Curtis H. Lam

A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+).

The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M2+)-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 ± 0.026) min−1. A pH rate profile analysis revealed pKas of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M2+-free conditions at 37°C.

A DNAzyme with Three Protein-Like Functional Groups: Enhancing Catalytic Efficiency of M2+-Independent RNA Cleavage

Catalytically efficient, sequence-specific RNA cleavage holds great therapeutic value for selective gene inactivation against viral infection and cancer. Towards that end, SELEX and related combinatorial methods of in vitro selection have been used to discover RNA-cleaving DNAzymes that have received considerable attention. In addition to therapeutic use in catalysing sequence-specific destruction of mRNA for targeted gene deactivation, applications of catalysis to sensing have also been suggested. 9–26] Like ribozymes, DNAzymes present a limited chemical repertoire compared to proteins. 28] Nevertheless, this inherent lack of functionality can be offset by divalent metal cations (M ). For RNA cleavage, Mg + , or other divalent cations (M), afford kcat/KM values that approach the limits of catalytic perfection (10 m 1 min ) ; however, such values are observed only at high concentrations of Mg + (e.g. , 10–25 mm). In contrast, at physiological Mg concentrations (0.2–0.8 mm), even the most efficient of DNAzymes such as Dz10-23 and Dz8-17 exhibit markedly reduced kcat/KM values that fall in the range of 10–10 m 1 min . [33] These findings highlight an inextricable link between high concentrations of Mg and catalytic efficiency. 35] By the same token, one must hypothesise that the scarcity of intracellular Mg limits the efficacy of DNAzymes in vivo. This catalytic shortcoming might be circumvented by selecting for DNAzymes with synthetically appended chemical functionalities that enhance the chemical repertoire of nucleic acid enzymes, which is otherwise impaired by a low physiological concentration of M . The generation of functionalized nucleic acids for in vitro selection involves the enzymatic polymerization of synthetically functionalized nucleoside triphosphates (dXTPs in which X is any given nucleobase). An abiding interest in this approach is highlighted in numerous studies in which synthetic nucleotides have been successfully incorporated for the discovery of highly functionalized nucleic acids, aptamers, RNA-based enzymes 53] and DNAzymes. Our initial attempt at enhancing the chemical repertoire of DNAzymes resulted in the DNAzyme Dz925-11, which required both the imidazole and amine-bearing modified nucleotides 1 and 2 (Scheme 1) for activity. Nevertheless, catalytic properties remained modest: whereas a reasonably fast rate of selfcleavage was observed (kobs ~0.3 min ), the optimum temperature for self cleavage remained depressed at 13 8C; further, although the cis-cleaving DNAzyme was engineered to cleave at a single ribonucleoside linkage with both multiple turnover and high sequence specificity, 59] both optimal temperature (25 8C) and kcat (0.03 min ) also remained rather low. Notably, attempts to reselect 2nd-generation catalysts from either 20 or 40 degenerate positions failed to provide any improvement and instead Dz925-11 and closely related sequence variants embedded within the N40 region were selected. This “null result” led to an important conclusion: modified dNTPs 1 and 2 found in Dz925-11 failed to provide more efficient catalysts, even when selections were attempted from longer libraries composed of 40 nucleotides (N40). Consequently, we hypothesised that further increasing chemical diversity by introducing a third modified nucleotide bearing a cationic guanidinium ion would increase the number of viable catalytic solutions to M -independent RNA cleavage and afford DNAzymes that could operate at higher temperature with improved catalytic properties. Thus, the simultaneous polymerization of dATP (8-histaminyl-dATP), dUTP (5-guanidinoallyl-dUTP) and dCTP (5-aminoallyl-dCTP; 1, 3 and 4 in Scheme 1) and subsequent in vitro selection from a library of 20 degenerate positions led to the discovery of Dz9-86, which catalysed M-independent cleavage at a single ribo ACHTUNGTRENNUNG(cytosine)phosphodiester linkage at much higher temperatures (37–45 8C) with a rate constant similar to that of Dz925-11 (kobs~0.15 min ) under physiological conditions (200 mm MCl 0.2 mm Mg , 37 8C). Despite these quantitative and qualitative improvements, we expected room for improvement in terms of rate constants for self-cleavage and multiple turnover. In particular, exploration of greater Scheme 1. Chemical structures of 1 (dATP), 2 (dUTP), 3 (dUTP) and 4 (dCTP).

Toward the Combinatorial Selection of Chemically Modified DNAzyme RNase A Mimics Active Against all-RNA Substrates

The convenient use of SELEX and related combinatorial methods of in vitro selection provides a formidable gateway for the generation of DNA enzymes, especially in the context of improving their potential as gene therapeutic agents. Here, we report on the selection of DNAzyme 12-91, a modified nucleic acid catalyst adorned with imidazole, ammonium, and guanidinium groups that provide for efficient M(2+)-independent cleavage of an all-RNA target sequence (kobs = 0.06 min(-1)). While Dz12-91 was selected for intramolecular cleavage of an all-RNA target, it surprisingly cleaves a target containing a lone ribocytosine unit with even greater efficiency (kobs = 0.27 min(-1)) than Dz9-86 (kobs = 0.13 min(-1)). The sequence composition of Dz12-91 bears a marked resemblance to that of Dz9-86 (kobs = 0.0014 min(-1) with an all-RNA substrate) that was selected from the same library to cleave a target containing a single ribonucleotide. However, small alterations in the sequence composition have a profound impact on the substrate preference and catalytic properties. Indeed, Dz12-91 displays the highest known rate enhancement for the M(2+)-independent cleavage of all-RNA targets. Hence, Dz12-91 represents a step toward the generation of potentially therapeutically active DNAzymes and further underscores the usefulness of modified triphosphates in selection experiments.

Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2–5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries.

In vitro selection of a DNAzyme with three modified nucleotides

Three modified nucleosides have been used in the in vitro selection of the self-cleaving DNAzyme 9-33. This DNAzyme operates in the absence of divalent metal cations with a first-order constant of approximately 0.05 min(-1).

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.

Abstract Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5′-GGT TGG TGT GGT TGG-3′ that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent’s aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.