LidNA, a miRNA inhibitor constructed with unmodified DNA, requires an xxxA insertion sequence in miRNA binding site for its potent inhibitory activity.

The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Structural insight into RNA synthesis by influenza D polymerase.

The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.

Specific interaction of zinc finger protein Com with RNA and the crystal structure of a self-complementary RNA duplex recognized by Com.

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com-RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.

Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3'-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.

Development of a SYBR-Green â quantitative PCR assay for the detection and genotyping of different hantaviruses.

Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR­Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR­Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

Influence of pendant chiral C(γ)-(alkylideneamino/guanidino) cationic side-chains of PNA backbone on hybridization with complementary DNA/RNA and cell permeability.

Intrinsically cationic and chiral C(γ)-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyleneguanidino (egd)-PNA with two carbon spacers from the backbone. The relative stabilization (ΔTm) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (aeg)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability.

Analysis of complementarity requirements for plant microRNA targeting using a Nicotiana benthamiana quantitative transient assay.

MicroRNAs (miRNAs) guide RNA-induced silencing complexes to target RNAs based on miRNA-target complementarity. Using a dual-luciferase based sensor system in Nicotiana benthamiana, we quantitatively assessed the relationship between miRNA-target complementarity and silencing efficacy measured at both the RNA and protein levels, using several conserved miRNAs and their known target sites from Arabidopsis thaliana. We found that naturally occurring sites have variable efficacies attributable to their complementarity patterns. We also observed that sites with a few mismatches to the miRNA 3' regions, which are common in plants, are often equally effective and sometimes more effective than perfectly matched sites. By contrast, mismatches to the miRNA 5' regions strongly reduce or eliminate repression efficacy but are nonetheless present in several natural sites, suggesting that in some cases, suboptimal miRNA efficacies are either tolerated or perhaps selected for. Central mismatches fully abolished repression efficacy in our system, but such sites then became effective miRNA target mimics. Complementarity patterns that are functional in animals (seed sites, 3'-supplementary sites, and centered sites) did not reliably confer repression, regardless of context (3'-untranslated region or open reading frame) or measurement type (RNA or protein levels). Overall, these data provide a robust and empirical foundation for understanding, predicting, and designing functional miRNA target sites in plants.

A 6'-fluoro-substituent in bicyclo-DNA increases affinity to complementary RNA presumably by CF­HC pseudohydrogen bonds.

The synthesis of a novel bicyclic thymidine analogue carrying a ß-fluoro substituent at C6' (6'F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1­2 °C compared to thymidine and +1­3 °C compared to a standard bc-T residue. Structural investigations of the 6'F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF­HC hydrogen bond. In contrast, structural investigations in solution by 1H and 13C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF­HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6'F-bcT nucleoside.

Synthesis and duplex-forming ability of oligonucleotides containing 4'-carboxythymidine analogs.

Oligonucleotides containing 4'-carboxy-, 4'-methoxycarbonyl-, 4'-carbamoyl-, and 4'-methylcarbamoyl-thymidines, and their 2'-methoxy, 2'-amino or 2'-acetamido analogs were prepared. Their duplex-forming ability with DNA and RNA complements was evaluated by UV melting experiments. Interestingly, 4'-carboxythymidine existing in the S-type sugar conformation was found to lead to an increase in the stability of the duplex formed with RNA complements compared to natural thymidine.

Overlapping genes coded in the 3'-to-5'-direction in mitochondrial genes and 3'-to-5' polymerization of non-complementary RNA by an 'invertase'.

Suppressor tRNAs induce expression of additional (off-frame) genes coded by stopless genetic codes without lengthening genomes, decreasing DNA replication costs. RNA 3'-to-5' polymerization by tRNAHis guanylyltransferase suggests further cryptic code: hypothetical 'invertases' polymerizing in the 3'-to-5' direction, advancing in the 5'-to-3' direction would produce non-complementary RNA templated by regular genes, with different coding properties. Assuming 'invertase' activity, BLAST analyses detect GenBank-stored RNA ESTs and proteins (some potentially coding for the hypothesized invertase) for human mitochondrial genes. These peptides' predicted secondary structures resemble their GenBank homologues'. 3'-to-5' EST lengths increase with their self-hybridization potential: Single-stranded RNA degradation perhaps limits 3'-to-5' elongation. Independent methods confirm predicted 3'-to-5' overlapping genes: (a) Presumed 3'-to-5' overlapping genes avoid codons belonging to circular codes; (b) Spontaneous replicational deamination (mutation) gradients occur at 3rd codon positions, unless these are involved in overlap coding, because mutations are counter selected in overlapping genes. Tests a and b converge on predicted 3'-to-5' gene expression levels. Highly expressed ones include also fewer stops, and mitochondrial genomes (in Primates and Drosophila) adapt to avoid dependence of 3'-to-5' coding upon antitermination tRNA activity. Secondary structure, circular code, gradient and coevolution analyses yield each clear positive results independently confirming each other. These positive results (including physical evidence for 3'-to-5' ESTs) indicate that 3'-to-5' coding and invertase activity is an a priori improbable working hypothesis that cannot be dismissed. Note that RNAs produced by invertases potentially produce triple-stranded DNA:RNA helices by antiparallel Hoogsteen pairings at physiological pH, as previously observed for mitochondrial genomes.

Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Bacterial type I toxin-antitoxin systems.

Toxin-antitoxin (TA) loci encode two-component systems that consist of a stable "toxin" whose ectopic overexpression either kills cells or confers growth stasis, and an unstable "antitoxin". TA systems have been initially discovered on plasmids, where they confer stability of maintenance through post-segregational killing (PSK). Plasmid loss results in rapid decrease of antitoxin levels, which allows the stable toxin to kill the plasmid-free cell. Later, TA systems were also found on bacterial and archaeal chromosomes, sometimes in staggering numbers. (1), (2) They are classified into five types depending on the nature and action of the antitoxin. In type I systems, the antitoxin is a small antisense RNA that base-pairs with the toxin encoding mRNA. By contrast, in type II systems, the antitoxin is a protein that interacts post-translationally with the toxin protein. The antitoxin in type III systems is a pseudoknot containing RNA that directly binds the toxin protein. (3), (4) In the recently proposed type IV systems, the protein antitoxin interferes with binding of the toxin to its target rather than inhibiting the toxin directly by binding, (5) whereas the antitoxin protein in type V systems cleaves the toxin-encoding mRNA. (6).

RDR6-mediated synthesis of complementary RNA is terminated by miRNA stably bound to template RNA.

RNA-dependent RNA polymerase RDR6 is involved in the biogenesis of plant trans-acting siRNAs. This process is initiated by miRNA-directed and Argonaute (AGO) protein-mediated cleavage of TAS gene transcripts. One of the cleavage products is converted by RDR6 to double-stranded (ds)RNA, the substrate for Dicer-like 4 (DCL4). Interestingly, TAS3 transcript contains two target sites for miR390::AGO7 complexes, 5'-non-cleavable and 3'-cleavable. Here we show that RDR6-mediated synthesis of complementary RNA starts at a third nucleotide of the cleaved TAS3 transcript and is terminated by the miR390::AGO7 complex stably bound to the non-cleavable site. Thus, the resulting dsRNA has a short, 2-nt, 3'-overhang and a long, 220-nt, 5'-overhang of the template strand. The short, but not long, overhang is optimal for DCL4 binding, which ensures dsRNA processing from one end into phased siRNA duplexes with 2-nt 3'-overhangs.

May cyclic nucleotides be a source for abiotic RNA synthesis?

Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2',3' and 3',5' cyclic forms. We have reported that 3',5' cyclic GMP and 3',5' cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.

Structural characterization of the viral and cRNA panhandle motifs from the infectious salmon anemia virus.

Infectious salmon anemia virus (ISAV) has emerged as a virus of great concern to the aquaculture industry since it can lead to highly contagious and lethal infections in farm-raised salmon populations. While little is known about the transcription/replication cycle of ISAV, initial evidence suggests that it follows molecular mechanisms similar to those found in other orthomyxoviruses, which include the highly pathogenic influenza A (inf A) virus. During the life cycle of orthomyxoviruses, a panhandle structure is formed by the pairing of the conserved 5' and 3' ends of each genomic RNA. This structural motif serves both as a promoter of the viral RNA (vRNA)-dependent RNA polymerase and as a regulatory element in the transcription/replication cycle. As a first step toward characterizing the structure of the ISAV panhandle, here we have determined the secondary structures of the vRNA and the cRNA panhandles on the basis of solution nuclear magnetic resonance (NMR) and thermal melting data. The vRNA panhandle is distinguished by three noncanonical U · G pairs and one U · U pair in two stem helices that are linked by a highly stacked internal loop. For the cRNA panhandle, a contiguous stem helix with a protonated C · A pair near the terminus and tandem downstream U · U pairs was found. The observed noncanonical base pairs and base stacking features of the ISAV RNA panhandle motif provide the first insight into structural features that may govern recognition by the viral RNA polymerase.

Duplex and triplex formation of mixed pyrimidine oligonucleotides with stacking of phenyl-triazole moieties in the major groove.

5-(1-Phenyl-1,2,3-triazol-4-yl)-2'-deoxycytidine was synthesized from a modified CuAAC protocol and incorporated into mixed pyrimidine oligonucleotide sequences together with the corresponding 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine. With consecutive incorporations of the two modified nucleosides, improved duplex formation with a complementary RNA and improved triplex formation with a complementary DNA duplex were observed. The improvement is due to π-π stacking of the phenyl-triazole moieties in the major groove. The strongest stacking and most pronounced positive influence on thermal stability was found in between the uridine analogues or with the cytidine analogue placed in the 3' direction to the uridine analogue. Modeling indicated a different orientation of the phenyl-triazole moieties in the major groove to account for the difference between the two nucleotides. The modified oligonucleotides were all found to be significantly stabilized toward nucleolytic degration.

Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

Locked nucleic acids: promising nucleic acid analogs for therapeutic applications.

Locked Nucleic Acid (LNA) is a unique nucleic-acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid-based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.