Synthesis and Enzymatic Characterization of Sugar-Modified Nucleoside Triphosphate Analogs.

Chemical modification of nucleic acids can be achieved by the enzymatic polymerization of modified nucleoside triphosphates (dN*TPs). This approach obviates some of the requirements and drawbacks imposed by the more traditional solid-phase synthesis of oligonucleotides. Here, we describe the protocol that is necessary to synthesize dN*TPs and evaluate their substrate acceptance by polymerases for their subsequent use in various applications including selection experiments to identify aptamers. The protocol is exemplified for a sugar-constrained nucleoside analog, 7',5'-bc-TTP.

Enzymatic Synthesis of 7',5'-Bicyclo-DNA Oligonucleotides.

The selection of artificial genetic polymers with tailor-made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo-DNA triphosphate (i.e., 7',5'-bc-TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7',5'-bc-TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA-dependent DNA synthesis and potentially also XNA-dependent XNA synthesis. However, DNA-dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7',5'-bc-TTP.

Generation of Aptamers with an Expanded Chemical Repertoire.

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).