Biological Chemistry of Nucleic Acids
Developing New and Useful RNA Catalysts.We have developed novel RNA catalysts, based on autocatalytic RNA group I introns, which can recombine RNA in predetermined ways. Our most advanced, the trans excision-spicing ribozyme, can excise out single nucleotide insertion mutations from transcripts inside bacterial cells. In this way, we have sequence-specifically repaired mutations in functional RNA transcripts. Questions that remain with this and other recently developed catalysts include how to develop them for optimal cellular activity, how they fold and function, how they can be exploited for the development of new biotechnology, and we then take these lessons to develop other useful catalysts. Methods used in these projects involve molecular biology (for example, PCR, cloning, site-directed mutagenesis, and gel electrophoresis).
Thermodynamics. We are working on a new project whereby we ascertain the effects of foreign molecules on the structure and function of nucleic acids. Areas of interest include the effects of cigarette smoke components on the stability of DNA and RNA duplexes. Methods used in this project involve UV spectroscopy (specifically thermal denaturation analysis). These types of projects are especially well suited for undergraduates, as well as graduate students who desire to become educators at liberal arts colleges.
Z-helical hairpins.We have found a novel DNA hairpin that adopts a Z-helical conformation as a function of the hairpin loop sequence (the DNA double helix is left-handed instead of the usual right-handed). Somehow, the structure of the loop is driving the stem into this unusual conformation. Therefore, we are working toward fully understanding the sequence and structure requirements of this molecule. Methods used in this project involve traditional biochemical techniques (for example, UV spectroscopy, CD spectroscopy, and in the very near future, NMR).
Scientific Programming. I am working on a new project that combines my interest in programming (using Microsoft Visual Studio 2005 – Visual Basic) with my knowledge of nucleic acids and molecular biology. The first program is a general purpose laboratory tool called STcalc, and the second project I am about to begin focuses on molecular biology and education. STcalc currently does three things. First, it will calculate serial dilutions. This is by far the most used application in my lab. Second, it will calculate thermodynamic parameters (like Tm, free energy) for small DNA and RNA duplexes. Third, it will calculate concentrations from nucleic acid absorbance measurements.
If you would like to use STcalc, please contact me. Note that although it has been extensively checked, you are responsible for making sure the data is correct. Please contact me to let me know if you use the program, would like additions or changes, or have problems.
- Dotson II, P. P., Johnson, A. K., and Testa, S. M. (2008) "Tetrahymena thermophila and Candida albicans Group I intron-derived ribozymes can catalyze the trans-excision-splicing reaction", Archives of Biochemistry and Biophysics.
- Dotson II, P. P., Sinha, J., and Testa, S. M. (2008) “A Pneumocystis carinii group I intron-derived ribozyme utilizes an endogenous guanosine as the first reaction step nucleophile in the trans excision-splicing reaction”, FEBS Journal, 275 (12), 3110-3122.
- Dotson II, P. P. Testa, S. M. (2006) "Group I Intron-Derived Ribozyme Recombination Reactions" Recent Developments in Nucleic Acids Research, 2(2006): 307-324 ISBN: 81-7895-192-4.
- Johnson, A. K., Sinha, J., Testa, S. M. (2005) "Ribozyme-Catalyzed Insertion of Targeted Sequences into RNAs", RNA 11, 897-905.
- Alexander, R. C., Baum, D. A., Testa, S. M. (2005) "5' Transcript Replacement in vitro Catalyzed by a Group I Intron-Derived Ribozyme", Biochemistry 44, 1067-1077.
- Bell, M. A., Sinha, J., Johnson, A. K., Testa, S. M. (2004) "Enhancing the Second Step of the Trans Excision-Splicing Reaction of a Group I Ribozyme by Exploiting P9.0 and P10 for Intermolecular Recognition", Biochemistry 41, 15327-15333.
- Disney, M. D., Testa, S. M., Turner, D. H. (2000) "Targeting a Pneumocystis carinii Group I Intron with Methylphosphonate Oligonucleotides: Backbone Charge is Not Required for Binding or Reactivity",Biochemistry 39, 6991-7000.
- Testa, S. M., Turner, D. H., Kierzek, R. (1999) "Thermodynamics of RNA-RNA Duplexes with 2- or 4-Thiouridines: Implications for Antisense Design and Targeting a Group I Intron", Biochemistry 38, 16655-16662.
- Testa, S. M, Gryaznov, S. M., Turner, D. H. (1999) "In Vitro Suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N3'->P5' phosphoramidate hexanucleotide", Proc. Natl. Acad. Sci. U.S.A. 96, 2734-2739.
- Testa, S. M., Gryaznov, S. M., Turner, D. H. (1998) "Antisense Binding Enhanced by Tertiary Interactions: Binding of Phosphorothioate and N3'-> P5' Phosphoramidate Hexanucleotides to the Catalytic Core of a Group I Ribozyme from the Mammalian Pathogen Pneumocystis carinii", Biochemistry 37, 9379-9385.
- Testa, S. M. , Haidaris, C. G., Gigliotti, F., Turner, D. H. (1997) "A Pneumocystis carinii Group I Intron Ribozyme that Does Not Require 2' OH Groups on its 5' Exon Mimic for Binding to the Catalytic Core", Biochemistry 36, 15303-15314.
- Profenno, L., Kierzek, R., Testa, S. M., Turner, D. H. (1997) "Guanosine Binds to the Tetrahymena Ribozyme in More than One Step, and Its 2' OH and the Nonbridging pro-Sp Phosphoryl Oxygen at the Cleavage Site Are Required for Productive Docking", Biochemistry 36, 12477-12485.
- Testa, S. M, Gilham, P. T. (1993) "Analysis of Oligonucleotide Structure using Hyperchromism Measurements at Long Wavelengths", Nucleic Acids Res. 21, 3907-3908.