Sarcin/Ricin Domain RNA Retains Its Structure Better Than A-RNA in Adaptively Biased Molecular Dynamics Simulations.

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.

In silico survey of the central conserved regions in viroids of the Pospiviroidae family for conserved asymmetric loop structures.

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 Pospiviroidae have 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 Pospiviroidae member 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 pKa of 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 Pospiviroidae member could form a motif that resembles an SRD in its CCR, and imply there could be undiscovered mimics of other RNA domains.

Enrichment of midsized RNAs with manganese chloride precipitation.

Enrichment of specific RNAs is important for RNA analysis. MnCl2 has been used previously to enrich viroid RNA fractions from total RNA from infected plants. We have expanded this method to show that MnCl2 can enrich single-stranded as well as structured RNAs of 450 nt and below from a total RNA preparation. We have applied this method to map the transcription start sites of a PSTVd transcript from total RNA from yeast under conditions where the RNA was previously undetectable.

Live-cell imaging of small nucleolar RNA tagged with the broccoli aptamer in yeast.

The development of the RNA 'vegetable' aptamers, Spinach and Broccoli, has simplified RNA imaging, especially in live cells. These RNA aptamers interact with a fluorophore (DFHBI or DFHBI-1T) to produce a green fluorescence signal. Although used in mammalian and Escherichia coli cells, the use of these aptamers in yeast has been limited. Here we describe how the Saccharomyces cerevisiae snoRNA, snR30, was tagged with the Spinach or the Broccoli aptamers and observed in live cells. The ability to observe aptamer fluorescence in polyacrylamide gels stained with a fluorophore or with a microplate reader can ease preliminary screening of the aptamers in different RNA scaffolds. In snR30 a tandem repeat of the Broccoli aptamer produced the best signal in vitro. Multiple factors in cell preparation were vital for obtaining a good fluorescence signal. These factors included the clearance of the native unmodified snR30, the amount and length of dye incubation and the rinsing of cells. In cells, the aptamers did not interfere with the structure or essential function of snR30, as the tagged RNA localized to the nucleolus and directed processing of ribosomal RNA in yeast. High-resolution images of the tagged snoRNA were obtained with live cells immobilized by a microcompressor.

Processing of Potato Spindle Tuber Viroid RNAs in Yeast, a Nonconventional Host.

Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for studying the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers, and a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Saccharomyces cerevisiae Of these, only one form, a construct that adopts a previously described tetraloop-containing conformation (TL), produces circles. TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' and 3' duplications, do not produce circles. The TL circles contain nonnative nucleotides resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, likely representing RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in various susceptibilities to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts.IMPORTANCE In higher eukaryotes, the majority of transcribed RNAs do not encode proteins. These noncoding RNAs are responsible for mRNA regulation, control of the expression of regulatory microRNAs, sensing of changes in the environment by use of riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world and, as such, would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and on host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

Assembly of supramolecular DNA complexes containing both G-quadruplexes and i-motifs by enhancing the G-repeat-bearing capacity of i-motifs.

The single-step assembly of supramolecular complexes containing both i-motifs and G-quadruplexes (G4s) is demonstrated. This can be achieved because the formation of four-stranded i-motifs appears to be little affected by certain terminal residues: a five-cytosine tetrameric i-motif can bear ten-base flanking residues. However, things become complex when different lengths of guanine-repeats are added at the 3' or 5' ends of the cytosine-repeats. Here, a series of oligomers d(XGiXC5X) and d(XC5XGiX) (X = A, T or none; i < 5) are designed to study the impact of G-repeats on the formation of tetrameric i-motifs. Our data demonstrate that tetramolecular i-motif structure can tolerate specific flanking G-repeats. Assemblies of these oligonucleotides are polymorphic, but may be controlled by solution pH and counter ion species. Importantly, we find that the sequences d(TGiAC5) can form the tetrameric i-motif in large quantities. This leads to the design of two oligonucleotides d(TG4AC7) and d(TGBrGGBrGAC7) that self-assemble to form quadruplex supramolecules under certain conditions. d(TG4AC7) forms supramolecules under acidic conditions in the presence of K+ that are mainly V-shaped or ring-like containing parallel G4s and antiparallel i-motifs. d(TGBrGGBrGAC7) forms long linear quadruplex wires under acidic conditions in the presence of Na+ that consist of both antiparallel G4s and i-motifs.

A mechanical microcompressor for high resolution imaging of motile specimens.

