Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA–protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA–protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

The advent of single-molecule force spectroscopy represents the introduction of forces, torques, and displacements as controlled variables in biochemistry. These methods afford the direct manipulation of individual molecules to interrogate the forces that hold together their structure, the forces and torques that these molecules generate in the course of their biochemical reactions, and the use of force, torque, and displacement as tools to investigate the mechanisms of these reactions. Because of their microscopic nature, the signals detected in these experiments are often dominated by fluctuations, which, in turn, play an important role in the mechanisms that underlie the operation of the molecular machines of the cell. Their direct observation and quantification in single-molecule experiments provide a unique window to investigate those mechanisms, as well as a convenient way to investigate fundamental new fluctuation theorems of statistical mechanics that bridge the equilibrium and non-equilibrium realms of this discipline. In this review we have concentrated on the developments that occurred in our laboratory on the characterization of biopolymers and of molecular machines of the central dogma. Accordingly, some important areas like the study of cytoskeletal motors have not been included. While we adopt at times an anecdotal perspective with the hope of conveying the personal circumstances in which these developments took place, we have made every effort, nonetheless, to include the most important developments that were taking place at the same time in other laboratories.

Biophysical studies have a very high impact on the understanding of internalization, molecular mechanisms, interactions, and localization of CPPs and CPP/cargo conjugates in live cells or in vivo. Biophysical studies are often first carried out in test-tube set-ups or in vitro, leading to the complicated in vivo systems. This review describes recent studies of CPP internalization, mechanisms, and localization. The multiple methods in these studies reveal different novel and important aspects and define the rules for CPP mechanisms, hopefully leading to their improved applicability to novel and safe therapies.

Synthetic peptide and peptido-mimetic filaments and tubes represent a diverse class of nanomaterials with a broad range of potential applications, such as drug delivery, vaccine development, synthetic catalyst design, encapsulation, and energy transduction. The structures of these filaments comprise supramolecular polymers based on helical arrangements of subunits that can be derived from self-assembly of monomers based on diverse structural motifs. In recent years, structural analyses of these materials at near-atomic resolution (NAR) have yielded critical insights into the relationship between sequence, local conformation, and higher-order structure and morphology. This structural information offers the opportunity for development of new tools to facilitate the predictable and reproducible de novo design of synthetic helical filaments. However, these studies have also revealed several significant impediments to the latter process – most notably, the common occurrence of structural polymorphism due to the lability of helical symmetry in structural space. This article summarizes the current state of knowledge on the structures of designed peptide and peptido-mimetic filamentous assemblies, with a focus on structures that have been solved to NAR for which reliable atomic models are available.

We demonstrate that reshaping of the dynamic, bulged-loop energy landscape of DNA triplet repeat ensembles by the presence of an abasic site alters repair outcomes by the APE1 enzyme. This phenomenon depends on the structural context of the lesion, despite the abasic site always having the same neighbors in sequence space. We employ this lesion-induced redistribution of DNA states and a kinetic trap to monitor different occupancies of the DNA bulge loop states. We show how such dynamic redistribution and associated differential occupancies of DNA states impact APE1 repair outcomes and APE1 induced interconversions. We correlate the differential biophysical properties of the dynamic, DNA ensemble states, with their ability to be recognized and processed as substrates by the APE1 DNA repair enzyme. Enzymatic digestions and biophysical characterizations reveal that APE1 cuts a fraction (10–12%) of the dynamic, rollameric substrates within the initial kinetic distribution. APE1 interactions also ‘induce’ rollamer redistribution from a kinetically trapped distribution to an equilibrium distribution, the latter not containing viable APE1 substrates. We distinguish between kinetically controlled ensemble (re)distributions of potential DNA substrates, versus thermodynamically controlled ensemble (re)distribution; features of importance to DNA regulation. We conclude that APE1 activity catalyzes/induces ensembles that represent the thermodynamically optimal loop distribution, yet which also are nonviable substrate states for abasic site cleavage by APE1. We propose that by inducing substrate redistributions in a dynamic energy landscape, the enzyme actually reduces the available substrate competent species for it to process, reflective of a regulatory mechanism for enzymatic self-repression. If this is a general phenomenon, such a consequence would have a profound impact on slowing down and/or misdirecting DNA repair within dynamic energy landscapes, as exemplified here within triplet repeat domains. In short, APE1-instigated redistribution of potential substrates induces a preferred pathway to an equilibrium ensemble of enzymatically incompetent states.

