Salmonella typhimurium detection and ablation using OmpD specific aptamer with non-magnetic and magnetic graphene oxide.

Foodborne diseases have increased in the last few years due to the increased consumption of packaged and contaminated food. Major foodborne bacteria cause diseases such as diarrhea, vomiting, and sometimes death. So, there is a need for early detection of foodborne bacteria as pre-existing detection techniques are time-taking and tedious. Aptamer has gained interest due to its high stability, specificity, and sensitivity. Here, aptamer has been developed against Salmonella Typhimurium through the Cell-Selex method, and to further find the reason for specificity and sensitivity, OmpD protein was isolated, and binding studies were done. Single molecular FRET experiment using aptamer and graphene oxide studies has also been done to understand the mechanism of FRET and subsequently used for target bacterial detection. Using this assay, Salmonella Typhimurium can be detected up to 10 CFU/mL. Further, Magnetic Graphene oxide was used to develop an assay to separate and ablate bacteria using 808 nm NIR where temperature increase was more than 60 °C within 30 s and has been shown by plating as well as a confocal live dead assay. Thus, using various techniques, bacteria can be detected and ablated using specific aptamer and Graphene oxide.

A single-molecule approach to unravel the molecular mechanism of the action of Deinococcus radiodurans RecD2 and its interaction with SSB and RecA in DNA repair.

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. They are responsible for resolving double-stranded DNA (dsDNA) into single-strands, which is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions to the organism's unusually high tolerance to gamma radiation and hydrogen peroxide. Although the results from X-ray Crystallography studies have revealed the structural characteristics of the protein, direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5' terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

Impact of Mutation on the Structural Stability and the Conformational Landscape of Inhibitor-Resistant TEM ß-Lactamase: A High-Performance Molecular Dynamics Simulation Study.

Gain-of-function mutations and structural adjustment toward ß-lactamase inhibitors in the TEM-type ß-lactamases among the uropathogenic E. coli (UPEC) culminate in treatment complications and demands detailed investigation. In this study, uncharacterized amino acid substitutions, M69L/I84V/W165G/V184A/V262I/N276S, in inhibitor-resistant TEM (IRT) ß-lactamase isolated from clinical UPEC were subjected to extensive molecular dynamics (EMD) simulations for 100 ns to estimate parameters such as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg), contour plot (Rg/RMSD), secondary structure element (SSE), etc. Residue interaction networks, principal component analysis (PCA), and correlation heatmaps were generated to predict the relation between functionally important atomic motions to uncover the structural stability of the mutants. To avoid the false positive conclusion of the simulation study, we performed three identically parameterize replicas of 100 ns each. Alterations in hydrophobic interactions resulted in conformation changes exhibited as comparable residue interaction networks. Besides, PCA and porcupine plot analysis based on the ensemble of structure from molecular dynamics trajectories revealed the collective atomic motions of the IRT variants that impart structural flexibility to their active site loop. This study conducted on IRT mutants that delineate intricate protein motions contributes to their stability and folding, which is an absolute necessity for designing candidate molecules owing to the clinical threat of emerging resistance against potent ß-lactam antibiotics.

Decoding the Structural Dynamics and Conformational Alternations of DNA Secondary Structures by Single-Molecule FRET Microspectroscopy.

In addition to the canonical double helix form, DNA is known to be extrapolated into several other secondary structural patterns involving themselves in inter- and intramolecular type hydrogen bonding. The secondary structures of nucleic acids go through several stages of multiple, complex, and interconvertible heterogeneous conformations. The journey of DNA through these conformers has significant importance and has been monitored thoroughly to establish qualitative and quantitative information about the transition between the unfolded, folded, misfolded, and partially folded states. During this structural interconversion, there always exist specific populations of intermediates, which are short-lived or sometimes even do not accumulate within a heterogeneous population and are challenging to characterize using conventional ensemble techniques. The single-molecule FRET(sm-FRET) microspectroscopic method has the advantages to overcome these limitations and monitors biological phenomena transpiring at a measurable high rate and balanced stochastically over time. Thus, tracing the time trajectory of a particular molecule enables direct measurement of the rate constant of each transition step, including the intermediates that are hidden in the ensemble level due to their low concentrations. This review is focused on the advantages of the employment of single-molecule Forster's resonance energy transfer (sm-FRET), which is worthwhile to access the dynamic architecture and structural transition of various secondary structures that DNA adopts, without letting the donor of one molecule to cross-talk with the acceptor of any other. We have emphasized the studies performed to explore the states of folding and unfolding of several nucleic acid secondary structures, for example, the DNA hairpin, Holliday junction, G-quadruplex, and i-motif.

