Richard H. Griffey

A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome.

The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell. Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field. Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator. Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons. Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site. One hundred forty-four are potentially novel non-translatable sRNA genes. Using total RNA isolated from E. coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization. Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.

Rapid Identification of Emerging Pathogens: Coronavirus

New surveillance approach can analyze >900 polymerase chain reactions per day.

Characterization of Oligonucleotide MetabolismIn Vivovia Liquid Chromatography/Electrospray Tandem Mass Spectrometry with a Quadrupole Ion Trap Mass Spectrometer

The pattern of nuclease degradation observed for an antisense phosphorothioate oligonucleotide in pig kidney was determined using liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) and LC/ESI-MS/MS with a quadrupole ion trap mass spectrometer. Metabolites were separated by length using reversed-phase high-performance liquid chromatography with aqueous hexafluoropropan-2-ol-triethylamine and a methanol gradient. The individual masses of metabolites in each LC peak were determined via deconvolution and converted into potential nucleotide compositions. The nucleotide composition was used to locate metabolites within the known oligomer sequence. The identity of metabolites was confirmed using on-line LC/MS/MS to generate fragment ions suitable for sequence verification. A limited number of shorter oligonucleotide fragments were observed, suggesting that metabolism in vivo may be sequence dependent.

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR.

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds l-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD50 of ca. 1 μM.

RNA as a small-molecule drug target: doubling the value of genomics.

Recent advances in the determination of RNA structure and function have led to new opportunities that will have a significant impact on the pharmaceutical industry. RNA, which, among other functions, serves as a messenger between DNA and proteins, was thought to be an entirely flexible molecule without significant structural complexity. However, recent studies have revealed a surprising intricacy in RNA structure. This observation unlocks opportunities for the pharmaceutical industry to target RNA with small molecules. Because both proteins and their specific mRNAs are potential drug-binding sites, the number of targets revealed from genome sequencing efforts is effectively doubled. Perhaps more importantly, drugs that bind to RNA might produce effects that cannot be achieved by drugs that bind to proteins.

Detection of base pair mismatches in duplex DNA and RNA oligonucleotides using electrospray mass spectrometry

The identify and location of base pair mismatches in non- covalent DNA:RNA duplexes are established using MS and MS-MS on a quadruple ion trap with electrospray ionization (ESI). MS-MS experiments on a 14mer duplex (D) with a single C:A base pair mismatch using lower activation energy results in selective cleavage of the mismatched A nucleobase, even in the presence of the wild-type duplex. The location of the mismatch base pair can be discerned via presence of the wild-type duplex. The location of the mismatch base pair can be discerned via selection of the (D-5H)5- ion and fragmentation of the backbone at that location in a n additional MS-MS experiment. Selective fragmentation is observed for C in a C-C mismatched base pair, which is very difficult to detect using chemical cleavage or E. coli mismatch binding protein. In an RNA:DNA duplex with a single base pair mismatch, the DNA base is removed without fragmentation of the RNA strand, greatly simplifying the interpretation of the resulting MS spectrum. A method is presented for detecting two DNA strands, for example a point mutation which generates an oncogenic phenotype, and the wild-type message. The results suggest that ESI-MS-MS may provide a rapid and selective method to identify and locate genetic mutations without the need for chemical degradation or protein binding followed by gel electrophoresis.

Molecular dynamics and NMR studies of single-stranded PNAs

Abstract Proton NMR spectroscopy and molecular dynamics simulations are employed to investigate the conformations of PNA monomers, a dimer and an octamer. The monomers exist as a 70:30 mixture of two amide rotamers interconverting slowly on the NMR time scale at 20 °C. In the major form, the side chain carbonyl group points toward the glycine, which places the methylene protons in proximity to the 2-aminoethyl protons. The minor form places its side chain carbonyl group away from the glycine, and the methylene protons are close in space to the glycine α protons. The PNA CT-dimer has multiple rotamers at 20 °C. In contrast, a NOESY spectrum taken from an octamer indicates only a single conformer in solution at 40 °C.

Diabetic Neuropathy: Structural Analysis of Nerve Hydration by Magnetic Resonance Spectroscopy

The water content of the sural nerve of diabetic patients was quantitatively defined by magnetic resonance proton imaging as a putative reflection of activity of the aldose-reductase pathway. Thirty-nine patients were evaluated, comparing group A, symptomatic diabetic men with sensory neuropathy; group B, similarly symptomatic diabetic men treated with aldose-reductase inhibition; group C, neurologically asymptomatic diabetic men; and group D, control nondiabetic men. Marked increase in hydration of the sural nerve was seen in more than half of the symptomatic diabetic patients. Two of 11 neurologically asymptomatic diabetics had increased nerve hydration, suggesting a presymptomatic alteration of the nerve. Symptomatic diabetics treated with aldose-reductase inhibitors had normal nerve water levels. Increased level of peripheral nerve water represents a new finding in diabetes mellitus. It seems to be related to aldose-reductase activity, involved in the development of neuropathy, and similar to events that occur in other target tissue in human diabetes.

Inhibitor-induced structural change in the HCV IRES domain IIa RNA

Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5′ untranslated region (5′ UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90° helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol. Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class.

Remarkable enhancement of binding affinity of Heterocycle-modified DNA to DNA and RNA. Synthesis, characterization and biophysical evaluation of N2-imidazolylpropylguanine and N2-imidazolylpropyl-2-aminoadenine modified oligonucleotides

Abstract Oligonucleotides containing novel N2-Imidazolylpropylguanine and N2-Imidazolylpropyl-2-aminoadenine moieties were synthesized and studied for their hybridization and biophysical properties. Interestingly, these heterocycle modified oligonucleotides showed a remarkable enhancement of heteroduplex binding affinity when hybridized to complementary DNA.