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Most biological fluids (such as blood, saliva, and lymph) in nature have certain viscoelasticity and are beginning to be used as the carrying fluids for viscoelastic microfluidics. However, the particle-focusing mechanisms in these new biological viscoelastic fluids are still unclear. In this work, the particle-focusing mechanisms in λ-DNA solutions were systematically explored. We first explored the particle focusing dynamics in a square cross-section under varied flow rates to uncover the effects of flow rate on particle focusing. Three focusing stages, from the classic five-position viscoelastic focusing to single-stream focusing and finally to multiplex-stream focusing, were clearly demonstrated. In addition, the particle focusing process along the channel length was demonstrated, and a first-fast-and-then-slow focusing process was clearly observed. Then, the effects of λ-DNA concentrations on particle focusing were explored and compared using the solutions with 0-25 ppm λ-DNA. Finally, we discussed the inferences of blockage ratio on particle focusing by changing the particle diameter and cross-sectional dimensions. Our work may provide a deeper understanding on the particle focusing mechanisms in biological viscoelastic fluids and lays a foundation for the subsequent particle counting and analysis and the development of low-cost portable flow cytometers., (© 2024 Wiley‐VCH GmbH.)

Bacterial resistance to conventional antibiotics stimulated the development of so-called "phage therapies" that rely on cell lysis, which is a process of destroying bacterial cells due to their infections by bacterial viruses. For λ bacteriophages, it is known that the critical role in this process is played by holin proteins that aggregate in cellular membranes before breaking them apart. While multiple experimental studies probed various aspects of cell lysis, the underlying molecular mechanisms remain not well understood. Here we investigate what physicochemical properties of holin proteins are the most relevant for these processes by employing statistical correlation analysis of cell lysis dynamics for different experimentally observed mutant species. Our findings reveal significant correlations between various physicochemical features and cell lysis dynamics. Notably, we uncover a strong inverse correlation between local hydrophobicity and cell lysis times, underscoring the crucial role of hydrophobic interactions in membrane disruption. Stimulated by these observations, a predictive model capable of explicitly estimating cell lysis times for any holin protein mutants based on their mean hydrophobicity values is developed. Our study not only provides important microscopic insights into cell lysis phenomena but also proposes specific routes to optimize medical and biotechnological applications of bacteriophages.

The S protein from bacteriophage lambda is a three-helix transmembrane protein produced by the prophage which accumulates in the host membrane during late gene expression. It is responsible for the first step in lysing the host cell at the end of the viral life cycle by multimerizing together to form large pores which permeabilize the host membrane to allow the escape of virions. Several previous studies have established a model for the assembly of holin into functional holes and the manner in which they pack together, but it is still not fully understood how the very rapid transition from monomer or dimer to multimeric pore occurs with such precise timing once the requisite threshold is reached. Here, site-directed spin labeling with a nitroxide label at introduced cysteine residues is used to corroborate existing topological data from a crosslinking study of the multimerized holin by EPR spectroscopy. CW-EPR spectral lineshape analysis and power saturation data are consistent with a three-helix topology with an unstructured C-terminal domain, as well as at least one interface on transmembrane domain 1 which is exposed to the lumen of the hole, and a highly constrained steric environment suggestive of a tight helical packing interface at transmembrane domain 2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redβ, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redβ to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century after its discovery, this combination of structural and computational observations has allowed us to propose molecular mechanisms for the actions of the model annealase Redβ, a defining member of the Redβ/RecT protein family., (© 2022. The Author(s).)