In order to obtain fine details in 3 dimensions (3D) over time, it is critical for motile biological specimens to be appropriately immobilized. Of the many immobilization options available, the mechanical microcompressor offers many benefits. Our device, previously described, achieves gentle flattening of a cell, allowing us to image finely detailed structures of numerous organelles and physiological processes in living cells. We have imaged protozoa and other small metazoans using differential interference contrast (DIC) microscopy, orientation-independent (OI) DIC, and real-time birefringence imaging using a video-enhanced polychromatic polscope. We also describe an enhancement of our previous design by engineering a new device where the coverslip mount is fashioned onto the top of the base; so the entire apparatus is accessible on top of the stage. The new location allows for easier manipulation of the mount when compressing or releasing a specimen on an inverted microscope. Using this improved design, we imaged immobilized bacteria, yeast, paramecia, and nematode worms and obtained an unprecedented view of cell and specimen details. A variety of microscopic techniques were used to obtain high resolution images of static and dynamic cellular and physiological events.

Binary Polymer Brushes of Strongly Immiscible Polymers.

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 coli bacterial seeding.

Influence of N-terminal hydrophobicity of cationic peptides on thermodynamics of their interaction with plasmid DNA.

There is a need to understand the thermodynamics of interaction of cationic peptides with DNA to design better peptide based non-viral gene delivery vectors. The main aim of this study was to understand the influence of N-terminal hydrophobicity of cationic amphiphilic peptides on thermodynamics of interaction with plasmid DNA. The model peptides used were TATPTD and TATPTDs modified at the N-terminal with hydrophobic amino acids. The thermodynamic binding data from isothermal titration calorimetry were compared with ethidium bromide analysis and ultrafiltration to correlate the binding parameters with the structural features of the various peptides used. It was observed that peptides having a smaller hydrophobic domain at the N-terminal have good DNA condensing ability compared with the ones with a longer hydrophobic domain. Calorimetry of peptides that reached saturation binding indicated that enthalpy and entropy are favorable for the interaction. Moreover, the interaction of these peptides with DNA appears to be predominantly electrostatic.

Structural features of the L-argininamide-binding DNA aptamer studied with ESI-FTMS.

The 24-mer DNA aptamer of Harada and Frankel (Harada, K.; Frankel, A. D. EMBO J. 1995, 14, 5798-5811) that binds L-argininamide (L-Arm) was studied by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). This DNA folds into a stem and loop such that the loop is able to engulf L-Arm. As controls, two derivatives of the same base composition, one with the same stem but a scrambled loop and the other with no ability to form a secondary structure, were studied. The two DNAs that could fold into stem-loop structures showed a more negatively charged distribution of ions than the linear control. This tendency was preserved in the presence of ligand; complexes expected to have more secondary structure had ions with more negative charges. Distinct species corresponding to no, one, and two bound L-Arm molecules were observed for each DNA. The fractional peak intensities were fit to a straightforward binding model and binding constants were obtained. Thus, ESI-FTMS can provide both qualitative and quantitative data regarding the structure of DNA and its interactions with noncovalent ligands.

Secondary structural characterization of oligonucleotide strands using electrospray ionization mass spectrometry.

Differences in charge state distributions of hairpin versus linear strands of oligonucleotides are analyzed using electrospray ionization mass spectrometry (ESI-MS) in the negative ion detection mode. It is observed that the linear structures show lower charge state distribution than the hairpin strands of the same composition. The concentration of ammonium acetate and the cone voltage are major factors that cause the shift of the negative ions in the charge states. The ESI data presented here are supported by UV spectra of strands acquired at 260 nm wavelength in aqueous ammonium acetate solution. We will show that the strands that demonstrate a higher charge state distribution in the gas phase also have a higher melting temperature in solution.

Discrete and continuous mathematical models of DNA branch migration.

DNA junctions, known as Holliday junctions, are intermediates in genetic recombination between DNAs. In this structure, two double-stranded DNA helices with similar sequence are joined at a branch point. The branch point can move along these helices when strands with the same sequence are exchanged. Such branch migration is modeled as a random walk. First, we model this process discretely, such that the motion of the branch is represented as transfer between discrete compartments. This is useful in analysing the results of DNA branch migration on junction comprised of synthetic oligonucleotides. The limit in which larger numbers of smaller steps go to continuous motion of the branch is also considered. We show that the behavior of the continuous system is very similar to that of the discrete system when there are more than just a few compartments. Thus, even branch migration on oligonucleotides can be viewed as a continuous process. One consequence of this is that a step size must be assumed when determining rate constants of branch migration. We compare migration where forward and backward movements of the branch are equally probable to biased migration where one direction is favored over the other. In the latter case larger differences between the discrete and continuous cases are predicted, but the differences are still small relative to the experimental error associated with experiments to measure branch migration in oligonucleotides.