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

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.

Whole cell-SELEX of aptamers with a tyrosine-like side chain against live bacteria.

In an effort to expand the binding and recognition capabilities of aptamers, a nucleoside triphosphate modified with a phenol that mimics the side chain of tyrosine was used in the selection of DNA aptamers against live bacteria. Of multiple modified aptamers that were isolated against Escherichia coli DH5α cells, one aptamer displays high selectivity and affinity for the target cells and is greatly enriched for phenol-modified dU nucleotides (dUy, 47.5%). When the same sequences are synthesized with TTP, no binding is observed. Taken together, these findings highlight the value of using modified nucleotide triphosphates in aptamer selections and portends success in SELEX against an array of whole cells as targets.

Systematic study of constraints imposed by modified nucleoside triphosphates with protein-like side chains for use in in vitro selection.

Successful selection of modified DNAzymes depends on the potential for modified nucleoside triphosphates (dNTPs) to replace their unmodified counterparts in enzyme catalyzed primer extension reactions and, once incorporated, to serve as template bases for information transfer prior to PCR amplification. To date, the most densely modified DNAzymes have been selected from three modified dNTPs: 8-histaminyl-deoxyadenosine (dAimTP), 5-guanidinoallyl-deoxyuridine (dUgaTP), and 5-aminoallyl-deoxycytidine (dCaaTP) to provide several RNA-cleaving DNAzymes with greatly enhanced rate constants compared to unmodified counterparts. Here we report biophysical and enzymatic properties of these three modified nucleosides in the context of specific oligonucleotide sequences to understand how these three modified nucleobases function in combinatorial selection. The base-pairing abilities of oligonucleotides bearing one or three modified nucleosides were investigated by thermal denaturation studies and as templates for enzymatic polymerization with both modified and unmodified dNTPs. While we address certain shortcomings in the use of modified dNTPs, we also provide key evidence of faithful incorporation and enzymatic read-out, which strongly supports their continued use in in vitro selection.

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.

A divalent metal-dependent self-cleaving DNAzyme with a tyrosine side chain.

The enzymatic incorporation of a phenol-modified 2'-deoxyuridine triphosphate gave rise to a modified DNA library that was subsequently used in an in vitro selection for ribophosphodiester-cleaving DNAzymes in the presence of divalent zinc and magnesium cations. After 11 rounds of selection, cloning and sequencing resulted in 14 distinct sequences, the most active of which was Dz11-17PheO. Dz11-17PheO self-cleaved an embedded ribocytidine with an observed rate constant of 0.20 ± 0.02 min(-1) in the presence of 10 mM Mg(2+) and 1 mM Zn(2+) at room temperature. The activity was inhibited at low concentrations of Hg(2+) cations and somewhat higher concentrations of Eu(3+) cations.

Protein-inspired modified DNAzymes: dramatic effects of shortening side-chain length of 8-imidazolyl modified deoxyadenosines in selecting RNaseA mimicking DNAzymes.

The discovery of imidazole/amine-functionalized DNAzymes that efficiently cleave RNA independently of divalent metal cations (M(2+)) and cofactors underscores the importance of expanding the catalytic repertoire with modified nucleosides. Considerable effort has gone into defining polymerase tolerances of various modified dNTPs for synthesizing and amplifying modified DNA. While long linkers are generally found to enhance incorporation and therefore increase sequence space, shorter linkers may reduce the entropic penalty paid for orienting catalytic functionality. Catalytic enhancement ultimately depends on both the functional group and appropriate linkage to the nucleobase. Whether a shorter linker provides enough catalytic enhancement to outweigh the cost of reduced polymerizability can only be determined by the outcome of the selection. Herein, we report the selection of DNAzyme 20-49 (Dz20-49), which depends on amine, guanidine, and imidazole-modified dNTPs. In contrast to previous selections where we used dA(ime)TP (8-(4-imidazolyl)ethylamino-2'-dATP), here we used dA(imm)TP (8-(4-imidazolyl)methylamino-2'-dATP), in which the linker arm is shortened by one methylene group. Although the most active clone, Dz20-49, was absolutely dependent on the incorporation of either dA(imm)p or dA(ime)p, it catalyzed cofactor independent self-cleavage with a rate constant of 3.1 ± 0.3 × 10(-3) min(-1), a value not dissimilar from unmodified catalysts and strikingly inferior to modified catalysts selected with dA(ime)TP. These results demonstrate that very subtle differences in modified nucleotide composition may dramatically effect DNAzyme selection.

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 (M(2+))-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 pK(a)'s 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 M(2+)-free conditions at 37 degrees C.

Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10-23 to enhance target affinity: implications for DNAzyme design.

Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg(2+)-dependent RNA-cleaving DNAzyme 10-23 (Dz10-23). As such, we have investigated the synthetic modification of Dz10-23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N'-biscyanoethoxycarbonyl)-guanidinoallyl-2'-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10-23. This particular modification was chosen as it had been used in the selection of Mg(2+)-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg(2+) for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10-23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg(2+)-dependent DNAzyme with enhanced catalytic activity.

Selection of PD1/PD-L1 X-Aptamers.

Specific, chemically modified aptamers (X-Aptamers) were identified against two immune checkpoint proteins, recombinant Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1). Selections were performed using a bead-based X-Aptamer (XA) library containing several different amino acid functional groups attached to dU at the 5-position. The binding affinities and specificities of the selected XA-PD1 and XA-PDL1 were validated by hPD-1 and hPD-L1 expression cells, as well as by binding to human pancreatic ductal adenocarcinoma tissue. The selected PD1 and PDL1 XAs can mimic antibody functions in in vitro assays.

A densely modified M2+-independent DNAzyme that cleaves RNA efficiently with multiple catalytic turnover.

Sequence-specific cleavage of RNA targets in the absence of a divalent metal cation (M2+) has been a long-standing goal in bioorganic chemistry. Herein, we report the in vitro selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (dAimTP), 5-guanidinoallyl-deoxyuridine (dUgaTP), and 5-aminoallyl-deoxycytidine (dCaaTP) along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A. Of several such M2+-free DNAymes, DNAzyme 7-38-32 cleaves a 19 nt all-RNA substrate with multiple-turnover, under simulated physiological conditions wherein only 0.5 mM Mg2+ was present, attaining values of kcat of 1.06 min-1 and a KM of 1.37 µM corresponding to a catalytic efficiency of ∼106 M-1 min-1. Therefore, Dz7-38-32 represents a promising candidate towards the development of therapeutically efficient DNAzymes.

X-Aptamer Selection and Validation.

Aptamers and second generation analogs, such as X-Aptamers (XAs), SOMAmers, locked nucleic acids (LNAs), and others are increasingly being used for molecular pathway targeting, biomarker discovery, or disease diagnosis by interacting with protein targets on the surface of cells or in solution. Such targeting is being used for imaging, diagnostic evaluation, interference of protein function, or delivery of therapeutic agents. Selection of aptamers using the original SELEX method is cumbersome and time-consuming, often requiring 10-15 rounds of selection, and provides aptamers with a limited number of functional groups, namely four bases of DNA or RNA, although newer SELEX methods have increased this diversity. In contrast, X-Aptamers provide an unlimited number of functional groups and thus are superior targeting agents. Here, we discuss the X-Aptamer selection process.

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.