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Less than one in thirty of the RNA sequences transcribed in humans are translated into protein. The noncoding RNA (ncRNA) functions in catalysis, structure, regulation, and more. However, for the most part, these functions are poorly characterized. RNA is modular and described by motifs that include helical A-RNA with canonical Watson–Crick base-pairing as well as structures with only noncanonical base pairs. Understanding the structure and dynamics of motifs will aid in deciphering functions of specific ncRNAs. We present computational studies on a standard sarcin/ricin domain (SRD), citrus bark cracking viroid SRD, as well as A-RNA. We have applied enhanced molecular dynamics techniques that construct an inverse free-energy surface (iFES) determined by collective variables that monitor base-pairing and backbone conformation. Each SRD RNA is flanked on each side by A-RNA, allowing comparison of the behavior of these motifs in the same molecule. The RNA iFESs have single peaks, indicating that the combined motifs should denature as a single cohesive unit, rather than by regional melting. Local root-mean-square deviation (RMSD) analysis and communication propensity (CProp, variance in distances between residue pairs) reveal distinct motif properties. Our analysis indicates that the standard SRD is more stable than the viroid SRD, which is more stable than A-RNA. Base pairs at SRD to A-RNA transitions have limited flexibility. Application of CProp reveals extraordinary stiffness of the SRD, allowing residues on opposite sides of the motif to sense each other’s motions.

Viroids are the smallest replicative pathogens, consisting of RNA circles (∼300 nucleotides) that require host machinery to replicate. Structural RNA elements recruit these host factors. Currently, many of these structural elements and the nature of their interactions are unknown. All Pospiviroidaehave homology in the central conserved region (CCR). The CCR of potato spindle tuber viroid (PSTVd) contains a sarcin/ricin domain (SRD), the only viroid structural element with an unequivocal replication role. We assumed that every member of this family uses this region to recruit host factors, and that each CCR has an SRD-like asymmetric loop within it. Potential SRD or SRD-like motifs were sought in the CCR of each Pospiviroidaemember as follows. Motif location in each CCR was predicted with MUSCLE alignment and Vienna RNAfold. Viroid-specific models of SRD-like motifs were built by superimposing noncanonical base pairs and nucleotides on a model of an SRD. The RNA geometry search engine FR3D was then used to find nucleotide groups close to the geometry suggested by this superimposition. Atomic resolution structures were assembled using the molecular visualization program Chimera, and the stability of each motif was assessed with molecular dynamics (MD). Some models required a protonated cytosine. To be stable within a cell, the pKaof that cytosine must be shifted up. Constant pH-replica exchange MD analysis showed such a shift in the proposed structures. These data show that every Pospiviroidaemember could form a motif that resembles an SRD in its CCR, and imply there could be undiscovered mimics of other RNA domains.

The phenomenon of microphase separation is an example of self-assembly in soft matter and has been observed in block copolymers (BCPs) and similar materials (i.e., supramolecular assemblies (SMAs) and homo/block copolymer blends (HBCs)). In this study, we use microphase separation to construct responsive polymer brushes that collapse to generate periodic surfaces. This is achieved by a chemical reaction between the minor block (10%, poly(4-vinylpyridine)) of the block copolymer and a substrate. The major block of polystyrene (PS) forms mosaic-like arrays of grafted patches that are 10–20 nm in size. Depending on the nature of the assembly (SMA, HBC, or neat BCP) and annealing method (exposure to vapors of different solvents or heating above the glass transition temperature), a range of “mosaic” brushes with different parameters can be obtained. Successive grafting of a secondary polymer (polyacrylamide, PAAm) results in the fabrication of binary polymer brushes (BPBs). Upon being exposed to specific selective solvents, BPBs may adopt different conformations. The surface tension and adhesion of the binary brush are governed by the polymer occupying the top stratum. The “mosaic” brush approach allows for a combination of strongly immiscible polymers in one brush. This facilitates substantial contrast in the surface properties upon switching, previously only possible for substrates composed of predetermined nanostructures. We also demonstrate a possible application of such PS/PAAm brushes in a tunable bioadhesion–bioadhesive (PS on top) or nonbioadhesive (PAAm on top) surface as revealed by Escherichia colibacterial seeding.

DNA branch migration is a fundamental process in genetic recombination. A new model system has been developed for studying branch migration in a small synthetic four-arm junction. A mathematical method for describing branch-point movement by discrete steps in such junctions is also presented. The key to our experimental system is the ability to fix the location of the branch point during the assembly of the junction with a reversible block. The block is provided by a short oligonucleotide that forms triplex DNA adjacent to the initial location branch point at low pH. Raising the pH causes the triplex strand to dissociate, making the branch point free to migrate. Once mobile, the branch point can run off the end of the junction. The time-course for this runoff is consistent with a random walk of the branch point. If it is assumed that one migration step moves the branch point one base-pair, the time-course gives a rate constant for one step of 1.4 second−1at 37°C in 10 mM MgCl2, 50 mM NaCl. These values are consistent with other measurements of non-enzymatic branch migration. We have also monitored the spread of the branch points directly with T4 endonuclease VII. Using EcoRI restriction endonuclease, we have shown that the binding of this protein to the arms of the junction essentially blocks branch migration through the binding site. In these experiments Ca2+replaces Mg2+, and the enzyme does not cleave the DNA. In vivothere must be a special process to get branch points to migrate past bound proteins.

Bacteriophage λ DNA integrates into the chromosome ofEscherichia coliby first forming an intasome at the phage attachment site on the phage DNA with the integrase Int and integration host factor. This intasome searches the host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactions to achieve integration. This study characterizes the weak interaction of the intasome andattB. The hypothesis that all of the proteins necessary for integration are brought to the reaction site by the intasome is given additional support by showing that the concentration of phage attachment site and notattB determines the optimal concentration of proteins for integration. The value of the dissocation constant of the complex formed between the intasome andattB is determined in two different ways. First, the rate of the integration reaction is measured as a function of theattB DNA concentration. The saturation constant reflects the dissociation constant of the complex. Second, a recomb ination reaction is inhibited by the inroduction of varying amounts of a secondattB with a sequence change that blocks recombination with this site. The inhibition constant reflects the dissociation constant of the intasome and alteredattB in this experiment. The two methods agree and give a dissociation constant of approximately 300 nM.attB contains two core binding sites for the intasome; it is shown that both are necessary for efficient capture. The value of the dissocation constants are considerably lower when a mutant integrase, IntE174K, is used. This increased affinity for core sites can explain how IntE174K can function in the absence of integration host factor. The inhibition constants also show dependence on the exact sequence of the inhibitingattB. Possible implications of this dependence are discussed.

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