Condensation and faithful separation of the genome are crucial for the cellular life cycle. During chromosome segregation, mechanical forces generated by the mitotic spindle pull apart the sister chromatids. The mechanical nature of this process has motivated a lot of research interest into the mechanical properties of mitotic chromosomes. Although their fundamental mechanical characteristics are known, it still remains unclear how these characteristics emerge from the structure of the mitotic chromosome. Recent advances in genomics, computational and super-resolution microscopy techniques have greatly promoted our understanding of the chromosomal structure and have motivated us to review the mechanical characteristics of chromosomes in light of the current structural insights. In this review, we will first introduce the current understanding of the chromosomal structure, before reviewing characteristic mechanical properties such as the Young's modulus and the bending modulus of mitotic chromosomes. Then we will address the approaches used to relate mechanical properties to the structure of chromosomes and we will also discuss how mechanical characterization can aid in elucidating their structure. Finally, future challenges, recent developments and emergent questions in this research field will be discussed.

DNA dynamics can only be understood by taking into account its complex mechanical behavior at different length scales. At the micrometer level, the mechanical properties of single DNA molecules have been well-characterized by polymer models and are commonly quantified by a persistence length of 50 nm (~150 bp). However, at the base pair level (~3.4 Å), the dynamics of DNA involves complex molecular mechanisms that are still being deciphered. Here, we review recent single-molecule experiments and molecular dynamics simulations that are providing novel insights into DNA mechanics from such a molecular perspective. We first discuss recent findings on sequence-dependent DNA mechanical properties, including sequences that resist mechanical stress and sequences that can accommodate strong deformations. We then comment on the intricate effects of cytosine methylation and DNA mismatches on DNA mechanics. Finally, we review recently reported differences in the mechanical properties of DNA and double-stranded RNA, the other double-helical carrier of genetic information. A thorough examination of the recent single-molecule literature permits establishing a set of general ‘rules’ that reasonably explain the mechanics of nucleic acids at the base pair level. These simple rules offer an improved description of certain biological systems and might serve as valuable guidelines for future design of DNA and RNA nanostructures.

Quantitative parameters for a two-state cooperative transition in duplex DNAs were finally obtained during the last 5 years. After a brief discussion of observations pertaining to the existence of the two-state equilibrium per se, the lengths, torsion, and bending elastic constants of the two states involved and the cooperativity parameter of the model are simply stated. Experimental tests of model predictions for the responses of DNA to small applied stretching, twisting, and bending stresses, and changes in temperature, ionic conditions, and sequence are described. The mechanism and significance of the large cooperativity, which enables significant DNA responses to such small perturbations, are also noted. The capacity of the model to resolve a number of long-standing and sometimes interconnected puzzles in the extant literature, including the origin of the broad pre-melting transition studied by numerous workers in the 1960s and 1970s, is demonstrated. Under certain conditions, the model predicts significant long-range attractive or repulsive interactions between hypothetical proteins with strong preferences for one or the other state that are bound to well-separated sites on the same DNA. A scenario is proposed for the activation of the ilvPG promoter on a supercoiled DNA by integration host factor.

CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam-induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. We examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution.

Desoxyribosenucleic acid, DNA, and cellulose molecules self-assemble in aqueous systems. This aggregation is the basis of the important functions of these biological macromolecules. Both DNA and cellulose have significant polar and nonpolar parts and there is a delicate balance between hydrophilic and hydrophobic interactions. The hydrophilic interactions related to net charges have been thoroughly studied and are well understood. On the other hand, the detailed roles of hydrogen bonding and hydrophobic interactions have remained controversial. It is found that the contributions of hydrophobic interactions in driving important processes, like the double-helix formation of DNA and the aqueous dissolution of cellulose, are dominating whereas the net contribution from hydrogen bonding is small. In reviewing the roles of different interactions for DNA and cellulose it is useful to compare with the self-assembly features of surfactants, the simplest case of amphiphilic molecules. Pertinent information on the amphiphilic character of cellulose and DNA can be obtained from the association with surfactants, as well as on modifying the hydrophobic interactions by additives.