Bacteriophage lambda is an excellent model system for studying capsid assembly of double-stranded DNA (dsDNA) bacteriophages, some dsDNA archaeal viruses, and herpesviruses. HK97 fold coat proteins initially assemble into a precursor capsid (procapsid) and subsequent genome packaging triggers morphological expansion of the shell. An auxiliary protein is required to stabilize the expanded capsid structure. To investigate the capsid maturation mechanism, we determined the cryo-electron microscopy structures of the bacteriophage lambda procapsid and mature capsid at 3.88 Å and 3.76 Å resolution, respectively. Besides primarily rigid body movements of common features of the major capsid protein gpE, large-scale structural rearrangements of other domains occur simultaneously. Assembly of intercapsomers within the procapsid is facilitated by layer-stacking effects at 3-fold vertices. Upon conformational expansion of the capsid shell, the missing top layer is fulfilled by cementing the gpD protein against the internal pressure of DNA packaging. Our structures illuminate the assembly mechanisms of dsDNA viruses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During the final step of the bacteriophage infection cycle, the cytoplasmic membrane of host cells is disrupted by small membrane proteins called holins. The function of holins in cell lysis is carried out by forming a highly ordered structure called lethal lesion, in which the accumulation of holins in the cytoplasmic membrane leads to the sudden opening of a hole in the middle of this oligomer. Previous studies showed that dimerization of holins is a necessary step to induce their higher order assembly. However, the molecular mechanism underlying the holin-mediated lesion formation is not well understood. In order to elucidate the functions of holin, we first computationally constructed a structural model for our testing system: the holin S105 from bacteriophage lambda. All atom molecular dynamic simulations were further applied to refine its structure and study its dynamics as well as interaction in lipid bilayer. Additional simulations on association between two holins provide supportive evidence to the argument that the C-terminal region of holin plays a critical role in regulating the dimerization. In detail, we found that the adhesion of specific nonpolar residues in transmembrane domain 3 (TMD3) in a polar environment serves as the driven force of dimerization. Our study therefore brings insights to the design of binding interfaces between holins, which can be potentially used to modulate the dynamics of lesion formation., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The ability to precisely control particle migration within microfluidic systems is essential for focusing, separating, counting, and detecting a wide range of biological species. To date, viscoelastic microfluidic systems have primarily been applied to the focusing, separation, and isolation of micrometer-sized species, with their use in nanoparticle manipulations being underdeveloped and underexplored, due to issues related to nanoparticle diffusivity and a need for extended channel lengths. To overcome such issues, we herein present sheathless oscillatory viscoelastic microfluidics as a method for focusing and separating both micrometer and sub-micrometer species. To highlight the efficacy of our approach, we segment our study into three size regimes, namely, micrometer (where characteristic particle dimensions are above 1 μm), sub-micrometer (where characteristic dimensions are between 1 μm and 100 nm), and nano (where characteristic dimensions are below 100 nm) regimes. Based on the ability to successfully manipulate particles in all these regimes, we demonstrate the successful isolation of p-bodies from biofluids (in the micrometer regime), the focusing of λ-DNA (in the sub-micrometer regime), and the focusing of extracellular vesicles (in the nanoregime). Finally, we characterize the physics underlying viscoelastic microflows using a dimensionless number that relates the lateral velocity (due to elastic effects) to the diffusion constant of the species within the viscoelastic carrier fluid. Based on the ability to precisely manipulate species in all three regimes, we expect that sheathless oscillatory viscoelastic microfluidics may be used to good effect in a range of biological and life science applications.

The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E'F' sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. 1 H N and 15 N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex., (© 2019 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Decades of crystallographic and NMR studies have produced canonical structural models of short DNA. However, no experimental method so far has been able to test these models in vivo, where DNA is long and constrained by interactions with membranes, proteins, and other molecules. Here, we employ high-resolution frequency-modulation AFM to image single long poly(dA)-poly(dT), poly(dG)-poly(dC), and lambda DNA molecules interacting with an underlying substrate that emulates the effect of biological constraints on molecular structure. We find systematic sequence-dependent variations in groove dimensions, indicating that the structure of DNA subject to realistic interactions may differ profoundly from canonical models. These findings highlight the value of AFM as a unique, single molecule characterization tool.

Metal-dependent DNA and RNA nucleases are enzymes that cleave nucleic acids with great efficiency and precision. These enzyme-mediated hydrolytic reactions are fundamental for the replication, repair, and storage of genetic information within the cell. Here, extensive classical and quantum-based free-energy molecular simulations show that a cation-π interaction is transiently formed in situ at the metal core of Bacteriophage-λ Exonuclease (Exo-λ), during catalysis. This noncovalent interaction (Lys131-Tyr154) triggers nucleophile activation for nucleotide excision. Then, our simulations also show the oscillatory dynamics and swinging of the newly formed cation-π dyad, whose conformational change may favor proton release from the cationic Lys131 to the bulk solution, thus restoring the precatalytic protonation state in Exo-λ. Altogether, we report on the novel mechanistic character of cation-π interactions for catalysis. Structural and bioinformatic analyses support that flexible orientation and transient formation of mobile cation-π interactions may represent a common catalytic strategy to promote nucleic acid hydrolysis in DNA and RNA nucleases.

