In vitro selection of ribozymes dependent on peptides for activity

Abstract

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide-and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

Identification of dynamical hinge points of the L1 ligase molecular switch

“…These virtual torsions span two evolutionarily conserved and restricted regions located in the three-way junction and a U 38 loop, the conserved residue that is postulated to be responsible for the allosteric control of the catalytic step (Robertson and Ellington 1999, 2000, 2001Robertson and Scott 2007;Robertson et al 2004), and they have been used in the present work as an index to follow the evolution of the conformational rearrangement and monitor important transitions. On the time scale of our simulations we were able to observe approximately two-thirds of the complete stem C's 80-Å conformational switch.…”

“…These torsions span regions that contain the evolutionarily conserved residues: U 37 , U 38 , and A 39 for u 37 and h 38 and the five-base motif (C 39 = G 18 G 37 A 38 C 17 /U 39 = A 18 U 37 G 38 U 17 ), and the neighboring U 19 for u 18 and u 44 (for a detailed statistical analysis of the conserved residues, see . Both U 38 and U 19 are of particular interest in the current work: U 38 contributes to the docking of stem C into stem A in the active conformation; mutation data demonstrate that it is critical for catalysis; and U 19 is evolutionarily conserved (97%), although its role in catalysis remains unclear (Robertson and Ellington 1999, 2000, 2001Robertson et al 2004).…”

“…It is possible to take advantage of this property to engineer new L1 ligase ribozymes by further selections that enable derivatives to be controlled by small molecules, peptides, or even proteins; in effect, creating artificial allosteric ribozymes (Robertson and Ellington 1999, 2000, 2001Robertson et al 2004).…”

Aptamer-based optical switch for biosensors

“…Aptamers have been shown to undergo liganddependent conformational changes and can be joined to ribozymes to create allosteric ribozymes (aptazymes). 86,87 To date almost all of these aptamer and aptazyme biosensors have been generated by empirical design 88,89 or in vitro selection, 21,86,90 however, algorithms for the prediction of nucleic acid secondary structure have advanced to the point where nucleic acid secondary structures can be rapidly enumerated based on nucleic acid sequence. 91 Therefore, the computational design methods for the generation of biosensors would be extremely valuable for a variety of reasons.…”

Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization

“…Analysis of the remaining clones revealed no active species. The winnowing of a functional population is generally incomplete in the early rounds of a selection, and the isolation of inactive species, whether aptamers or ribozymes, in parallel with active ones is frequently observed (Robertson et al 2004). In the case of the current selection, the co-isolation of inactive variants may have been facilitated by the equilibration or release of dsDNA templates during the labeling procedure prior to sorting.…”