The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Our laboratory studies the nucleic acids, RNA and DNA. In these efforts, we use concepts and techniques from biochemistry, chemical biology, and organic chemistry. We study DNA as a catalyst (deoxyribozyme) for bioorganic chemical reactions; we apply double-stranded DNA as a structural constraint for nanotechnology; we investigate the thermodynamics and kinetics of RNA folding; and we pursue allosteric nucleic acid enzymes (aptazymes) as sensors for practical detection of environmental toxins.

(1) DNA enzymes as catalysts for bioorganic reactions. Catalytic RNAs play important roles in fundamental biochemistry. As a means to explore the catalytic power of nucleic acids, and as a practical experimental approach to catalyze desirable bioorganic reactions, we investigate DNA enzymes (deoxyribozymes). Many of our efforts in this area have focused on deoxyribozymes that ligate RNA, which are used for synthesizing large RNA molecules that have site-specific modifications or interesting and biochemically relevant topologies (e.g., branched and lariat RNA). We are currently turning our attention to deoxyribozymes for bioorganic reactions other than RNA ligation. For example, we seek DNA enzymes that can join nucleic acids to proteins, thereby allowing formation of oligonucleotide-peptide conjugates, which may have utility for drug delivery and for encoding purposes. We also seek DNA enzymes that can join sugars to proteins. The resulting glycopeptides are very difficult to synthesize by other means, and they are integral to many normal and pathological biochemical processes.

(2) Double-helical DNA as a structural constraint. Double-stranded DNA has many favorable properties that make it an excellent candidate for a nanoscale structural constraint. We investigate double-stranded DNA to constrain and control RNA folding. The basic concept is that incorporation of two complementary DNA strands into RNA-one strand at each of two different positions-allows imposition of a duplex DNA constraint onto the RNA. This constraint may either stabilize or destabilize the properly folded RNA structure. We have shown that a disruption in RNA structure due to a DNA duplex can be reversed either by addition of free DNA oligonucleotides that interfere with duplex formation or by degradation of the duplex scaffold using deoxyribonuclease (DNase). We are working to modulate the DNA scaffold in other ways, including addition of specific protein restriction enzymes; cleavage of a disulfide tether between the duplex and RNA; photocleavage of the tether between the duplex and RNA; and binding of a ligand to a portion of the DNA sequence that forms the constraint. We are excited about using double-stranded DNA as a constraint not only for controlling RNA folding but for other nanotechnology applications as well.

(3) RNA folding. RNA conformational changes are intimately related to the roles of RNA in biochemistry. Fluorescence is a sensitive tool for examining the folding of many biomolecules including RNA. We are developing new chromophores and fluorescence techniques to monitor RNA folding. In particular, we are attempting to identify for RNA what tryptophan provides for proteins: a general fluorescence probe for RNA structure and folding. We develop the synthetic procedures to attach chromophores such as pyrene to RNA. We then study the folding of RNA in which individual fluorescent probes are placed specifically at various nucleotide positions and on variable-length tethers. From these studies we seek a fundamental understanding of RNA folding landscapes.

A major challenge is to understand how RNA structures change through time; that is, to understand how RNA folds. Our approach is to use covalent constraints to force RNA into specific misfolded conformations and to study the folding transitions that occur when these constraints are suddenly released. We use photochemically cleavable constraints, which allows the use of light as the initiator of RNA folding. These studies take three forms. First, we covalently append a bulky group onto one specific position of an RNA to cause misfolding; photochemical removal of the group initiates RNA folding. Second, we attach a covalent tether across two positions of the RNA, with a photocleavable linker within the tether. Photocleavage breaks the tether and induces RNA folding. Finally, we attach across two positions a tether that incorporates a photoisomerizable group such as azobenzene, which permits reversible photochemical switching of RNA folding.

(4) Allosteric nucleic acid enzymes as practical sensors for environmental toxins. The basic concept is to develop allosteric nucleic acid enzymes ("aptazymes"), for which the catalytic activity is regulated by the binding of a ligand at a site remote from the active site. When the change in catalytic activity upon ligand binding is coupled with a useful form of signal readout, such as that based on fluorescence, then a practical sensor device has been created. In collaboration with two other research groups on the UIUC campus, we are creating aptazyme sensors for agriculturally important toxins such as those produced by Fusarium and Aspergillus fungi that infect corn, grass, peanuts, and other crops.