Guanosine Residues Research Articles

Synthetic Oligodeoxynucleotides Containing Suppressive TTAGGG Motifs Inhibit AIM2 Inflammasome Activation

AbstractSynthetic oligodeoxynucleotides (ODNs) comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. In this study, we demonstrate that such suppressive ODN abrogate activation of cytosolic nucleic acid–sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNF-α, and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1–dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and murine CMV. Inhibition was dependent on A151’s phosphorothioate backbone, whereas substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.

Development of dansyl-modified oligonucleotide probes responding to structural changes in a duplex

We have synthesized a nonnucleoside amidite block of dansyl fluorophore to prepare dansyl-modified oligonucleotides (ONTs). The fluorescence intensities of dansyl-ONT specifically increased by the presence of adjacent guanosine residues but, significantly reduced in a dansyl-flipping duplex. These changes were caused by solvatochromism effect due to the number of guanine which is hydrophobic functional group and the external environment of dansyl group. The fluorescence intensities could be plotted as a function of the ONTs concentrations and the increase in the fluorescence was observed to equimolar concentrations of target DNA. This duplex exhibited higher melting temperature relative to the corresponding duplexes containing other base pairs. Similar changes in fluorescence could be detected upon hybridization with complementary RNAs. Thus, the dansyl-modified ONTs provide sequence specific fluorescent probe of DNA and RNA.

Mitigation of gamma-radiation induced abasic sites in genomic DNA by dietary nicotinamide supplementation: Metabolic up-regulation of NAD+ biosynthesis

The search for non-toxic radio-protective drugs has yielded many potential agents but most of these compounds have certain amount of toxicity. The objective of the present study was to investigate dietary nicotinamide enrichment dependent adaptive response to potential cytotoxic effect of 60Co γ-radiation. To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on control diet (CD) and nicotinamide supplemented diet (NSD). After 6 weeks of CD and NSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activities of enzymes involved in NAD+ biosynthesis. Increased activities of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNAT) were observed up to 48h post-irradiation in NSD fed irradiated mice. Concomitant with increase in liver NAMPT and NMNAT activities, NAD+ levels were replenished in NSD fed and irradiated animals. However, NAMPT and NMNAT-mediated NAD+ biosynthesis and ATP levels were severely compromised in liver of CD fed irradiated mice. Another major finding of these studies revealed that under γ-radiation stress, dietary nicotinamide supplementation might induce higher and long-lasting poly(ADP)-ribose polymerase 1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG) activities in NSD fed animals compared to CD fed animals. To investigate liver DNA damage, number of apurinic/apyrimidinic sites (AP sites) and level of 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) residues were quantified. A significant increase in liver DNA AP sites and 8-oxo-dG levels with concomitant increase in caspase-3 was observed in CD fed and irradiated animals compared to NSD fed and irradiated mice. In conclusion present studies show that under γ-radiation stress conditions, dietary nicotinamide supplementation restores DNA excision repair activity via prolonged activation of PARP1 and PARG activities. Present results clearly indicated that hepatic NAD+ replenishment might be a novel and potent approach to reduce radiation injury.

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp.

We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments.

Exploring GpG bases next to anticodon in tRNA subsets

Transfer RNA (tRNA) structure, modifications and functions are evolutionary and established in bacteria, archaea and eukaryotes. Typically the tRNA modifications are indispensable for its stability and are required for decoding the mRNA into amino acids for protein synthesis. A conserved methylation has been located on the anticodon loop specifically at the 37th position and it is next to the anticodon bases. This modification is called as m1G37 and it is catalyzed by tRNA (m1G37) methyltransferase (TrmD). It is deciphered that G37 positions occur on few additional amino acids specific tRNA subsets in bacteria. Furthermore, Archaea and Eukaryotes have more number of tRNA subsets which contains G37 position next to the anticodon and the G residue are located at different positions such as G36, G37, G38, 39, and G40. In eight bacterial species, G (guanosine) residues are presents at the 37th and 38th position except three tRNA subsets having G residues at 36th and 39th positions. Therefore we propose that m1G37 modification may be feasible at 36th, 37th, 38th, 39th and 40th positions next to the anticodon of tRNAs. Collectively, methylation at G residues close to the anticodon may be possible at different positions and without restriction of anticodon 3rd base A, C, U or G.

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.