Vital insights into prokaryotic genome compaction by nucleoid-associated protein (NAP) and illustration of DNA flexure angles at single-molecule resolution.

Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP), well known for its DNA bending ability. Although the IHF induced bending states of DNA have been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Binding of IHF leads to introduction of two kinks in the dsDNA that allowed us to come up with a quadrilateral model. The findings have further been extended by calculating the angles of flexibility, that gives the idea of the degree of dynamicity of the nucleoprotein complex. We have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a 'Slacked-Dynamic' state throughout the observation window where many of them have switched between multiple 'Wobbling States' in the course of attainment of packaged form. This study opens up an opportunity to improve the understanding of the functions of other nucleoid-associated proteins (NAPs) by complementing the previous detailed atomic-level structural analysis, which eventually will allow accessibility towards a better hypothesis.

Direct observation of effect of crowding induced macromolecular hydration on molecular breathing in the stem of Fork-DNA by single-molecule FRET microspectroscopy.

The perpetually changing cellular conditions, nucleotide sequence, and environmental effects including osmotic stress have multiple effects on DNA, leading to several conformational alternations and subsequently influencing their activity, too. In this work, single-molecule FRET microspectroscopy has been employed to monitor the breathing dynamics as an effect of molecular crowding in the stem region of Fork-DNA. The structural integrity greatly alters with the presence or absence of nucleotide overhangs and on the nature and concentration of the crowding agent, thus affecting the stability of the stem region and hence the forked DNA. The multiple hydrogen bonds and hydrophobic interactions between the polynucleotide strands appear to be altered with osmotic crowding. This induces increased flexibility in the double helix and allows DNA to breath. The conformational alternation of the DNA happens in nanometer resolution, that is been monitored by the change in the FRET efficiency between the dyes attached to two different strands of the DNA. The nature and molecular weight of crowding agents control the degree of spatial breathing in the stem of Fork-DNA. These constant fluctuations between the entropically favorable partially folded structures to an enthalpically favorable folded structure are not only valuable for elucidating nucleic acid structure but might play an important role in enzyme kinetics.

Real-Time Monitoring of the Multistate Conformational Dynamics of Polypurine Reverse Hoogsteen Hairpin To Capture Their Triplex-Affinity for Gene Silencing by smFRET Microspectroscopy.

Single-molecule Förster resonance energy transfer microspectroscopic methods are employed for real-time monitoring and to gain deeper insights into the formation of the polypurine reverse Hoogsteen hairpin (PPRH) and its triplex-forming activity. The heterogeneity in the behavior of individual PPRHs has been documented, and it is seen that the degree of anharmonic plasticity of the antiparallel hairpin is stabilized by the formation of reverse Hoogsteen (RH) bonds. While being involved in the hairpin formation, they flip reversibly between the open and closed conformations, irrespective of the concentration of ions present in their microenvironment. However, the nature of the cation present in the buffer plays a crucial role in determining the structural stability. The Watson Crick (WC) bonds are found to be more dynamic in the triplex compared to that of the RH base pairs, indicating the involvement of progressive WC bonds during the triplex motif formation by the PPRH. The majority of the intact triplex DNA attained a semifolded relaxed state before progressing toward a tightly folded state, emphasizing the fact that the folding mechanism pursues an ambiguous path in the mode of acquiring the final step of the triple helix motif. Moreover, the presence of triplex-forming sequences in the regulatory regions of the genome further provides an intricate link between the experimental results and sequence occurrence.

Direct observation of the external force mediated conformational dynamics of an IHF bound Holliday junction.

