Nucleic acids and proteins are abundant linear biopolymers in living organisms. Much of my research focused on developing new antisense agents targeting an important class of nucleic acids known as ribonucleic acids (RNA). A number of such antisense agents have been developed over the years, including peptide nucleic acids (PNAs) – nucleic acid analogs that utilize 2-aminoethylglycine linkages in place of the negatively charged phosphodiester nucleic acid backbone. A key feature of PNAs is their ability to tightly bind complementary RNA sequences and either block or alter protein synthesis. One potential limitation of PNAs as an antisense agent, however, is that at least one equivalent of PNA is required for each target RNA inhibited. This is particularly problematic for translational suppression given the multiple copies of RNAs present in cells. One way to overcome this limitation is an enzymatic degradation antisense strategy. Conjugating PNA with RNA cleavage motifs has been shown to enhance the rate of RNA hydrolysis. Chapters 2 and 3 are focused on our attempts to develop an RNA-cleaving PNA based on this approach.
Chapter 2 describes the initial design, synthesis, and the RNA cleavage activity of this new class of RNA-cleaving PNA. Polyamines are known RNA cleavage agents. Two different PNA monomers harboring a diamine cleavage arm were incorporated into PNA oligomers via a modified solid-phase peptide synthesis. The resulting PNAs retain the high binding affinities of the parent PNAs, but also exhibit the ability to cleave complementary RNA sequences. The extent of cleavage appears to depend on the position of the diamine scissor. A PNA oligomer harboring the synthetic PNA-diamine monomer at its N-terminus (the 3’-side of the complementary RNA) exhibits a high RNA cleavage efficiency. However, a PNA-diamine adduct with the cleavage arm in the middle of the PNA oligomer cleaves its RNA target only partially. An “in-line attack” hypothesis to explain the reduced cleavage activity of the internal diamine group is proposed, as are potential strategies to overcome this limitation.
Chapter 3 explores one strategy to overcome the poor cleavage activity of our PNA oligomers with internal diamine cleavage sites. The strategy explored is based on the assumption that the extremely stable PNA-RNA helix structure hinders the internal PNA-diamine catalyzed RNA cleavage reaction. A potential solution is suggested by the RNA AAUAA bulge region of the self-cleavage group I intron. The bulge region allows RNA to adopt an “in-line attack” conformation on the backbone, thereby facilitating the cleavage reaction. In contrast, the PNA-RNA duplex lacks such a bulge, and appears to adopt a “cleavage-resistant” conformation. To explore whether introducing bulges into the RNA cleavage site could enhance the cleavage rate, the RNA cleavage activity of a PNA-diamine oligomer was examined for four different complementary RNA substrates – a parent complementary RNA strand, as well as those containing different A, C, and AC bulges. Notably, enhanced cleavage of RNAs with bulge-loops by the PNA-diamines is observed. These findings are consistent with the hypothesis that a perturbation of the enzyme-substrate ground state configuration may enhance the RNA cleavage rate.
Chapter 4 details a distinct project focused on the structure of a new protein – the Methanosarcina barkeri trimethylamine methyltransferase (MttB). The MttB plays a critical role in the growth of M. barkeri on trimethylamine. What makes this enzyme unusual is that, like other methylamine methyltransferases found in M. barkeri, this enzyme contains an in-frame UAG (amber) codon that is ultimately used to incorporate pyrrolysine, the 22nd genetically-encoded amino acid found in nature. The X-ray crystal structure of trimethylamine methyltransferase (MttB) is reported. The protein exists as a homohexamer with the D3 symmetry, and each subunit adopts a TIM-barrel fold. The structure is compared to the other structurally characterized pyrrolysine containing protein, M. barkeri monomethylamine methyltransferase (MtmB), as well as a non-pyrrolysine exhibiting MttB homology.