Intrinsic Acid-Base Properties of a Hexa-2′-deoxynucleoside Pentaphosphate, d(ApGpGpCpCpT): Neighboring Effects and Isomeric Equilibria

The intrinsic acid-base properties of the hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT) [=(A1∙G2∙G3∙C4∙C5∙T6)=(HNPP)⁵⁻] have been determined by ¹H NMR shift experiments. The pKa values of the individual sites of the adenosine (A), guanosine (G), cytidine (C), and thymidine (T) residues were measured in water under single-strand conditions (i.e., 10% D₂O, 47 °C, I=0.1 M, NaClO₄). These results quantify the release of H⁺ from the two (N7)H⁺ (G∙G), the two (N3)H⁺ (C∙C), and the (N1)H⁺ (A) units, as well as from the two (N1)H (G∙G) and the (N3)H (T) sites. Based on measurements with 2'-deoxynucleosides at 25 °C and 47 °C, they were transferred to pKa values valid in water at 25 °C and I=0.1 M. Intramolecular stacks between the nucleobases A1 and G2 as well as most likely also between G2 and G3 are formed. For HNPP three pKa clusters occur, that is those encompassing the pKa values of 2.44, 2.97, and 3.71 of G2(N7)H⁺, G3(N7)H⁺, and A1(N1)H⁺, respectively, with overlapping buffer regions. The tautomer populations were estimated, giving for the release of a single proton from five-fold protonated H₅(HNPP)(±) , the tautomers (G2)N7, (G3)N7, and (A1)N1 with formation degrees of about 74, 22, and 4%, respectively. Tautomer distributions reveal pathways for proton-donating as well as for proton-accepting reactions both being expected to be fast and to occur practically at no "cost". The eight pKa values for H₅(HNPP)(±) are compared with data for nucleosides and nucleotides, revealing that the nucleoside residues are in part affected very differently by their neighbors. In addition, the intrinsic acidity constants for the RNA derivative r(A1∙G2∙G3∙C4∙C5∙U6), where U=uridine, were calculated. Finally, the effect of metal ions on the pKa values of nucleobase sites is briefly discussed because in this way deprotonation reactions can easily be shifted to the physiological pH range.

Structural characterization of the major DNA‐DNA cross‐link formed by epichlorohydrin

Epichlorohydrin (ECH) is a bifunctional alkylating agent used extensively as an industrial intermediate, a laboratory reagent, and as an insecticide. ECH is classified as a probable human carcinogen since occupational exposure to ECH has been linked to elevated cancer risk for industrial workers. The ability of ECH to form DNA‐DNA cross‐links by consecutive alkylation of two nucleobases within a DNA molecule leads to the possibility of both cytotoxic and promutagenic properties. Previous work provided evidence for the formation of interstrand ECH‐DEB cross‐links involving deoxyguanosine residues within 5′‐GC and 5′‐GGC sites. However, the covalent structure of ECH‐induced DNA cross‐links has not been determined. In the present study, ECH interstrand cross‐links within synthetic DNA oligonucleotides containing a central 5′‐GGC site will be purified and characterized via electrospray ionization mass spectroscopy. This project was supported by grants from the National Center for Research Resources (5P20RR016463–12) and the National Institute of General Medical Sciences (8 P20 GM103423–12) from the National Institutes of Health.

A guanosine-centric mechanism for RNA chaperone function.

RNA chaperones are ubiquitous, heterogeneous proteins essential for RNA structural biogenesis and function. We investigated the mechanism of chaperone-mediated RNA folding by following the time-resolved dimerization of the packaging domain of a retroviral RNA at nucleotide resolution. In the absence of the nucleocapsid (NC) chaperone, dimerization proceeded through multiple, slow-folding intermediates. In the presence of NC, dimerization occurred rapidly through a single structural intermediate. The RNA binding domain of heterogeneous nuclear ribonucleoprotein A1 protein, a structurally unrelated chaperone, also accelerated dimerization. Both chaperones interacted primarily with guanosine residues. Replacing guanosine with more weakly pairing inosine yielded an RNA that folded rapidly without a facilitating chaperone. These results show that RNA chaperones can simplify RNA folding landscapes by weakening intramolecular interactions involving guanosine and explain many RNA chaperone activities.

