Date of Award
Doctor of Philosophy (PhD)
Dr. Thomas Neilson
A variety of short oligoribonucleotide sequences were synthesized using the phosphotriester chemical synthesis developed in Neilson's laboratory. These sequences were designed to incorporate a variety of features that would aid in the study of perturbations of helix structure and stability. Variable temperature proton nuclear magnetic resonance (NMR) spectroscopy was used in this study and provides a powerful technique for the study of nucleic acid conformations and for the investigation of the effects of the mispairing and single stranded regions caused by the helix imperfections introduced.
The assignment procedure for NMR spectra was improved through the study of a series of related sequences. This study determined the effects that the addition of a nucleotide to the terminus of a sequence or the insertion of a nucleotide into the middle of a sequence would have on the chemical shifts from the rest of the sequence.
A series of self-complementary pentaribonucleotides, with a central non-base paired opposition (AGXCU, where X ≡ A, G, C or U), was studied to determine the effects of small loops on duplex stability. In contrast to earlier results, these pentamers formed stable duplexes when X ≡ A or C, although the duplex Tm's were significantly, reduced. These sequences also provided the opportunity to study the sequence effects of adjacent internal G.C base pairs on duplex stability when the middle base pair was A·U, G·C or G.U. Comparison with earlier results using corresponding A·U base pair neighbours, demonstrated the enhanced stabilization of G·C base pairs.
The effects of terminal non-base paired (dangling) adenosines were more closely investigated and found to contribute an average of 11°c to the thermal stability of the duplex formed. This study also demonstrated that 5'-dangling adenosines contribute less to overall stability than do 3'-dangling adenosines. However, this effect did display some sequence dependence.
The triribonucleotide GpCpA was the first trimer shown to form a stable RNA duplex (Tm = 33°C). The duplex consisted of two G·C base pairs and two 3'-dangling adenosines and had a stability equal to that of the tetramer duplex UpGpCpA with four Watson-Crick base pairs.
The influence of base stacking on duplex formation was studied and it was discovered that the direction of base stacking had a definite influence on helix stability. Stacking in the 5'→3' direction was more favourable to duplex formation than stacking in the 3'→5' direction.
Lastly, the significance of invariant adenosines at position 14 and 21, in the D-stem of tRNA, was investigated through a model study that suggested the adenosines contributed to D-stem stability.
Most of the results presented in this thesis have been published or accepted for publication in:
1. Jeremy R. Everett, Donald W. Hughes, Russell A. Bell, Dirk Alkema, Thomas Neilson and Paul J. Romaniuk. "Nearest-Neighbour and Next-Nearest-Neighbour Effects in the Proton NMR Spectra of the Oligoribonucleotides ApGpX and CpApX." (1980) Biopolymers 19, 557.
2. Thomas Neilson, Paul J. Romaniuk, Dirk Alkema, Donald W. Hughes, Jeremy R. Everett and Russell A. Bell. "The Effects of Base Sequence on the Stability of Short Ribonucleic Acid Duplexes." (1980) Nucleic Acids Res. Sym. Series No. 7, 293.
3. Dirk Alkema, R.A. Bell, P.A. Hader and T. Neilson. "Triplet GpCpA Forms a Stable RNA Duplex." (1981) J. Am. Chem. Soc. 103, 2866.
4. Russell A. Bell, Jeremy R. Everett, Donald W. Hughes, Dirk Alkema, Paul Hader, Thomas Neilson and Paul J. Romaniuk. "Nearest-Neighbour and Next-Nearest-Neighbour Effects in the Proton NMR Spectra of the Oligoribonucleotides ApXpG, CpXpG, CpApXpUpG, ApGpXpC and ApGpXpCpU." (1981) Biopolymers 20, 1383.
5. Dirk Alkema, Russell A. Bell, Paul A. Hader and Thomas Neilson. "Short RNA duplex Stability: Contribution from non-base paired residues to the direction of stacking." (1981) in "Biomolecular Stereodynamics" R.H. Sarma, ed., Adenine Press, Elmsford, NY., p. 417.
6. Dirk Alkema, Russell A. Bell, Paul A. Hader and Thomas Neilson. "Invariant Adenosine Residues Stabilize tRNA D-steins." (1982) Accepted for publication in FEBS Letters.
7. Dirk Alkema, Paul A. Hader, Russell A. Bell and Thomas Neilson. "Effects of Flanking G·U· Base Pairs on Internal Watson-Crick, G·U and Non-Bonded Base Pairs Within a Short RNA Duplex." (1982) Accepted for publication in-Biochemistry.
Two additional publications will appear shortly:
1. Paul A. Hader, Thomas Neilson, Dirk Alkema, Eric C. Kofoid and M.C. Ganoza. "Sequencing of Short RNA Oligomers by Proton Nuclear Magnetic Resonance." (1982) Accepted for publication in FEBS Letters.
2. Paul A. Hader, Dirk Alkema, Russell A. Bell and Thomas Neilson. "Parameters for Proton Chemical Shift Prediction in Oligoribonucleotides." (1982) J. Chem. Comm. (in press).
Alkema, Dirk, "Perturbations of Short RNA Helices" (1982). Open Access Dissertations and Theses. Paper 1571.