No abstract

Aptamers are single-stranded DNA or RNA molecules capable of tightly binding to specific targets. These functional nucleic acids are obtained by an in vitro Darwinian evolution method coined SELEX (Systematic Evolution of Ligands by EXponential enrichment). Compared to their proteinaceous counterparts, aptamers offer a number of advantages including a low immunogenicity, a relative ease of large-scale synthesis at affordable costs with little or no batch-to-batch variation, physical stability, and facile chemical modification. These alluring properties have propelled aptamers into the forefront of numerous practical applications such as the development of therapeutic and diagnostic agents as well as the construction of biosensing platforms. However, commercial success of aptamers still proceeds at a weak pace. The main factors responsible for this delay are the susceptibility of aptamers to degradation by nucleases, their rapid renal filtration, suboptimal thermal stability, and the lack of functional group diversity. Here, we describe the different chemical methods available to mitigate these shortcomings. Particularly, we describe the chemical post-SELEX processing of aptamers to include functional groups as well as the inclusion of modified nucleoside triphosphates into the SELEX protocol. These methods will be illustrated with successful examples of chemically modified aptamers used as drug delivery systems, in therapeutic applications, and as biosensing devices.

While Nature harnesses RNA and DNA to store, read and write genetic information, the inherent programmability, synthetic accessibility and wide functionality of these nucleic acids make them attractive tools for use in a vast array of applications.

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 pKa'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 M2+-free conditions at 37°C.

Deoxyribozymes or DNAzymes are single-stranded catalytic DNA molecules that are obtained by combinatorial in vitro selection methods. Initially conceived to function as gene silencing agents, the scope of DNAzymes has rapidly expanded into diverse fields, including biosensing, diagnostics, logic gate operations, and the development of novel synthetic and biological tools. In this review, an overview of all the different chemical reactions catalyzed by DNAzymes is given with an emphasis on RNA cleavage and the use of non-nucleosidic substrates. The use of modified nucleoside triphosphates (dN*TPs) to expand the chemical space to be explored in selection experiments and ultimately to generate DNAzymes with an expanded chemical repertoire is also highlighted.

Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.

The terminal deoxynucleotidyl transferase (TdT) belongs to the X family of DNA polymerases. This unusual polymerase catalyzes the template‐independent addition of random nucleotides on 3′‐overhangs during V(D)J recombination. The biological function and intrinsic biochemical properties of the TdT have spurred the development of numerous oligonucleotide‐based tools and methods, especially if combined with modified nucleoside triphosphates. Herein, we summarize the different applications stemming from the incorporation of modified nucleotides by the TdT. The structural, mechanistic, and biochemical properties of this polymerase are also discussed.

Abstract:The enzymatic co-polymerization of modified nucleoside triphosphates (dN*TPs and N*TPs) is a versatile method for the expansion and exploration of expanded chemical space in SELEX and related combinatorial methods of in vitro selection. This strategy can be exploited to generate aptamers with improved or hitherto unknown properties. In this review, we discuss the nature of the functionalities appended to nucleoside triphosphates and their impact on selection experiments. The properties of the resulting modified aptamers will be described, particularly those integrated in the fields of biomolecular diagnostics, therapeutics, and in the expansion of genetic systems (XNAs).