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.

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

Sequence-specific cleavage of RNA targets in the absence of a divalent metal cation (M) has been a long-standing goal in bioorganic chemistry. Herein, we report the selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (dATP), 5-guanidinoallyl-deoxyuridine (dUTP), and 5-aminoallyl-deoxycytidine (dCTP) along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A.

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.

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.

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 (dU, 47.5%).

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 (dATP), 5-guanidinoallyl-deoxyuridine (dUTP), and 5-aminoallyl-deoxycytidine (dCTP) 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.

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.

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)).

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.

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.

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.

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.