Cryo-electron microscopy (cryo-EM) has become the technique of choice for structural biology of macromolecular assemblies, after the ‘resolution revolution’ that has occurred in this field since 2012. With a suitable instrument, an appropriate electron detector and, last but not least, a cooperative sample it is now possible to collect images from which macromolecular structures can be determined to better than 2 Å resolution, where reliable atomic models can be built. By electron tomography and sub-tomogram averaging of cryo-samples, it is also possible to reconstruct subcellular structures to sub-nanometre resolution. This review describes the infrastructure that is needed to achieve this goal. Ideally, a cryo-EM lab will have a dedicated 300 kV electron microscope for data recording and a 200 kV instrument for screening cryo-samples, both with direct electron detectors, and at least one 120 kV EM for negative-stain screening at room temperature. Added to this should be ancillary equipment for specimen preparation, including a light microscope, carbon coater, plasma cleaner, glow discharge unit, a device for fast, robotic sample freezing, liquid nitrogen storage Dewars and a ready supply of clean liquid nitrogen. In practice, of course, the available budget will determine the number and types of microscopes and how elaborate the lab can be. The cryo-EM lab should be designed with adequate space for the electron microscopes and ancillary equipment, and should allow for sufficient storage space. Each electron microscope room should be connected to the image-processing computers by fibre-optic cables for the rapid transfer of large datasets. The cryo-EM lab should be overseen by a facility manager whose responsibilities include the day-to-day tasks to ensure that all microscopes are operating perfectly, organising service and repairs to minimise downtime, and controlling the budget. Large facilities will require additional support staff who help to oversee the operation of the facility and instruct new users.

X-ray crystallography enables detailed structural studies of proteins to understand and modulate their function. Conducting crystallographic experiments at cryogenic temperatures has practical benefits but potentially limits the identification of functionally important alternative protein conformations that can be revealed only at room temperature (RT). This review discusses practical aspects of preparing, acquiring, and analyzing X-ray crystallography data at RT to demystify preconceived impracticalities that freeze progress of routine RT data collection at synchrotron sources. Examples are presented as conceptual and experimental templates to enable the design of RT-inspired studies; they illustrate the diversity and utility of gaining novel insights into protein conformational landscapes. An integrative view of protein conformational dynamics enables opportunities to advance basic and biomedical research.

When the iconic DNA genetic code is expressed in terms of energy differentials, one observes that information embedded in chemical sequences, including some biological outcomes, correlate with distinctive free energy profiles. Specifically, we find correlations between codon usage and codon free energy, suggestive of a thermodynamic selection for codon usage. We also find correlations between what are considered ancient amino acids and high codon free energy values. Such correlations may be reflective of the sequence-based genetic code fundamentally mapping as an energy code. In such a perspective, one can envision the genetic code as composed of interlocking thermodynamic cycles that allow codons to ‘evolve’ from each other through a series of sequential transitions and transversions, which are influenced by an energy landscape modulated by both thermodynamic and kinetic factors. As such, early evolution of the genetic code may have been driven, in part, by differential energetics, as opposed exclusively by the functionality of any gene product. In such a scenario, evolutionary pressures can, in part, derive from the optimization of biophysical properties (e.g. relative stabilities and relative rates), in addition to the classic perspective of being driven by a phenotypical adaptive advantage (natural selection). Such differential energy mapping of the genetic code, as well as larger genomic domains, may reflect an energetically resolved and evolved genomic landscape, consistent with a type of differential, energy-driven ‘molecular Darwinism’. It should not be surprising that evolution of the code was influenced by differential energetics, as thermodynamics is the most general and universal branch of science that operates over all time and length scales.