The lysis-lysogeny switch in E. coli due to infection from lambda phage has been extensively studied and explained by scientists of molecular biology. The bacterium either survives with the viral strand of deoxyribonucleic acid (DNA) or dies producing hundreds of viruses for propagation of infection. Many proteins transcribed after infection by λ phage take part in determining the fate of the bacterium, but two proteins that play a key role in this regard are the cI and cro dimers, which are transcribed off the viral DNA. This paper presents a novel modeling mechanism for the lysis-lysogeny switch, by transferring the interactions of the main proteins, the lambda right operator and promoter regions and the ribonucleic acid (RNA) polymerase, to a finite state machine (FSM), to determine cell fate. The FSM, and thus derived is implemented in field-programmable gate array (FPGA), and simulations have been run in random conditions. A Markov model has been created for the same mechanism. Steady state analysis has been conducted for the transition matrix of the Markov model, and the results have been generated to show the steady state probability of lysis with various model values. In this paper, it is hoped to lay down guidelines to convert biological processes into computing machines.

Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 10 3 CFU·mL -1 . The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence. Graphical abstract Schematic presentation of the lateral flow-based fluorometric detection of DNA amplicons using conjugates of scCro DNA binding protein with (A) carbon nanoparticles, (B) HRP and (C) HRP and carbon nanoparticles.

The authors report on a simplified approach to encapsulate upconversion nanoparticles (UCNPs) in polystyrene spheres by mini-emulsion polymerisation. The resulting particles (PS-UCNP) are hydrophilic, stable and suitable for biomolecular recognition and biosensing applications. Also, a strategy was developed for bioconjugation of antibodies onto the surface of the PS-UCNPs by using the bifunctional fusion protein linker-protein G (LPG). LPG mediates the functionalisation of PS-UCNPs with antibodies against digoxigenin allowing for specific labelling of convective PCR (cPCR) amplicons. Lambda DNA was amplified using cPCR on a heat block for 30 min using the digoxigenin labelled forward and biotin labelled reverse primers. The antibody functionalised PS-UCNPs bind to the digoxigenin end of the cPCR amplicons. Finally, the streptavidin labelled magnetic beads were used to selectively capture the PS-UCNP-labelled cPCR amplicons and the upconversion signal was detected at 537 nm under 980 nm excitation. This sandwich approach enables direct recognition of the target lambda DNA with a detection limit of 10 3 copies μL -1 . The upconversion signal decreased proportionally to the concentration of the lambda DNA with a linear response between 10 7 and 10 3 copies of DNA. Graphical abstract Schematic representation of polystyrene-encapsulated upconversion nanoparticles (PS-UCNPs) prepared by mini-emulsion polymerisation. The PS-UCNPs were functionalised with anti-digoxigenin antibody using the fusion protein linker-protein G (LPG). Detection of digoxigenin-labelled amplicons is achieved (a) by using the antibody-functionalised LPG@PS-UCNP labels; (b) magnetic separation, and (c) 980 nm laser light for detection of the green upconversion luminescence peaking at 537 nm.

RNA-protein interactions permeate biology. Transcription, translation, and splicing all hinge on the recognition of structured RNA elements by RNA-binding proteins. Models of RNA-protein interactions are generally limited to short linear motifs and structures because of the vast sequence sampling required to access longer elements. Here, we develop an integrated approach that calculates global pairwise interaction scores from in vitro selection and high-throughput sequencing. We examine four RNA-binding proteins of phage, viral, and human origin. Our approach reveals regulatory motifs, discriminates between regulated and non-regulated RNAs within their native genomic context, and correctly predicts the consequence of mutational events on binding activity. We design binding elements that improve binding activity in cells and infer mutational pathways that reveal permissive versus disruptive evolutionary trajectories between regulated motifs. These coupling landscapes are broadly applicable for the discovery and characterization of protein-RNA recognition at single nucleotide resolution.

Protein folding/unfolding can be analyzed experimentally at a local scale by monitoring the physical properties of local probes as a function of the temperature, for example, the distance between fluorophores or the values of chemical shifts of backbone atoms. Here, the analytical Lifson-Roig model for the helix-coil transition is modified to analyze local thermal unfolding of the fast-folder W protein of bacteriophage lambda (gpW) simulated by all-atom molecular dynamics (MD) simulations in explicit solvent at 15 different temperatures. The protein structure is described by the coarse-grained dihedral angles (γ) and bond angles (θ) built between successive C α -C α virtual bonds. Each (γ,θ) pair serves as a local probe of protein unfolding. Local native/non-native states are defined for each pair of (γ,θ) angles by analyzing the free-energy landscapes Δ G(γ,θ) computed from MD trajectories. The three local elementary equilibrium constants of the model are extracted for each (γ,θ) pair along the sequence from MD simulations, and the model predictions are compared to the MD data. Using only the local equilibrium constants as an input, we show that the local denaturation curves computed from the model partition function fit their MD simulated counterparts in a satisfying manner without any adjustment. In the model and MD simulations, gpW unfolds gradually between 320 and 340 K, with an average native percentage decreasing from 0.8 (320 K) to 0.2 (340 K). In the prism of the model, there is no stable structure at the local scale in this 20 K unfolding temperature range. The enthalpy change upon local unfolding computed from the model and from MD trajectories suggests that the unfolded state between 320 and 340 K corresponds to a dynamical equilibrium between a large ensemble of constantly changing structures. The present results confirm the downhill unfolding of gpW, which does not obey a two-state global folding/unfolding model, and shed light on the interpretation of local denaturation curves.

