24:2805] that the dystrophin rod domain lacks the arrangement of sites required for lateral self-association, and that dystrophin, unlike the other known proteins of the spectrin superfamily, may thus exist as a monomer.

D-glucose metabolism in cross-linked pancreatic islets.

D-glucose metabolism was investigated in pancreatic islets that underwent cross-linking of intracellular proteins by dimethyl suberimidate. Both the utilization of D-[5-3H]glucose and oxidation of D-[U-14C]glucose were much more severely affected at high (16.7 mM) than at low (2.8 mM) concentration of the hexose, when comparing cross-linked to control islets. The preferential stimulation of D-[U-14C]glucose oxidation relative to D-[5-3H]glucose utilization, resulting from an increase in hexose concentration, was not abolished, however, in cross-linked islets. Likewise, the activation of phosphofructokinase in glucose-stimulated islets did not appear to be impaired in cross-linked islets, the islet content in tritiated acidic metabolites generated from D-[5-3H]glucose failing to be increased in cross-linked islets. It is proposed, therefore, that the impaired responsiveness of cross-linked islets to a rise in D-glucose concentration might be attributable to an altered regulation of glucokinase, as possibly resulting from a missing interaction of the enzyme with GLUT-2.

Polyacrylamide-based redox polymer for connecting redox centers of enzymes to electrodes.

Enzyme electrodes based on complexing a water-soluble copolymer of acrylamide and vinylimidazole with [Os(dmebpy)2C1]+/2+ (dmebpy = 4,4'-dimethyl-2,2'-bipyridine) and cross-linking with oxidases by water-soluble cross-linkers are described. The potential of the polyacrylamide-based redox polymer is +55 mV (SCE), a typical electron diffusion coefficient (De) in the redox hydrogel that results from its cross-linking is (1.3 +/- 0.1) x 10(-9) cm2/s. The properties of the enzyme electrodes formed when this redox hydrogel "wired" horseradish peroxidase (HRP), lactate oxidase (LOx) or glucose oxidase (GOx) depended on the thickness of the hydrogel film, the chemistry of their cross-linking, and their enzyme content. At the wired HRP electrodes, H2O2 was electrocatalytically reduced to water at 0.0 V (SCE). Lactate and glucose were electrocatalytically oxidized at 0.16 V (SCE). The GOx electrodes, when made with 140 micrograms/cm2 thick polymer films, were selective for glucose in the presence of physiological concentrations of urate and ascorbate.

Arrangement of phosphatidylethanolamine molecular species in Escherichia coli membranes and reconstituted lipids as determined by dimethyl suberimidate cross-linking of nearest neighbor lipids.

Dimethylsuberimidate cross-linking has been used to determine the arrangement of phosphatidylethanolamine (PE) molecular species in Escherichia coli membranes. No large deviations from random mixing were found in wild-type strain AB1623, either in whole cells or in extracted lipids which were reconstituted into multilamellar vesicles. These results suggest, first, that there is little difference in the PE molecular species composition of the three lipid monolayers (the inner and outer monolayers of the inner membrane and the inner monolayer of the outer membrane) which contain significant amounts of PE. Secondly, the results suggest that the molecular species within each monolayer and in the extracted lipids are arranged close to randomly with no tendency for like molecular species to cluster. E. coli strain L8-2, which has a defect in beta-oxidation and a temperature-sensitive mutation in total fatty acid synthesis, was grown on cis-vaccenate (cis-11,12- octadecenate) to enrich the cells in divaccenoyl PE. Again, in whole cells or in lipids extracted from whole cells and reconstituted into multilamellar vesicles, the species were close to randomly arranged. However, a consistent, slight tendency of divaccenoyl species to pair with like species as compared to pairing with the second most common species, vaccenoyl, palmitoleoyl PE, was noted in both extracted lipids and in whole cells.

Interactive intermediates are formed during the urea unfolding of rhodanese.

Structural transitions have been studied on the pathway for urea denaturation of rhodanese. Unlike guanidinium hydrochloride, urea gives no visible precipitation. Increasing urea concentrations cause a transition in which the enzyme activity is completely lost by 4.5 M urea, and there is a shift of the intrinsic fluorescence maximum from 335 nm for the native enzyme to 350 nm. There is a maximum exposure of organized hydrophobic surfaces at 4.5 M urea as reported by the fluorescence of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid. Above 4.5 M urea, this probe reports the progressive loss of organized hydrophobic surfaces. The polarization of the intrinsic fluorescence falls with increasing urea concentrations in a complex transition showing that rhodanese flexibility increases in at least two phases. Rhodanese becomes increasingly susceptible to digestion by subtilisin between 3.5 and 4.5 M urea, giving rise to large fragments. At urea concentrations > 5 M, rhodanese is completely digested. There is a small increase in the rate of sulfhydryl accessibility between 3.5 and 4.5 M urea, but there is a large increase in the sulfhydryl accessibility above 4.5 M urea. Dimethyl suberimidate cross-linking shows the presence of associated species in 3-5 M urea, but there are few cross-linkable species at lower or higher urea concentrations. These results are consistent with a model in which urea unfolding of rhodanese is associated with the initial production of a species having organized regions of structure with exposed hydrophobic surfaces separated by flexible elements.

Characterization of the lysogenic repressor (c) from transposable Mu-like bacteriophage D108.

The c gene products from related, transposable phages Mu and D108 encode lysogenic repressors which negatively regulate transcription and transposition. Using the gel shift assay to monitor c-operator specific DNA-binding activity, the 19.5 kDa D108 c repressor was purified to homogeneity. Sequence analysis of the N-terminus confirmed the identity of the purified protein as the repressor and ascribed its ATG initiation codon to base pair 864 from the D108 left end. Analytical gel filtration and dimethyl suberimidate cross-linking of repressor at 0.1-0.5 microM concentrations revealed that the repressor protein could form oligomers in the absence of its DNA substrate. From DNase I footprinting and gel mobility shift analyses, the D108 repressor only bound to two operators (O1 and O2) which, as in Mu, flank an Integration Host Factor (IHF) binding site. In contrast to Mu, an O3 site in D108 was not found. Moreover, D108 repressor first bound operator O2, while occupancy of O1 required higher protein concentrations. The implications of these results on the D108 regulatory system are discussed.