Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate.

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

Shaping Rolling Circle Amplification Products into DNA Nanoparticles by Incorporation of Modified Nucleotides and Their Application to In Vitro and In Vivo Delivery of a Photosensitizer.

Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.

New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments.

5-Ethynyl-2'-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.