Long-read genomic applications, such as genome mapping in nanochannels, require long DNA that is free of small-DNA impurities. We have developed a chip-based system based on entropic trapping that can simultaneously concentrate and purify a long DNA sample under the alternating application of an applied pressure (for sample injection) and an electric field (for filtration and concentration). In contrast, short DNA tends to pass through the filter owing to its comparatively weak entropic penalty for entering the nanoslit. The single-stage prototype developed here, which operates in a continuous pulsatile manner, achieves selectivities of up to 3.5 for λ-phage DNA (48.5 kilobase pairs) compared to a 2 kilobase pair standard based on experimental data for the fraction filtered using pure samples of each species. The device is fabricated in fused silica using standard clean-room methods, making it compatible for integration with long-read genomics technologies.

Methods to fuse multiple DNA fragments are extremely useful in synthetic biology and protein engineering. Here, we report a gold nanoparticle-mediated simultaneous overlap extension-PCR (AuNP-mediated SOE-PCR) method that enables the fusion of multiple DNA fragments simultaneously with their amplification in a single reaction using typical PCR conditions. Using greater concentrations of rTaq DNA polymerase and AuNPs significantly improves the performance of SOE-PCR especially for the fusion of more than three DNA fragments. We show that up to six lambda DNA fragments can be simultaneously fused by AuNP-mediated SOE-PCR., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Virus-like particles (VLPs) are stable protein cages derived from virus coats. They have been used extensively as biomolecular platforms, e.g., nanocarriers or vaccines, but a convenient in situ technique is lacking for tracking functional status. Here, we present a simple way to monitor disassembly of 19 F-labeled VLPs derived from bacteriophage Qβ by 19 F NMR. Analysis of resonances, under a range of conditions, allowed determination not only of the particle as fully assembled but also as disassembled, as well as detection of a degraded state upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

The O protein is a crucial factor initiating the DNA replication of lambdoid bacteriophage. Efficient DNA replication of Shiga toxin-converting phage is necessary for effective production of Shiga toxin - main virulence factor of STEC strains. We developed an improved protocol for overproduction, bacterial cell lysis and purification of λO protein. With use of this method we have also isolated O proteins of Stx-phage P27 and 933W that were never purified before. Purified proteins were tested for their DNA binding activity and revealed a sequence specific interactions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Complex double-stranded DNA viruses utilize a terminase enzyme to package their genomes into a preassembled procapsid shell. DNA packaging triggers a major conformational change in the proteins assembled into the shell and most often subsequent addition of a decoration protein that is required to stabilize the structure. In bacteriophage λ, DNA packaging drives a procapsid expansion transition to afford a larger but fragile shell. The gpD decoration protein adds to the expanded shell as trimeric spikes at each of the 140 three-fold axes. The spikes provide mechanical strength to the shell such that it can withstand the tremendous internal forces generated by the packaged DNA in addition to environmental insults. Hydrophobic, electrostatic, and aromatic-proline noncovalent interactions have been proposed to mediate gpD trimer spike assembly at the expanded shell surface. Here, we directly examine each of these interactions and demonstrate that hydrophobic interactions play the dominant role. In the course of this study, we unexpectedly found that Trp308 in the λ major capsid protein (gpE) plays a critical role in shell assembly. The gpE-W308A mutation affords a soluble, natively folded protein that does not further assemble into a procapsid shell, despite the fact that it retains binding interactions with the scaffolding protein, the shell assembly chaparone protein. The data support a model in which the λ procapsid shell assembles via cooperative interaction of monomeric capsid proteins, as observed in the herpesviruses and phages such as P22. The significance of the results with respect to capsid assembly, maturation, and stability is discussed.

