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The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol(DV) of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol(DV) contains a specific priming site for adenosine 5'-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn(2+) is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol(DV) ensures the conservation of the 5'-adenosine by strongly discriminating against viral templates containing an erroneous 3'-end nucleotide in the presence of Mg(2+). In the presence of Mn(2+), NS5Pol(DV) is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.

The United States is currently experiencing an opioid crisis, with more than 47,000 deaths in 2017 due to opioid overdoses. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS), which require expensive instrumentation and extensive sample preparation. Our aim was to develop a portable point-of-care device that can be used for the instant detection of opioids in body fluids. Here, we reported the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in the blood using a homogeneous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified using a high throughput one-bead one-compound (OBOC) combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), that are coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescent activation under a confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify the peptide-beads that fluoresce in the presence of an anti-morphine antibody, but lost fluorescence when the free morphine was present. After the positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were then synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for the detection of morphine in plasma. We demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for the detection of illicit drugs and cancer biomarkers in body fluids.

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.