Nucleoside triphosphates--building blocks for the modification of nucleic acids

Abstract

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.

Scheme 1

Yoshikawa method for the synthesis of nucleoside triphosphates (B = modified or natural nucleobase; R = H, OH, or modification).

Scheme 2

Ludwig-Eckstein synthetic approach (B = modified or natural nucleobase; R = H, OH, OAc or modification).

Figure 1

Chemical structures of the proline-containing analogues dU^tPTP (9); dU^cPTP (10); and dU^FPTP (11); the urea modified dU^BpuTP (12); and the sulfonamide functionalized dU^BsTP (13) [51].

Scheme 3

The Borch approach for the synthesis of nucleoside triphosphates (B = modified or natural nucleobase; R = H, OH, or modification).

Scheme 4

Synthesis of modified dNTPs via direct aqueous Sonogashira (compound 19a) or Suzuki (compound 19b) coupling reactions (R₁ = H, OH, or modification; R₂ = functional group) [56].

Figure 2

(a) Chemical structures of AMP-PCP 20 and (α,β)(β,γ)-bisCF₂ dTTP 21; (b) Schematic representation of the single-turnover kinetic assay using the modified dGTP and DNA polymerase β (X = CH₂, CHF, CF₂, CCl₂, or O; M = C or T) [87].

Figure 3

(a) Chemical structures of dT^spinTP 22 and dT^dendTP 23; (b) Close-up view of the X-ray structure of the DNA polymerase KlenTaq with the modified triphosphate dT^spinTP 22, picture taken from reference [96].

Figure 4

Sequence and hypothetical 2D structure of Dz16.2-11 (bold-face U’s indicate the position of the modified nucleoside; the arrow shows the cleavage site within the RNA substrate) [109].

Figure 5

(a) Chemical structures of dA^imTP 24, dC^aaTP 25, dU^gaTP 26, and the phenol-modified dUTP 27; (b) Sequence and hypothetical 2D structure of Dz9-86 (bold-face A’s, U’s, and C’s indicate the position of the modified nucleosides) [112].

Figure 6

(a) Chemical structure of the amine-modified dUTP; (b) Sequence and hypothetical 2D structure of aptamer T5 (bold-face U’s indicate the position of the modified nucleosides) [134]; (c) Chemical structure of the carboxamide-modified dUTPs [40].