Most breast cancers express estrogen receptor (ER) α, and the antiestrogen drug tamoxifen has been widely used for their treatment. Unfortunately, up to half of all ERα-positive tumors have intrinsic or acquired endocrine therapy resistance. Our recent studies revealed that the ER coactivator Mediator Subunit 1 (MED1) plays a critical role in tamoxifen resistance through cross-talk with HER2. Herein, we assembled a three-way junction (3-WJ) pRNA-HER2apt-siMED1 nanoparticle to target HER2-overexpressing human breast cancer via an HER2 RNA aptamer to silence MED1 expression. We found that these ultracompact RNA nanoparticles are very stable under RNase A, serum, and 8 M urea conditions. These nanoparticles specifically bound to HER2-overexpressing breast cancer cells, efficiently depleted MED1 expression, and significantly decreased ERα-mediated gene transcription, whereas point mutations of the HER2 RNA aptamer on these nanoparticles abolished such functions. The RNA nanoparticles not only reduced the growth, metastasis, and mammosphere formation of the HER2-overexpressing breast cancer cells but also sensitized them to tamoxifen treatment. These biosafe nanoparticles efficiently targeted and penetrated into HER2-overexpressing tumors after systemic administration in orthotopic xenograft mouse models. In addition to their ability to greatly inhibit tumor growth and metastasis, these nanoparticles also led to a dramatic reduction in the stem cell content of breast tumors when combined with tamoxifen treatment in vivo. Overall, we have generated multifunctional RNA nanoparticles that specifically targeted HER2-overexpressing human breast cancer, silenced MED1, and overcame tamoxifen resistance.

The DNA mismatch repair (MMR) system corrects errors that occur during DNA replication. MMR needs the coordinated and highly dynamic assembly of repair enzymes at the site of the lesion. By visualizing transient intermediates of these assemblies, single-molecule approaches have shed critical insights into the mechanisms of MMR. These studies frequently require long (>20kb) DNA substrates with lesions and other extrahelical structures inserted at defined positions. DNA derived from bacteriophage λ (λ-DNA) is a high quality long (48.5kb) DNA substrate that is frequently used in single-molecule studies. Here we provide detailed protocols for site-specific incorporation of recombinant sequences and extrahelical structures into λ-DNA. We also describe how to assemble DNA curtains, and how to collect and analyze single-molecule observations of lesion recognition by MMR proteins diffusing on these DNA curtains. These protocols will facilitate future single-molecule studies of DNA transcription, replication, and repair., (© 2017 Elsevier Inc. All rights reserved.)

Bioincorporation of the methionine analogue S-(2-fluoroethyl)-l-homocysteine (l-MFE) into bacteriophage lysozyme overproduced in Escherichia coli results not only in the expected l-MFE incorporation but surprisingly substantial l-vinthionine incorporation into the labeled lysozymes. Synthetic l-vinthionine itself however is not activated by purified Escherichia coli methionyl-tRNA synthetase. The indirect preparation of vinthionine-containing proteins has the potential to be an alternate strategy to prepare vinyl thioether moieties for click chemistry applications on proteins.

We show experimentally that an inexpensive glass microcapillary can accumulate λ-phage DNA at its tip and deliver the DNA into the capillary using a combination of electro-osmotic flow, pressure-driven flow, and electrophoresis. We develop an efficient simulation model based on the electrokinetic equations and the finite-element method to explain this phenomenon. As a proof of concept for the generality of this trapping mechanism we use our numerical model to explore the effect of the salt concentration, the capillary surface charge, the applied voltage, the pressure difference, and the mobility of the analyte molecules. Our results indicate that the simple microcapillary system has the potential to capture a wide range of analyte molecules based on their electrophoretic mobility that extends well beyond our experimental example of λ-phage DNA. Our method for separation and preconcentration of analytes therefore has implications for the development of low-cost lab-on-a-chip devices.

A complete understanding of the cellular pathways involved in viral infections will ultimately require a diverse arsenal of experimental techniques, including methods for tracking individual viruses and their interactions with the host. Here we demonstrate the use of holographic microscopy to track the position, orientation, and DNA content of unlabeled bacteriophages (phages) in solution near a planar, functionalized glass surface. We simultaneously track over 100 individual λ phages at a rate of 100 Hz across a 33 μm × 33 μm portion of the surface. The technique determines the in-plane motion of the phage to nanometer precision, and the height of the phage above the surface to 100 nm precision. Additionally, we track the DNA content of individual phages as they eject their genome following the addition of detergent-solubilized LamB receptor. The technique determines the fraction of DNA remaining in the phage to within 10% of the total 48.5 kilobase pairs. Analysis of the data reveals that under certain conditions, λ phages move along the surface with their heads down and intermittently stick to the surface by their tails, causing them to stand up. Furthermore, we find that in buffer containing high concentrations of both monovalent and divalent salts, λ phages eject their entire DNA in about 7 s. Taken together, these measurements highlight the potential of holographic microscopy to resolve the fast kinetics of the early stages of phage infection.

