Structural flexibility and the thermodynamics of helix exchange constrain attenuation and allosteric activation of hammerhead ribozyme TRAPs.
Perturbations of precleavage equilibria in RNA-cleaving ribozymes can be exploited to control cleavage kinetics. In the targeted ribozyme-attenuated probes (TRAP) design, antisense and attenuator sequences are appended onto the catalytic core of a ribozyme or deoxyribozyme. The attenuator anneals to conserved bases in the catalytic core to form an inactive conformation, which is activated upon binding of a sense strand oligonucleotide to the antisense module. In this work, the apparent Michaelis-Menton constant (K'm) for the binding of the RNA substrate to the ribozyme is shown to be within a factor of 2 for a number of constructs whose observed cleavage rates varied by several 100-fold. These observations rule out models of allosteric regulation based on modulation of substrate binding affinity, instead favoring a model in which regulation arises from equilibration between the active and inactive conformations of the TRAP. Free energies of formation for isolated helices that are exchanged during this reequilibration were determined from the concentration dependence of optical melt data. These values established that the thermodynamic stabilities of sense-antisense duplexes and of the attenuator-core duplexes correlate with observed rates of cleavage. Notably reduced cleavage rates are observed for TRAP ribozymes with extended antisense sequences, suggesting that tight binding of attenuator to the core is assisted by a long antisense portion. A construct with a 25-nucleotide antisense showed greater than 730-fold activation upon annealing with a 20-nucleotide DNA sense strand oligo, representing the greatest activation observed to date for the TRAP design.
Saksmerprome
Vanvimon
V
Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, USA.
Burke
Donald H
DH
D000494:Allosteric Regulation; D001483:Base Sequence; D004789:Enzyme Activation; D004794:Enzyme Repression; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D011485:Protein Binding; D015347:RNA Probes / Q000737:chemistry*; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry*; D013379:Substrate Specificity; D013816:Thermodynamics
D009692:Nucleic Acid Heteroduplexes; D016376:Oligonucleotides, Antisense; D015347:RNA Probes; D016337:RNA, Catalytic; C427204:hammerhead ribozyme