In situ electrochemical evaluation of anticancer drug temozolomide and its metabolites–DNA interaction

Temozolomide (TMZ) is an antineoplastic alkylating agent with activity against serious and aggressive types of brain tumours. It has been postulated that TMZ exerts its antitumor activity via its spontaneous degradation at physiological pH. The in vitro evaluation of the interaction of TMZ and its final metabolites, 5-aminoimidazole-4-carboxamide (AIC) and methyldiazonium ion, with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues. The results obtained revealed the decrease of the dsDNA oxidation peaks with incubation time, showing that TMZ and AIC/methyldiazonium ion interact with dsDNA causing its condensation. Furthermore, the experiments of the in situ TMZ and AIC/methyldiazonium ion-dsDNA interaction using the multilayer dsDNA-electrochemical biosensor confirmed the condensation of dsDNA caused by these species and showed evidence for a specific interaction between the guanosine residues and TMZ metabolites, since free guanine oxidation peak was detected. The oxidative damage caused to DNA bases by TMZ metabolites was also detected electrochemically by monitoring the appearance of the 8-oxoguanine/2,8-dyhydroxyadenine oxidation peaks. Nondenaturing agarose gel electrophoresis of AIC/methyldiazonium ion-dsDNA samples confirmed the occurrence of dsDNA condensation and oxidative damage observed in the electrochemical results. The importance of the dsDNA-electrochemical biosensor in the in situ evaluation of TMZ-dsDNA interactions is clearly demonstrated.

Steady‐state and time‐resolved fluorescence of tetramethylrhodamine attached to DNA: correlation with DNA sequences

Time-resolved fluorescence as well as steady-state absorption and fluorescence were detected in order to study the interactions between tetramethylrhodamine (TAMRA) and DNA when TAMRA was covalently labeled on single- and double-stranded oligonucleotides. Fluorescence intensity quenching and lifetime changes were characterized and correlated with different DNA sequences. The results demonstrated that the photoinduced electron transfer interaction between guanosine residues and TAMRA introduced a short lifetime fluorescence component when guanosine residues were at the TAMRA-attached terminal of the DNA sequences. The discrepancy of two-state and three-state models in previous studies was due to the DNA sequence selection and sensitivity of techniques used to detect the short lifetime component. The results will help the design of fluorescence-based experiments related to a dye labeled probe.

A new fluorometric assay for the study of DNA-binding and 3′-processing activities of retroviral integrases and its use for screening of HIV-1 integrase inhibitors

Fluorometry using a substrate DNA labeled with a single fluorophore (6-carboxyfluorescein) at the 3'-end of the processed strand was shown to be a useful tool for monitoring DNA-binding and 3'-processing activities of HIV-1 and PFV integrases (INs). The DNA binding to either of the INs resulted in a fluorescence signal decrease, which is likely due to the fluorescence quenching by aromatic amino acids located near the 3'-end of the processed strand. The fluorescence deviations upon the 3'-processing strongly depended on the sequence of the fluorescein-labeled terminus of the substrate DNA. In the case of HIV-1 IN, a time-dependent fluorescence decrease was detected. Since it correlated with the rate of 3'-processing resulted in the labeled GT dinucleotide accumulation, it might be explained by the fluorescein quenching by a guanosine residue in the single-stranded dinucleotide. The 3'-processing catalyzed by PFV IN led to the fluorescence enhancement. We ascribed it to the migration of the cleaved AT dinucleotide conjugated with fluorescein away from the amino acids that could quench its fluorescence. The fluorescence-based assay was used for the search of new HIV-1 IN inhibitors. Some bisphosphonate derivatives, which are known to block the phosphorolytic activity of HIV-1 reverse transcriptase, were shown to inhibit HIV-1 IN at micromolar concentrations. This property makes bisphosphonates promising agents for the development of HIV-1 inhibitors affecting two viral enzymes.

RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H2O2 or by the Fe(II)/ascorbate/H2O2 system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H2O2 relative to the Fe(II)/ascorbate/H2O2 system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H2O2 concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H2O2, it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA–cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.

Detection and Typing of Viruses Using Broadly Sensitive Cocktail-PCR and Mass Spectrometric Cataloging: Demonstration with Dengue Virus

Virus detection and taxonomic identification of serotypes, strains, or genotypes provide important information relevant for diagnosis, and for the epidemiological characterization and tracking of new strains in an endemic region. In the specific case of dengue virus, rapid serotype identification can also be useful in the treatment of secondary infections that may cause the more severe dengue hemorrhagic fever and dengue shock syndrome. In this work, dengue virus was used as a model to test a new approach of combining broadly sensitive RT-PCR amplification of nearly any virus strain with subsequent serotype- and finer-level identification by mass spectrometry. PCR primers were appended with promoter sequences, such that the resulting PCR products could be transcribed into RNA. RNA fragments generated by guanosine-specific RNase T(1) digestion were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Viral serotypes were identified by comparing the pattern of observed fragment masses to a mass database. The database was created by computationally fragmenting 2517 dengue strains after each guanosine residue using the same primers. Computationally, all 2517 strains in the mass database were correctly identified at the serotype level from the predicted PCR product. The methodology was successfully demonstrated experimentally by identifying the serotypes of eight test strains using mosquito cell cultures infected with strains of all four serotypes and with full-length cDNA clones.