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone's motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging.

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, term edattH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a "synaptosome" complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1-177, and a flexible C-terminal "tail", residues 178-261. Here, we quantitatively compare the binding of the full-length protein (Redβ(FL)) and the N-terminal domain (Redβ(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Redβ(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ(177) binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182-261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Redβ(FL) and Redβ(177) to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fluorescence microscopy is a field of growing applications in various fields as it can be used to study immunofluorescence,fluorescent proteins techniques, live-cell imaging and many more. The present investigation is based on this single molecule observation technique to observe the effect of N-arylhydroxamic acids on λ plasmid DNA and linear Bacteriophage T7 DNA. The compounds under present investigation include N-1-naphthyl-o-methylbenzohydroxamic acid,N-1-naphthyl-p-methylbenzohydroxamic acid, N-1-naphthyl ethoxy benzo hydroxamic acid, N-1-naphthylphenylacetohydroxamic acid and N-1-naphthyl valero hydroxamic acid. The hydrodynamic radius, RH of λ DNA and long-axis length, l of T7 DNA were determined from the direct observation of Brownian motion of the DNA molecules in the absence and presence of hydroxamic acids. Folding transition was observed for λ DNA as well as T7 DNA in the presence of naphthyl hydroxamic acids. N-1-naphthylvalerohydroxamic acid was found to be most effective.

Viruses use spatial control of constituent proteins as a means of manipulating and evading host immune systems. Similarly, precise spatial control of proteins encapsulated or presented on designed nanoparticles has the potential to biomimetically amplify or shield biological interactions. Previously, we have shown the ability to encapsulate a wide range of guest proteins within the virus-like particle (VLP) from Salmonella typhimurium bacteriophage P22, including antigenic proteins from human pathogens such as influenza. Expanding on this robust encapsulation strategy, we have used the trimeric decoration protein (Dec) from bacteriophage L as a means of controlled exterior presentation on the mature P22 VLP, to which it binds with high affinity. Through genetic fusion to the C-terminus of the Dec protein, either the 17 kDa soluble region of murine CD40L or a minimal peptide designed from the binding region of the "self-marker" CD47 was independently presented on the P22 VLP capsid exterior. Both candidates retained function when presented as a Dec-fusion. Binding of the Dec domain to the P22 capsid was minimally changed across designed constructs, as measured by surface plasmon resonance, demonstrating the broad utility of this presentation strategy. Dec-mediated presentation offers a robust, modular means of decorating the exposed exterior of the P22 capsid in order to further orchestrate responses to internally functionalized VLPs within biological systems.

The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,β)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

This paper demonstrates that it is possible to trap and release a super paramagnetic micro bead by fixing three super paramagnetic micro beads in a triangular array at the sensitive end of a micro cantilever, and by simply switching on/off an external magnetic field. To provide evidence of this principle we trap a micro bead that is attached to the free end of single DNA molecule and that has been previously fixed at the other end to a glass surface, using the standard sample preparation protocol of magnetic tweezers assays. The switching process is reversible which preserves the integrity of the tethered molecule, and a local force applied over the tethered bead excludes the neighbouring beads from the magnetic trap. We have developed a quadrature phase interferometer which is able to perform under fluid environments to accurately measure small deflections, which permits the exploration of DNA elasticity. Our results agree with measurements from magnetic tweezer assays performed under similar conditions. Furthermore, compared to the magnetic tweezer methodology, the combination of the magnetic trap with a suitable measurement system for cantilever deflection, allows for the exploration of a wide range of forces using a local method that has an improved temporal resolution.