We have investigated the isomerization dynamics and plausible energy landscape of 4-way Holliday junctions (4WHJs) bound to integration host factor (IHF, a DNA binding protein), considering the effect of applied external force, by single-molecule FRET methods. A slowing down of the forward as well as the backward rates of the isomerization process of the protein bound 4WHJ has been observed under the influence of an external force, which indicates an imposed restriction on the conformational switching. This has also been reflected by an increase in rigidity, as observed from the increase in the single-molecule FRET (smFRET)-anisotropy values (0.270 ± 0.012 to 0.360 ± 0.008). The application of an external force has assisted the conformational transitions to share the unstacked open structure intermediate, with different rate-limiting steps and a huge induced variation in the energy landscape. Furthermore, the associated landscape of the 4WHJ is visualized in terms of rarely interconverting states embedded into the two isoforms by using nonlinear dynamics analysis, which shows that the chaoticity of the system increases at intermediate force (0.4 to 1.6 pN). The identification of chaos in our investigation provides useful information for a comprehensive explanation of the origin of the complex behavior of the system, which effectively helps us to perceive the dynamics of IHF bound 4WHJs under the influence of external force, and also demonstrates the applicability of nonlinear dynamics analysis in the field of biology.

Direct observation of breathing dynamics at the mismatch induced DNA bubble with nanometre accuracy: a smFRET study.

The detailed conformational dynamics of the melted region in double-stranded DNA has been studied using a combination of ensemble and single-molecule FRET techniques. We monitored the millisecond time scale fluctuation kinetics of the two strands at the bubble region that varies with the size of the bubble. As the individual strands at the melting bubble behave as single-stranded DNA, and hence fluctuate dynamically to attain energetically favored configurations, the rates of these fluctuations increase with increase in the bubble size. In different short DNAs under investigation, the two strands never cross each other to form a knot, irrespective of the number of base pair mismatches present. Rather, they prefer to stay apart from each other, as the size of the bubble increases and follow exactly an opposite trend for bubbles of smaller size. The range within which the bubble strands fluctuate are monitored with great accuracy in the nanometre resolution from the single-molecule FRET measurements. The shape of the bubble that plays a crucial role in determining the activity of the DNA was speculated. These results shall be useful in quantifying the chemical processes within DNA as well as to develop a deeper understanding of the activity of the DNA due to induced mismatches.

Single-Molecule FRET Studies of the Hybridization Mechanism during Noncovalent Adsorption and Desorption of DNA on Graphene Oxide.

Remarkable observations on the adsorption and desorption mechanisms of single-stranded oligonucleotides and the hybridization of double-stranded DNA (ds-DNA) on a graphene oxide (GO) surface have been made using ensemble and single-molecule fluorescence methods. Probe and target DNAs labeled individually with fluorescence resonance energy transfer (FRET) pairs and having similar adsorption affinities toward the GO surface are used to provide detailed insights into the hybridization mechanism. Single-molecule FRET results reveal an "in situ" DNA hybridization mechanism, i.e., hybridization between the probe and target DNAs to form a ds-DNA, and simultaneous desorption from the GO surface thereafter. These results also demonstrate that the electrostatic interaction between DNA and GO is of little importance to the overall theory of interaction and the largest effects are from solvation forces, specifically the hydrophobic effect. This investigation improves the fundamental understanding of the DNA hybridization dynamics on the GO surface, opening new windows in the field of biophysics as well as in sensing and therapeutic applications.

Conformational Changes Followed by Complete Unzipping of DNA Double Helix by Charge-Tuned Gold Nanoparticles.

The complete unzipping of DNA double helix by small size gold nanoparticles having weakly positive surface charge has been monitored using ensemble and single molecule fluorescence resonance energy transfer (smFRET) techniques. We believe, as the gold nanoparticles have positive charge on the surface, the DNA and nanoparticles were pulled together to form two single strands. The positively charged ligands on the nanoparticles attached to the DNA, and the hydrophobic ligands of the nanoparticles became tangled with each other, pulling the nanoparticles into clusters. At the same time, the nanoparticles pulled the DNA apart. The conformational changes followed by unzipping have been investigated for long DNA (calf thymus DNA) as well as for short DNA (∼40 base pair) using ensemble methods like circular dichroism (CD) spectroscopy, fluorescence intercalation assay, viscometric method, and single molecule FRET imaging. This observation not only reveals a new aspect in the field of nano-bio interface but also provides additional information about DNA dynamics.

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.

In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC·PCNA·DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC·PCNA·DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC·PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polδ only one species converts to the RFC·PCNA·DNA·polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.

Codon-dependent tRNA fluctuations monitored with fluorescence polarization.