Guanine Bases in DNA G-Quadruplex Adopt Nonplanar Geometries Owing to Solvation and Base Pairing

The effect of base pairing and solvation on pyramidalization of the glycosidic nitrogen found in the residues of parallel G-quadruplex with NDB ID UDF062 is analyzed and explained with theoretical calculations. The extent of the pyramidalization depends on the local geometry of the 2'-deoxyguanosine residues, namely on their glycosidic torsion and sugar pucker, which are predetermined by the 3D-architecture of G-quadruplex. Pyramidal inversion of the glycosidic nitrogen found in 2'-deoxyguanosines of G-quadruplex is induced owing to site-specifically coordinated solvent. Different adiabatic structural constraints used for fixing the base-to-sugar orientation of 2'-deoxyguanosine in geometry optimizations result in different extents of pyramidalization and induce pyramidal inversion of the glycosidic nitrogen. These model geometry constraints helped us analyze the effect of real constraints represented by explicit molecular environment of selected residues of the G-quadruplex. The maximal extent of the glycosidic nitrogen pyramidalization found in the high-resolution crystal structure corresponds to the calculation to deformation energy of only 1 kcal mol(-1). The out-of-plane deformations of nucleobases thus provide a way for compensating the site-specific external environmental stress on the G-quadruplex.

The Butter Flavorant, Diacetyl, Forms a Covalent Adduct with 2-Deoxyguanosine, Uncoils DNA, and Leads to Cell Death

Diacetyl (DA), a natural butter flavorant, is a causative agent for the lung disease obliterative bronchiolitis. Mutagenic properties of 1,2-dicarbonyls have previously been empirically linked to their possible interaction with DNA nucleobases. This study for the first time identifies chemically the adduct of DA with 2-deoxyguanosine. Selective reactivity of DA with 5'-TTTGTTTTT-3' over 5'-TTTTTTTTT-3' indicated its propensity to modify specifically the guanosine residue. Treatment of plasmid DNA, pBR322, with DA induced changes in electrophoretic mobility that are typical of ternary structure disruption. Such DNA nucleobase interaction of DA translated into increased apoptosis in DA-treated SH-SY5Y cells in a dose-dependent manner (IC(50) = 0.114 ± 0.0421 mM). The traditional carbonyl scavengers metformin, 2-thiobarbituric acid, and d-penicillamine protected cells from DA toxicity in proportion to their rates of reaction with DA, with d-penicillamine causing a maximal increase in the IC(50) to 5.23 ± 0.0992 mM when co-incubated with DA.

Prevalence of the IVS1(+1)G->A and 35delG mutations in the GJB2 gene of Turkish patients with nonsyndromic hearing loss

GJB2 encodes connexin 26, a gap junction protein that is assumed to be a component of the potassium recycling pathway in the inner ear. Loss or malfunction of these gap junctions, as might be reflected by mutations in GJB2, may disrupt potassium movement from the hair cells through the supporting cell network to the endolymph, leading to hearing impairment. One mutation, the deletion of 1 guanosine residue from a stretch of 6 between nucleotide positions 30 and 35 (35delG) at codon 10, is the most common deafness-causing allelic variant of GJB2 in sporadic patients and autosomal recessive families. Mutations in the GJB2 gene represent the most common cause of autosomal recessive, sensorineural hearing loss. The 35delG mutation accounts for more than two-thirds of identified mutations. In this study, mutations, especially in the connexin 26 (GJB2), connexin 30 (GJB6), and 12srRNA genes, among 173 unrelated patients with prelingual nonsyndromic autosomal recessive deafness were screened and investigated by using the polymerase chain reaction-based restriction fragment length polymorphism (PCR-based RFLP), single-strand conformation polymorphism (SSCP), and sequence analysis methods. In patients with severe to profound hearing loss, 2 different mutations and 1 polymorphism (35delG and IVS1(+1)G->A mutations and V153I polymorphism) were found. The 35delG mutation was detected as the most common pathogenic allele among the Turkish patients and accounted for 50% of all mutant GJB2 alleles. The 35delG and IVS1+1G->A mutations in the Cx26 gene were detected with total allele frequencies of 16.47% and 4.33%, respectively, and the V153 polymorphism was found in a heterozygous state at an allele frequency of 3.47%. However, the 342-kb deletion in the Cx30 gene and mitochondrial (mt)1555A->G in the 12srRNA gene mutations could not be detected among the studied patients.

