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Título: RNA folding : synthesis, structure and biological properties of hairpins based on 2',5'-linked RNA loops
Autores: Hannoush, Rami Nabil
Fecha: 2002
Publicador: McGill University - MCGILL
Fuente:
Tipo: Electronic Thesis or Dissertation
Tema: Biology, Molecular.
Chemistry, Biochemistry.
Descripción: Ribonucleic acids (RNA) are polymers of ribonucleotides linked together by 3',5'-phosphodiester linkages and play a prominent role in protein biosynthesis. Much less common are ribonucleic acids based on 2',5'-phosphodiester linkages (2',5'-RNA). In this study, the synthesis, physical and biological properties of several 2' ,5'-linked oligoribonucleotides are described.
One aspect of this thesis deals with the synthesis and structural studies of 2',5'-dodecaribonucleotides. Their base sequence was designed to promote intramolecular folding by base pairing leading to the formation of "hairpin loops". The hairpins consist of a tetranucleotide loop and a 4 base-pair duplex (stem). The thermodynamic stability and conformation of these hairpins were assessed by UV, CD and NMR spectroscopy. Melting experiments (UV) revealed that with a few exceptions, hairpins comprised of 2',5'-(UUCG) loops exhibit greater thermodynamic stability (e.g. Tm) than the corresponding hairpins with 3' ,5'-linked loops. The 'extra' stability imparted by the 2',5'-UUCG) loop was found to be virtually independent of the sugar composition of the stem. For example, the 2',5'-tetraloop stabilizes hairpins whether their duplex stem is based on double-stranded DNA or RNA. In contrast, the 3',5-tetraloop stabilizes hairpins only when their stem duplex is double-stranded RNA. NMR studies revealed that the 2',5'-tetraloop (UUCG) is self-stabilized by a wobble U·G base pair, extensive base stacking and sugar-base contacts. A more striking feature is the protrusion of the cytosine residue into the solvent without participating in any of the loop stabilizing interactions. This identifies the 2',5 '-linked (UUCG) loop as a novel structural motif and provides the first demonstration that 2',5' can fold back on itself to form a hairpin structure of unusual thermodynamic stability.
The ability of hairpin structures to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase (RT) was also evaluated. Four potent hairpins based on 3',5'- and 2' ,5'-RNA were able to inhibit the RNase H activity of HIV-1 reverse transcriptase, a key enzyme for the proliferation of the human immunodeficiency virus (HIV-1). The polymerase activity of HIV-1 RT was not affected by this class of oligonucleotides. The hairpins were identified from a nucleic acid library synthesized via diversity-oriented solid-phase synthesis. These compounds represent the first examples of hairpins, 12 nucleotides in length, that inhibit specifically the RNase H activity of HIV-1 RT without affecting its polymerase activity.
Another study in this work dealt with yeast RNase III (Rnt1p), an enzyme implicated in the mechanism of action of RNA interference (RNAi). Through these investigations, it was discovered that Rntlp cleaves the DNA strand of DNA:RNA hybrids. This was totally unexpected since Rntlp, like other RNase III enzymes, was thought to be specific only towards the cleavage of double-stranded RNA. These studies also increased our knowledge about the mechanism by which the enzyme cleaves the target RNA (or DNA) strand and suggest that the vicinal 2'-hydroxyl group on the ribose sugar does not participate directly in the cleavage reaction.
Finally, the formation of C-tetrad structures (i-motif) was induced through the design and synthesis of extra-stable hairpin loops containing deoxycytidine rich stems. The corresponding hairpins containing ribocytidine folded into duplexes rather than C-tetrad structures. These studies lead to the first detection and identification of antiparallel 2',5 '-RNA duplexes based on hemiprotonated C·C+ base pairs.
Idioma: en