De novo gene synthesis by an antiviral reverse transcriptase.

Defense-associated reverse transcriptase (DRT) systems perform DNA synthesis to protect bacteria against viral infection, but the identities and functions of their DNA products remain largely unknown. We show that DRT2 systems encode an unprecedented immune pathway that involves de novo gene synthesis through rolling circle reverse transcription of a noncoding RNA (ncRNA). Programmed template jumping on the ncRNA generates a concatemeric cDNA, which becomes double-stranded upon viral infection.

De novo gene synthesis by an antiviral reverse transcriptase.

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA).

Mechanism of target site selection by type V-K CRISPR-associated transposases.

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments.

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins.

Bioorthogonal chemistry-based RNA labeling technologies: evolution and current state.

To understand the structure and ensuing function of RNA in various cellular processes, researchers greatly rely on traditional as well as contemporary labeling technologies to devise efficient biochemical and biophysical platforms. In this context, bioorthogonal chemistry based on chemoselective reactions that work under biologically benign conditions has emerged as a state-of-the-art labeling technology for functionalizing biopolymers. Implementation of this technology on sugar, protein, lipid and DNA is fairly well established.

Responsive fluorescent nucleotides serve as efficient substrates to probe terminal uridylyl transferase.

We repurposed a terminal uridylyl transferase enzyme to site-specifically label RNA with microenvironment sensing fluorescent nucleotide mimics, which in turn provided direct read-outs to estimate the binding affinities of the enzyme to RNA and nucleotide substrates. This enzyme-probe system provides insights into the catalytic cycle, and can facilitate the development of discovery platforms to identify robust enzyme inhibitors.

Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9.

Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques.

Impact of Regulatory Guidance on Evaluating Cardiovascular Risk of New Glucose-Lowering Therapies to Treat Type 2 Diabetes Mellitus: Lessons Learned and Future Directions.

Responding to concerns about the potential for increased risk of adverse cardiovascular outcomes, specifically myocardial infarction, associated with certain glucose-lowering therapies, the US Food and Drug Administration and the Committee for Medicinal Products for Human Use of the European Medicines Agency issued guidance to the pharmaceutical industry in 2008. Glucose-lowering therapies were granted regulatory approval primarily from smaller studies that have demonstrated reductions in glycated hemoglobin concentration. Such studies were overall underpowered and of insufficient duration to show any effect on cardiovascular outcomes.

A mechanism-based computational model to capture the interconnections among epithelial-mesenchymal transition, cancer stem cells and Notch-Jagged signaling.

Epithelial-mesenchymal transition (EMT) and cancer stem cell (CSCs) formation are two fundamental and well-studied processes contributing to cancer metastasis and tumor relapse. Cells can undergo a partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype or a complete EMT to attain a mesenchymal one. Similarly, cells can reversibly gain or lose 'stemness'.

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA.

The development of modular and efficient methods to functionalize RNA with biophysical probes is very important in advancing the understanding of the structural and functional relevance of RNA in various cellular events. Herein, we demonstrate a two-step bioorthogonal chemical functionalization approach for the conjugation of multiple probes onto RNA transcripts using a 5-vinyl-modified uridine nucleotide analog (VUTP). VUTP, containing a structurally noninvasive and versatile chemoselective handle, was efficiently incorporated into RNA transcripts by in vitro transcription reactions.

Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry.

Recent developments in RNA labeling technology have provided viable tools to analyze RNA synthesis, processing and function in cell-free and cellular environments. Notably, emerging methodologies based on posttranscriptional chemical labeling by using bioorthogonal chemistry have enabled the visualization and profiling of exogenous and endogenous RNA transcripts. In this review, we first give an overview of different RNA labeling strategies based on chemical as well as genetically encoded systems.

A base-modified PNA-graphene oxide platform as a turn-on fluorescence sensor for the detection of human telomeric repeats.

Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths.

Raman spectroscopy of proteins and nucleoproteins.

A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side-chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three-dimensional structure, intramolecular dynamics, and intermolecular interactions.

Thermoresponsive nanocomposite double network hydrogels.

The utility and efficacy of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogels as smart materials is limited by their physical properties. In this study, we sought to design PNIPAAm nanocomposite hydrogels which displayed enhanced mechanical properties as well as deswelling-reswelling kinetics but without reducing equilibrium swelling or altering the convenient volume phase transition temperature (VPTT) of PNIPAAm. PNIPAAm hydrogels were formed as double networks (DN) comprised of a tightly crosslinked 1st network and a loosely crosslinked 2nd network.

A structural model for the single-stranded DNA genome of filamentous bacteriophage Pf1.

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament ( approximately 2000 x 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue alpha-helical subunit. The subunit alpha-helices, which are inclined at a small average angle ( approximately 16 degrees ) from the virion axis, are arranged compactly around the DNA core.

Raman tensors and their application in structural studies of biological systems.

The Raman scattering of a molecule is generated by interactions of its electrons with incident light. The electric vector of the Raman scattered light is related to the electric vector of the incident light through a characteristic Raman tensor. A unique Raman tensor exists for each Raman-active molecular vibrational mode.

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra.

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell.

Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit.

Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2.

Raman spectroscopy of proteins.

A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side -chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three-dimensional structure, intramolecular dynamics, and intermolecular interactions.

Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation.

IHF and HU are small basic proteins of eubacteria that bind as homodimers to double-stranded DNA and bend the duplex to promote architectures required for gene regulation. These architectural proteins share a common alpha/beta fold but exhibit different nucleic acid binding surfaces and distinct functional roles. With respect to DNA-binding specificity, for example, IHF is sequence specific, while HU is not.

The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution.

The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein-protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data revealed that a four-domain isoform of Eap is a monomer in solution.

Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22.

Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42-kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation.

Impact of in vitro assembly defects on in vivo function of the phage P22 portal.

The podovirus P22, which infects O-antigen strains of Salmonella, incorporates a dsDNA translocating channel (portal dodecamer) at a unique vertex of the icosahedral capsid. The portal subunit (gp1, 82.7 kDa) exhibits multiple S-Hcdots, three dots, centeredX hydrogen bonding states for cysteines 153, 173, 283 and 516 and these interactions are strongly perturbed by portal ring formation.