We have revealed a connection between DNA-nanoparticle (NP) binding and in vitro DNA damage induced by citrate- and branched polyethylenimine-coated silver nanoparticles (c-AgNPs and b-AgNPs) as well as graphene oxide (GO) nanosheets. All three types of nanostructures triggered an early onset of DNA melting, where the extent of the melting point shift depends upon both the type and concentration of the NPs. Specifically, at a DNA/NP weight ratio of 1.1/1, the melting temperature of lambda DNA dropped from 94 °C down to 76 °C, 60 °C, and room temperature for GO, c-AgNPs and b-AgNPs, respectively. Consistently, dynamic light scattering revealed that the largest changes in DNA hydrodynamic size were also associated with the binding of b-AgNPs. Upon introduction to cells, b-AgNPs also exhibited the highest cytotoxicity, at the half-maximal inhibitory (IC50) concentrations of 3.2, 2.9, and 5.2 mg/L for B and T-lymphocyte cell lines and primary lymphocytes, compared to the values of 13.4, 12.2, and 12.5 mg/L for c-AgNPs and 331, 251, and 120 mg/L for GO nanosheets, respectively. At cytotoxic concentrations, all NPs elicited elevated genotoxicities via the increased number of micronuclei in the lymphocyte cells. However, b-AgNPs also induced micronuclei at subtoxic concentrations starting from 0.1 mg/L, likely due to their stronger cellular adhesion and internalization, as well as their subsequent interference with normal DNA synthesis or chromosome segregation during the cell cycle. This study facilitates our understanding of the effects of NP chemical composition, surface charge, and morphology on DNA stability and genotoxicity, with implications ranging from nanotoxicology to nanobiotechnology and nanomedicine.

DNA in cells is heavily covered with all types of proteins that regulate its genetic activity. Detection of DNA-bound proteins is a challenge that is well suited to solid-state nanopores as they provide a linear readout of the DNA and DNA-protein volume in the pore constriction along the entire length of a molecule. Here, we demonstrate that we can realize the detection of even individual DNA-bound proteins at the single-DNA-molecule level using solid-state nanopores. We introduce and use a new model system of anti-DNA antibodies bound to lambda phage DNA. This system provides several advantages since the antibodies bind individually, tolerate high salt concentrations, and will, because of their positive charge, not translocate through the pore unless bound to the DNA. Translocation of DNA-antibody samples reveals the presence of short 12 μs current spikes within the DNA traces, with amplitudes that are about 4.5 times larger than that of dsDNA, which are associated with individual antibodies. We conclude that transient interactions between the pore and the antibodies are the primary mechanism by which bound antibodies are observed. This work provides a proof-of-concept for how nanopores could be used for future sensing applications.

Bacteriophage λ is a classical model object in molecular biology, but little is still known on the physical properties of its DNA and regulatory elements. A study was made of the electrostatic properties of phage λ DNA and regulatory elements. A global electrostatic potential distribution along the phage genome was found to be nonuniform with main regulatory elements being located in a limited region with a high potential. The RNA polymerase binding frequency on the linearized phage chromosome directly correlates with its local potential. Strong promoters of the phage and its host Escherichia coli have distinct electrostatic upstream elements, which differ in nucleotide sequence. Attachment and recombination sites of phage λ and its host have a higher potential, which possibly facilitates their recognition by integrase. Phage λ and host Rho-independent terminators have a symmetrical M-shaped potential profile, which only slightly depends on the annotated terminator palindrome length, and occur in a region with a substantially higher potential, which may cause polymerase retention, facilitating the formation of a terminator hairpin in RNA. It was concluded that virtually all elements of phage λ genome have potential distribution specifics, which are related to their structural properties and may play a role in their biological function. The global potential distribution along the phage genome reflects the architecture of the regulation of its transcription and integration in the host genome.

Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nanopores can probe the structure of biopolymers in solution; however, diffusion makes it difficult to study the same molecule for extended periods. Here we report devices that entropically trap single DNA molecules in a 6.2-femtolitre cage near a solid-state nanopore. We electrophoretically inject DNA molecules into the cage through the nanopore, pause for preset times and then drive the DNA back out through the nanopore. The saturating recapture time and high recapture probability after long pauses, their agreement with a convection-diffusion model and the observation of trapped DNA under fluorescence microscopy all confirm that the cage stably traps DNA. Meanwhile, the cages have 200 nm openings that make them permeable to small molecules, like the restriction endonuclease we use to sequence-specifically cut trapped DNA into fragments whose number and sizes are analysed upon exiting through the nanopore. Entropic cages thus serve as reactors for chemically modifying single DNA molecules.

