RRM
2009 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
The HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein, catalyzing the rearrangement of nucleic acids into thermodynamically more stable structures. It plays role in essentially every step of the retroviral replication cycle. NC is involved in dimerization of the RNA genome, RNA packaging, tRNA primer annealing and various strand-exchange reactions. We previously developed a novel quadruplex formation assay to study the role of NC in facilitating invasion of short DNA duplexes. Other researchers have developed various DNA enzymes (DNAzymes) to cleave RNA in a sequence-specific manner, which could be used in degradation and inactivation of mRNAs of interest. However, DNAzyme activity is limited by stable secondary structures of mRNAs, which should be invaded prior to cleavage. In the present work we investigate role of NC in invasion of nucleic acid secondary structures by complementary sequences. To study NC’s ability to facilitate invasion of DNA quadruplexes, a thrombin binding aptamer, GGTTGGTGTGGTTGG, was investigated in the presence of K+ ions. The reaction was studied as a function of temperature, which revealed that in the absence of NC quadruplex invasion follows a bimolecular pathway with initial partial annealing of the complementary strand. In the presence of NC, the reaction appears to be monomolecular wherein complete unfolding of quadruplex occurs prior to strand invasion. We also studied of invasion of RNA hairpins by DNAzymes and subsequent RNA cleavage. This study showed that in the absence of NC, the DNAzyme is able to invade RNA hairpins with 6-7 bp stems, while in the presence of NC unfolding and cleavage of significantly more stable 10 bp hairpins could be achieved. Taken together, these studies demonstrated that NC has a potential to be used in antisense therapy to facilitate invasion and cleavage of structured mRNA by DNAzymes.
Keywords: HIV-1 nucleocapsid protein, antisense therapy, DNAzymes
