Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.

Peptide aptamers have primarily been used as research tools to manipulate protein function and study regulatory networks. However, they also find multiple applications in therapeutic research, from target identification and validation to drug discovery. Because of their unbiased combinatorial nature, peptide aptamers interrogate the biological significance of numerous molecular surfaces on target proteins. Their use enables the identification and validation of some of these surfaces as interesting therapeutic targets to pursue. Peptide aptamers can subsequently be used to guide the discovery of small molecule drugs specific for these molecular surfaces.Here, we present a high-throughput screening assay that identifies small molecules that displace interactions between proteins and their cognate peptide aptamers. AptaScreen is a duplex yeast two-hybrid assay featuring two luciferase reporter genes. It can be performed in 96- or 384-well plates and can be fully automated.