In this study, we analyze correlations of vibrational motion on the surface of a small globular protein and in its hydration shell. In contrast to single particle hydration water dynamics, which are perturbed by interactions with the protein solute only in the first few hydration layers, we find that correlated, collective motions extend into the surrounding solvent on a 10 Å length scale, specifically at far-infrared frequencies below 100 cm(-1). As a function of frequency, we analyze the distribution of correlated longitudinal motions in the three-dimensional environment of the protein solute, as well as in the vicinity of different protein-water interfaces. An anisotropic distribution of these correlations is observed, which is related to specific protein-water vibrations and interactions at the interfaces, as well as flexibilities of solvent exposed sites. Our results show that coupling of protein and water dynamics leaves a three-dimensional imprint in the collective dynamics of its hydration shell, and we discuss potential implications for biomolecular function, e.g., molecular recognition and binding, and the dynamical coupling of proteins to their native solvation environment.

Nanoparticle technologies provide a powerful tool for the development of reagents for use in both therapeutic and diagnostic, or "theragnostic" biomedical applications. Two broad classes of particles are under development, viral and synthetic systems, each with their respective strengths and limitations. Here we adapt the phage lambda system to construct modular "designer" nanoparticles that blend these two approaches. We have constructed a variety of modified "decoration" proteins that allow site-specific modification of the shell with both protein and nonproteinaceous ligands including small molecules, carbohydrates, and synthetic display ligands. We show that the chimeric proteins can be used to simultaneously decorate the shell in a tunable surface density to afford particles that are physically homogeneous and that can be manufactured to display a variety of ligands in a defined composition. These designer nanoparticles set the stage for development of lambda as a theragnostic nanoparticle system.

This paper presents a microfluidic method for precise control of the size and polydispersity of surfactant-DNA nanoparticles. A mixture of surfactant and DNA dispersed in 35% ethanol is focused between two streams of pure water in a microfluidic channel. As a result, a rapid change of solvent quality takes place in the central stream, and the surfactant-bound DNA molecules undergo a fast coil-globule transition. By adjusting the concentrations of DNA and surfactant, fine-tuning of the nanoparticle size, down to a hydrodynamic diameter of 70 nm with a polydispersity index below 0.2, can be achieved with a good reproducibility.

Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single-molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ-DNA undergoes a solid-to-fluid-like disordering transition as a function of temperature, resulting locally in less densely packed DNA, reducing DNA-DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.

Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

Interactions of proteins with nucleic acids arise at all levels of cellular function, from chromosomal packing to biological regulation. These interactions can be analyzed in a high-throughput fashion by immobilizing the DNA sequences of interest, possibly numbering in the thousands, at discrete locations on a solid support and identifying those sequences that a protein analyte binds. Ideally, such surface assays would use unlabeled analyte to simplify protocols and avoid the possibility of perturbing the protein/DNA interaction. The present study compares three electrochemical modalities for simultaneously detecting binding of unlabeled transcription factor proteins to immobilized DNA duplexes based on (i) changes in the duplex diffusive motions, (ii) variations in the surface potential, and (iii) variations in the interfacial charging impedance, all of which can be conveniently derived from AC voltammetry traces. Cro protein from bacteriophage lambda is used as a model transcription factor. Specific binding of protein was successfully detected through modalities (i) and (ii), but not (iii). The effectiveness of these techniques is compared as a function of sampling frequency and protein concentration. Binding of 15 kDa Cro slowed down rotational diffusion of immobilized duplexes approximately 3-fold, and induced up to 5 mV changes in the surface potential. Moreover, by assessing Cro binding to bacteriophage operators of variable affinity, the study illustrates how contrast between specific and nonspecific interactions impacts detection.

The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force-extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force-extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.

The properties of channel-confined semiflexible polymers are determined by a complicated interplay of chain stiffness and excluded volume effects. Using Pruned-Enriched Rosenbluth Method (PERM) simulations, we study the equilibrium properties of channel-confined polymers by systematically controlling chain stiffness and excluded volume. Our calculations of chain extension and confinement free energy for freely jointed chains with and without excluded volume show excellent agreement with theoretical predictions. For ideal wormlike chains, the extension is seen to crossover from Odijk behavior in strong confinement to zero-stretching, bulk-like behavior in weak confinement. In contrast, for self-avoiding wormlike chains, we always observe that the linear scaling of the extension with the contour length is valid in the long-chain limit irrespective of the regime of confinement, owing to the coexistence of stiffness and excluded volume effects. We further propose that the long-chain limit for the extension corresponds to chain lengths wherein the projection of the end-to-end distance along the axis of the channel is nearly equal to the mean span parallel to the axis. For DNA in nanochannels, this limit was identified using PERM simulations out to molecular weights of more than 1 megabase pairs; the molecular weight of λ-DNA is found to exhibit nearly asymptotic fractional extension for channels sizes used commonly in experiments.