Stability of RNA tertiary structure elements

In addition to acting as a transmitter of genetic information, RNAs often exert biological functions and are the terminal gene products. These RNAs have to fold into defined 3D structures to achieve their function. While the formation of RNA secodary structure elements is well understood and their structure prediction is often correct, the formation of tertiary structure elements is still not well understood. Computer-based prediction of RNA tertiary structures is impossible due to the lack of knowledge about the thermodynamic parameters of RNA tertiary structure interactions. We propose to study the stability of RNA tertiary structure elements both in inter- as well as in intra-molecular interactions. The intermolecular RNA-RNA interactions will be analysed using the human HIV dimerisation initiation site (DIS). The HIV genome is packed into its capsid as a dimer, whereby the dimerisation occurs via base pairing over a short complementary sequence. The dimerisation process is initiated via formation of a hairpin loop-loop structure called "the kissing complex". We will systematically determine the thermodynamic parameters for HIV-type DIS loop-loop complexes as well as kinetically access the formation of the extended complex initiated by the kissing loop. To be able to do this, we developed a ribozyme-coupled system, whereby the extended complex folds into a ribozyme leading to cleavage of the targeted RNA. We want to develop antisense ribozymes, which initiate annealing via kissing complexes. The intramolecular tertiary structure interactions will be studied using the self-splicing td group I intron. Folding of this RNA will be studied both in vivo and in vitro, thereby assessing the contribution of single interactions to the folding pathway as well as to the overall structural stability of the molecule. We will introduce mutations into the intron RNA, which disrupt tertiary structure interactions and determine their impact on the melting temperature of the structure and on the folding pathway. Understanding the mechanism of RNA-RNA interactions (called RNA annealing) should enable the design of therapeutic RNAs targetted against disease causing RNA molecules. The novelty in the approach presented here is the strategy of initiating annealing via a kissing loop. He hope, that this strategy will improve the efficiency of antisense ribozymes.

Most living organisms store their genetic information in DNA (Deoxyribonucleic acid), but a few viruses store this information in form of RNA (ribonucleic acid), like the retrovirus HIV. These RNA genomes encode complex information not only to produce the essential proteins, but also the primary, secondary and tertiary structures of the viral RNA encode many signals, which are essential for viral replication. Understanding the function and structure of these signals might reveal targets for potential therapy against the viruses. We are interested in understanding the three-dimensional structure of RNA molecules and we used the dimerisation initiation signal of the HIV-1 (HIV-1 DIS) as a model system. This DIS domain is essential for viral replication and for packaging the virus as a "diploid" genome into viral particles. We determined the thermodynamic parameters for HIV-1 DIS-like loop-loop interactions both in vitro and within living cells. These parameters should help understand the very basic principles that govern RNA folding and the stability of RNA tertiary structures.