During protein synthesis dictated by the codon sequence of messenger RNA, the ribosome selects aminoacyl-tRNA (aa-tRNA) with high accuracy, the exact mechanism of which remains elusive. By using a single-molecule fluorescence resonance energy transfer method coupled with fluorescence emission anisotropy, we provide evidence of random thermal motion of tRNAs within the ribosome in nanosecond timescale that we refer to as fluctuations. Our results indicate that cognate aa-tRNA fluctuates less frequently than near-cognate. This is counterintuitive because cognate aa-tRNA is expected to fluctuate more frequently to reach the ribosomal A-site faster than near-cognate. In addition, cognate aa-tRNA occupies the same position in the ribosome as near-cognate. These results argue for a mechanism which guides cognate aa-tRNA more accurately toward the A-site as compared to near-cognate. We suggest that a basis for this mechanism is the induced fit of the 30S subunit upon cognate aa-tRNA binding. Our single-molecule fluorescence resonance energy transfer time traces also point to a mechanistic model for GTP hydrolysis on elongation factor Tu mediated by aa-tRNA.

Self-assembly of Yttrium-containing lacunary polyoxotungstate macroanions in solution with controllable supramolecular structure size by pH or solvent content.

The self-assembly and the formation of "Blackberry" type supramolecular structures for a type of Yttrium-containing polyoxometalate (K 15Na 6(H 3O) 9[(PY 2W 10O 38) 4(W 3O 14)].9H 2O, or {P 4Y 8W 43}) macroanions is characterized by using static and dynamic light scattering techniques. {P 4Y 8W 43} macroions are found to form hollow, spherical, single-layer "blackberry" structures in water and water-acetone mixed solvents. Very interestingly, the blackberry size can be accurately controlled by either changing acetone content in water-acetone mixed solvents, or by changing solution pH in aqueous solution. The blackberry size increases with decreasing pH (lower charge density) or higher acetone content in the mixed solvent (lower dielectric constant) and the blackberry size can change in responding to the change of external conditions. This indicates that the {P 4Y 8W 43} macroanions possess the properties of both "strong electrolyte type" and "weak electrolyte type" macroions, as we outlined previously. This is due to the special chemical feature of such clusters, which can be treated as Na 2HPO 4-type electrolytes in solution. The kinetics of the blackberry formation can be controlled by temperature.

Effect of increased hydrophobicity on the binding of two model amphiphilic chlorin drugs for photodynamic therapy with blood plasma and its components.

The binding of serum albumin and lipoprotein with chlorin p(6) and purpurin 18, two structurally related chlorins, has been studied to understand the role for these proteins as endogenous carriers for these drugs. As a drug carrier a protein may aid in selective delivery of a drug to a tumor region. Binding with serum albumin may result in accumulation of the drug in the stroma of the tumor cell and lead to a reduction of cellular uptake of photosensitizers. However, it is possible that this factor may not be a problem for cellular uptake of chlorin p(6) and purpurin 18 by the tumor tissues, since it binds more efficiently with low-density lipoprotein when it become more lipophilic, indicating that the principal carriers for these molecules are lipoproteins. Since the tumor tissues contain numerous lipoprotein receptors, chlorin p(6) and purpurin 18 could be internalized more efficiently in tumor cells.

Chlorin p6 as a fluorescent probe for the investigation of surfactant-cyclodextrin interactions.

Cyclodextrins (CD) are often proposed as potential vehicles in targeted drug delivery. However, if the membrane structure is disrupted by CD, then it cannot be considered to be a good drug delivery vehicle. When an extrinsic fluorescence probe is used to monitor such interactions, there are no less than three possible equilibria that can operate simultaneously: surfactant-cyclodextrin, surfactant-fluorophore and cyclodextrin-fluorophore. The fluorescence intensity/lifetime might be affected by all these and so, the results depend strongly on the fluorophore used as well as the nature of the surfactant. This aspect highlights the importance of the suitability of the fluorescence probe to be used to study complicated systems and interaction. In the present work, chlorin p6, prepared from chlorophyll from spinach leaves, has been used as the fluorescence probe to investigate the interaction between alpha-CD and beta-CD with the neutral surfactants Triton X 100 (TX 100) and cetyl trimethyl ammonium bromide (CTAB). The fluorophore is found to be a sensitive one for the study of the interaction of alpha, beta and gamma-CD with the surfactants TX 100 and CTAB. It is found that contrary to earlier reports, a complex between alpha-CD and TX 100 is formed, even though the binding constant is not very high. This observation can be obtained with chlorin p6, which does not bind to the CDs, but not with a fluorophore, which binds to the CD as well and thus complicates the situation as the binding with CD is stronger than that between TX 100 and alpha-CD as compared to that between TNS and CD.