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Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0–78.4) for IgM, 88.2% (95% CI: 64.4–98.0) for IgG, and 100% (95% CI: 78.4–100) for IgM/IgG, at 99.8% (95% CI: 99.2–100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0–3.0) and 0.4% (95% CI: 0–2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas.

To study the cleavage mechanism ofbacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp-or RNase P is an essential structure-specific endoribonuclease that generates the mature 5' ends of tRNAs. In vitro, RNA subunits of bacterial RNase P enzymes were shown to be catalytically active in the absence of the protein subunit (1). Processing of precursor tRNAs (ptRNAs) by RNase P is an essentially irreversible reaction yielding 3'-OH and 5'-phosphate termini. A solvent hydroxide is thought to act as the nucleophile in an SN2 in-line displacement mechanism (2, 3).To gain a deeper insight into the cleavage mechanism by Escherichia coli RNase P RNA, we have synthesized ptRNA substrates carrying a single Rp-or Sp-phosphorothioate modification at the RNase P cleavage site. The diastereomeric substrates were analyzed for gel-resolvable binding to RNase P RNA and were studied in single turnover experiments in the presence of different divalent metal ions, such as Mg2+, Mn2+, and Cd2+. The following results were obtained: (i) the Spdiastereomer moderately affected ptRNA ground state binding, while the Rp-diastereomer had no effect; (ii) cleavage by RNase P RNA involves direct metal ion coordination to the pro-Rp oxygen; (iii) there is no specific role for Mg2+ at the pro-Sp oxygen; and (iv) cleavage of the Rp-diastereomeric substrate has a lower cooperative dependence (nH = 1.8) upon[Cd2+] than cleavage of the unmodified substrate upon [Mg2+] (nH = 3.3). Implications for the unique RNase P RNA cleavage mechanism are discussed in the context of previously proposed mechanistic models (2-4). MATERIALS AND METHODS Separation and Identification of Diastereomeric 13-Mers.Oligoribonucleotides of identical sequence (13-mers), either unmodified or carrying a single phosphorothiote modification (5'-CCCUUUCsGCGGGA), were prepared by solid-phase synthesis essentially as described (5, 6). Oligoribonucleotides were purified by reversed-phase HPLC on an ODS C18 Beckman Ultrasphere column at 45°C (5). The material of the peak containing the two diastereomeric 13-mers was vacuumdried, and the Rp-and Sp-diastereomers were separated by a second reversed-phase run at 4°C. The assignment of configuration of the two diastereomeric 13-mers was accomplished by digestion with the stereoselective enzymes snake venom phosphodiesterase I or nuclease P1 essentially as described (6). Phosphorothioate-specific iodine hydrolysis was performed essentially as described recently (7).Assembly of the ptRNAGIY. Chemically synthesized RNA oligomers were phosphorylated at their 5' termini by T4 polynucleotide kinase; T7 transcripts were synthesized in the presence of excess 5'-GMP to obtain 5'-monophosphates (7). Modified or unmodified 13-mers, a second 11-nt RNA oligonucleotide (5'-GUAGCUCAGUC-3', obtained either by chemical RNA synthesis or T7 transcription), and the 3'-portion of the tRNA (obtained by T7 transcription, starting at G+ 18; see Fig. 1) were annealed to a bridging DNA oligonucleotide (complementary to...

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Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA. In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect. A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects. Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant. Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C. These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rpphosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E.coli RNase P RNAs were separated into tRNA binding and nonbinding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, AM, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E.coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

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Background:The noninvasive detection of RNA tumor markers in body fluids represents an attractive diagnostic option, but diagnostic performance of tissue-derived markers is often poorer when measured in body fluids rather than in tumors. We aimed to develop a procedure for measurement of tumor RNA in urine that would minimize donor-dependent influences on the results. Methods: RNA isolated from urinary cell pellet, celldepleted fraction, and whole urine was quantified by reverse transcription quantitative-PCR. The donordependent influence of urine background on individual steps of the standardized procedure was analyzed using an external RNA standard. Using a test set of samples from 61 patients with bladder cancer and 37 healthy donors, we compared 4 putative RNA tumor markers identified in whole urine with 5 established, tissuederived RNA tumor markers for the detection of bladder cancer. Results: Of the markers analyzed by this system, the RNA ratio of v-ets erythroblastosis virus E26 oncogene homolog 2 (avian; ETS2) to urokinase plasminogen activator (uPA) enabled the most specific (100%) and sensitive (75.4%) detection of bladder cancer from whole urine, with an area under the curve of 0.929 (95% CI 0.882-0.976).