Kinetic Analysis of 3′–5′ Nucleotide Addition Catalyzed by Eukaryotic tRNAHis Guanylyltransferase

The tRNA(His) guanylyltransferase (Thg1) catalyzes the incorporation of a single guanosine residue at the -1 position (G(-1)) of tRNA(His), using an unusual 3'-5' nucleotidyl transfer reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs), which catalyze tRNA repair and editing, are the only known enzymes that add nucleotides in the 3'-5' direction. Thg1 enzymes share no identifiable sequence similarity with any other known enzyme family that could be used to suggest the mechanism for catalysis of the unusual 3'-5' addition reaction. The high-resolution crystal structure of human Thg1 revealed remarkable structural similarity between canonical DNA/RNA polymerases and eukaryotic Thg1; nevertheless, questions regarding the molecular mechanism of 3'-5' nucleotide addition remain. Here, we use transient kinetics to measure the pseudo-first-order forward rate constants for the three steps of the G(-1) addition reaction catalyzed by yeast Thg1: adenylylation of the 5' end of the tRNA (k(aden)), nucleotidyl transfer (k(ntrans)), and removal of pyrophosphate from the G(-1)-containing tRNA (k(ppase)). This kinetic framework, in conjunction with the crystal structure of nucleotide-bound Thg1, suggests a likely role for two-metal ion chemistry in all three chemical steps of the G(-1) addition reaction. Furthermore, we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, and R133) that participate primarily in the nucleotidyl transfer step of the reaction. These data provide a foundation for understanding the mechanism of 3'-5' nucleotide addition in tRNA(His) maturation.

Evidence for Macrocyclic Lactone Anthelmintic Resistance in Dirofilaria immitis

Reports of loss-of-efficacy (LOE) events in dogs infected with Dirofilaria immitis despite adherence to accepted prophylaxis regimens with a macrocyclic lactone anthelmintic are attracting considerable attention. It is crucially important to distinguish among several possible causes for these LOE reports, one of which is the evolution of resistance to these drugs in heartworms. We review here recent evidence at the molecular level that supports the hypothesis that parasites derived from LOE cases have experienced a strong selection event and that these populations are characterized by very high frequencies of single-nucleotide polymorphisms in a D. immitis gene encoding a P-glycoprotein transporter, comprised of homozygous guanosine residues at 2 locations ("GG-GG" genotype). Furthermore, an infected dog adopted to Canada from the southern United States harbored a microfilarial population that was insensitive to very high doses of macrocyclic lactones and was characterized by a high frequency of the GG-GG genotype associated with LOE cases. We propose that this case be defined as a drug-resistant heartworm infection and suggest that a simple assay for the existence of resistant parasites is a 7-day microfilariae suppression test, which can be performed in a veterinary clinic as part of an effort to document the geographic distribution of this phenotype.

Applications of RNA structure analysis to retroviral packaging and anti-retroviral therapeutic discovery

Our laboratory is blending new RNA chemistries, largely directed at the ribose 2'-hydroxyl group, with quantitative interpretation to create new kinds of chemical microscopes for accurate RNA structure analysis. This seminar will focus on two examples from retroviral biology. All retroviral RNAs contain a packaging element that directs the genome into nascent virus particles through a mechanism that has been unclear. Using SHAPE, we probed the structure of murine leukemia virus RNA inside virus particles and identified the binding site for the nucleocapsid domain of Gag. The RNA structural context is critical: High-affinity binding requires two specific UCUG-UR-UCUG motifs flanked by base-paired elements. Mutation of four guanosine residues in these two motifs - only 4 nucleotides per genomic RNA - reduced packaging to non-specific levels. These motifs only form in genomic RNA dimers; thus analysis of authentic full-length RNA was critical. This work illustrates how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements. In the second example, we discovered strong correlations between shRNA-mediated inhibition of HIV-1 replication in a cell-based assay and both the energetic cost of breaking secondary structures and the overall annealing energy between guide strand and folded target RNA. These correlations were only apparent when the energetic cost of disrupting pre-existing structure in the HIV-1 RNA was calculated using a secondary structure model derived from SHAPE probing information. We verified these correlations by design and evaluation of additional shRNAs and could achieve consistent inhibition of HIV-1 production by 90%. This approach emphasizes the profound, but ultimately predictable, role of RNA structure in tuning RNAi function and is likely to prove broadly useful in understanding large-scale biological recognition involving RNA.