Allosteric Control of Ribozyme Catalysis by Using DNA Constraints

Covalent, crosslinking tethers have previously been attached to nucleic acids for control of structure and catalysis. Surprising results have been found, such as a study with the Tetrahymena group I intron ribozyme in which crosslinks that should substantially disrupt structure nevertheless did not suppress catalysis nearly as much as expected. As an alternative approach to control macromolecular structure, we recently described the use of covalently attached DNA strands as constraints on RNA conformation. When two complementary DNA strands are attached to a large and foldable RNA, DNA duplex formation can compete with native RNA structure. This can destabilize RNA folding (by >6 kcalmol 1 in one instance) because the DNA duplex must be disrupted in order for the RNA to fold properly. The integrity of the DNA constraint can be modulated by added enzymes, oligonucleotides, or smallmolecule ligands that cleave or interact with the DNA strands. These studies suggested the possibility that the catalytic activity of a ribozyme, and not merely the structure of a foldable RNA, could be controlled by strategic attachment of DNA strands. If this can be established, then we anticipate that the DNA constraint approach will be useful for studying RNA structure–function relationships that involve catalysis and not only folding. Here, we report the identification of a new deoxyribozyme for attachment of DNA to RNA, which considerably aids the synthetic procedure. Using this deoxyribozyme, we describe the successful application of DNA constraints to control the catalytic activity of the hammerhead ribozyme. We provide initial data that allow us to understand the structural basis of catalytic control in terms of modulation of tertiary structure but not secondary structure. To facilitate synthetic access to DNA-derivatized RNA, which previously required a laborious procedure that depended on assembly of numerous RNA fragments, we used in vitro selection to identify new deoxyribozymes that ligate DNA to RNA. We previously reported the 7S11 deoxyribozyme, which ligates an RNA 2’-hydroxyl group to a 5’-triphosphorylated or 5’adenylated RNA (Figure 1A). Although 7S11 and an improved variant, 10DM24, show modest activity when 5’-adenylated DNA is used in place of analogous RNA, the ligation yield was impractically low when examined with an RNA substrate that differed from the arbitrary sequence used during the original selection procedure (data not shown). Therefore, we performed a new selection experiment directly with 5’-adenylated DNA. With a branch-site adenosine that provided the 2’-hydroxyl group in the RNA substrate, we iterated selection rounds (50 mm CHES, pH 9.0, 40 mm MgCl2, 150 mm NaCl, and 2 mm KCl at 37 8C) using an incubation time as low as merely 1 min in the later rounds. The pool activity was 34% at round 9, after which individual deoxyribozymes were cloned and characterized. One of these deoxyribozymes, 9FQ4 (Figure 1B), was examined further. Deoxyribozyme 9FQ4 had relatively good RNA sequence tolerance, in that many changes to the RNA nucleotides other than the branch-site adenosine were accepted with good ligation rate and yield for model substrates. However, the branch-site nucleotide itself could not be changed from adenosine; of the other three nucleotides, only C but neither G nor U gave a potentially useful yield. For the 5’-adenylated DNA substrate, changes to all nucleotides other than the 5’-terminal nucleotide were tolerated well. The 5’-nucleotide of the DNA could be G or A with high ligation rate and yield, or C with low activity, whereas 5’-T was not tolerated. We surveyed the applicability of 9FQ4 for attachment of DNA to RNA using the biologically derived P4-P6 RNA domain, the sequence of which is unrelated to the substrates used during selection. P4-P6 is an independently folding domain of the Tetrahymena group I intron RNA, and it was the basis for our first experiments with DNA constraints. Ten adenosine nucleotides throughout P4-P6 were chosen for testing on the basis of the surface exposure of their 2’-hydroxyl groups; exposed hydroxyls could presumably have DNA attached without inherently perturbing the RNA structure. Using 9FQ4 deoxyribozyme with appropriate binding arms to target the desired adenosine 2’-hydroxyl groups, we found that four out of the Figure 1. Branched nucleic acid formation by deoxyribozymes. A) The 7S11 deoxyribozyme, which forms branched RNA. The indicated nucleotides form a fourth Watson–Crick paired region denoted P4. The leaving group is pyrophosphate (PPi). B) The new 9FQ4 deoxyribozyme, which attaches 5’-adenylated DNA to RNA. The leaving group is adenosine 5’-monophosphate (pA=AMP).