Biological Chemistry of Nucleic Acids

If you are interested in doing graduate or undergraduate research, please stop by my office.

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.

STcalc can be downloaded by clicking here (.zip). 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", Nucleic Acids Research .
• Dotson II, P. P., Frommeyer, K. N., and Testa, S. M. (2008) "Ribozyme Mediated Trans Insertion-Splicing of Modified Oligonucleotides into RNA", 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”, Biochemistry 47, 4780-4787.
• Dotson II, P. P., Sinha, J., and Testa, S. M. (2008) "Kinetic characterization of the first step of the ribozyme-catalyzed 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", Biochemistry 44, 10702-10710.
• Baum, D. A. & Testa, S. M. (2005) "In Vivo Excision of a Single Targeted Nucleotide from an mRNA by a Trans Excision-Splicing Ribozyme", 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, 7796-7804.
• Baum, D. A., Sinha, J., & Testa, S. M. (2005) "Molecular Recognition in a Trans Excision-Splicing Ribozyme: Non-Watson-Crick Base Pairs at the 5' Splice Site and wG at the 3' Splice Site Can Play a Role in Determining the Binding Register of Reaction Substrates", 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 43, 4323-4331.
• Alexander, R. C., Johnson, A. K., Thorpe, J. A., Gevedon, T., & Testa, S. M. (2003) "Canonical Nucleosides Can Be Utilized by T4 DNA Ligase as Universal Template Bases at Ligation Junctions", Nucleic Acids Res. 31, 3208-3216.
• Johnson, A. K., Baum, D. A., Tye, J., Bell, M. A., & Testa, S. M. (2003) "Molecular Recognition Properties of IGS-Mediated Reactions Catalyzed by a Pneumocystis carinii Group I Intron", Nucleic Acids Res. 31, 1921-1934.
• Bell, M. A., Johnson, A. K., & Testa, S. M. (2002) "Ribozyme-Catalyzed Excision of Targeted Sequences from within RNAs", 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.