Rationally Designed Allosteric Variants of Hammerhead Ribozymes Responsive to the HIV-1 Tat Protein

Hammerhead ribozymes that are subject to allosteric control by small molecule and oligonucleotide effectors have been reported recently. Rational design has been an effective strategy for the creation of these ribozymes, which incorporate structurally interdependent hammerhead motifs and effector-binding sequences. In this paper we report the rational design of the first protein-responsive allosteric ribozymes that are regulated by the HIV-1 Tat. The TAR-Tat interaction of HIV-1 has the interesting feature that both Tat and arginine are able to bind to and bring about comparable conformational changes in the TAR loop. Here we describe the construction of two classes of TAR-modified hammerhead ribozymes and their response to Tat protein and to its derivatives. Instances of both allosteric activation and inhibition were found. Interestingly, the activation response was stimulated by both Tat and argininamide while the inhibitory response was stimulated by Tat and by its derivative peptide, ADP1, but not by argininamide. Overall, the extent of allosteric response in our ribozymes was modest relative to those reported for ribozymes with small molecule effectors. Future work utilizing combinatorial approaches along with elements of rational design should reveal the means by which highly efficient, protein-mediated allostery of ribozymes may be achieved.