8 research outputs found
RNA binding properties of conserved protein subunits of human RNase P
Human nuclear RNase P is required for transcription and processing of tRNA. This catalytic RNP has an H1 RNA moiety associated with ten distinct protein subunits. Five (Rpp20, Rpp21, Rpp25, Rpp29 and Pop5) out of eight of these protein subunits, prepared in refolded recombinant forms, bind to H1 RNA in vitro. Rpp20 and Rpp25 bind jointly to H1 RNA, even though each protein can interact independently with this transcript. Nuclease footprinting analysis reveals that Rpp20 and Rpp25 recognize overlapping regions in the P2 and P3 domains of H1 RNA. Rpp21 and Rpp29, which are sufficient for reconstitution of the endonucleolytic activity, bind to separate regions in the catalytic domain of H1 RNA. Common themes and discrepancies in the RNA-protein interactions between human nuclear RNase P and its related yeast and archaeal counterparts provide a rationale for the assembly of the fully active form of this enzyme
Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1
Background: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reversetranscription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. Methodology/Significant Findings: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. Conclusion: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides
Morpholino oligonucleotides do not participate perfectly in standard Watson-Crick complexes with RNA
RNase P from E. coli will cleave a RNA at a site designated in a complex with an external guide sequence (EGS). The location of the site is determined by the Watson-Crick complementary sequence that can be formed between the RNA and the EGS. Morpholino oligonucleotides (PMOs) that have the same base sequences as any particular EGS will not direct cleavage by RNase P of the target RNA at the expected site in three mRNAs. Instead, cleavage occurs at a secondary site that does not correspond exactly to the expected Watson-Crick sequence in the PMO. This cleavage in the mRNA for a drug resistance gene, CAT mRNA, is at least second order in the concentration of the PMOs, but the mechanism is not understood yet and might be more complicated than a simple second-order reaction. EGSs and PMOs inhibit the reactions of each other effectively in a competitive fashion. A basic peptide attached to the PMO (PPMO) is more effective because of its binding properties to the mRNA as a substrate. However, a PMO is just as efficient as a PPMO on a mRNA that is mutated so that the canonical W-C site has been altered. The altered mRNA is not recognizable by effective extensive W-C pairing to an EGS or PMO. The complex of a PMO on a mutated mRNA as a substrate shows that the dimensions of the modified oligonucleotide cannot be the same as a naked piece of single-stranded RNA
Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum
Azepines and Piperidines with Dual Norepinephrine Dopamine Uptake Inhibition and Antidepressant Activity
Herein, we describe the discovery
of inhibitors of norepinephrine
(NET) and dopamine (DAT) transporters with reduced activity relative
to serotonin transporters (SERT). Two compounds, <b>8b</b> and <b>21a</b>, along with nomifensine were tested in a rodent receptor
occupancy study and demonstrated dose-dependent displacement of radiolabeled
NET and DAT ligands. These compounds were efficacious in a rat forced
swim assay (model of depression) and also had activity in rat spontaneous
locomotion assay