Aminomodified Nucleobases:  Functionalized Nucleoside Triphosphates Applicable for SELEX

Abstract

5-Aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased T(m) and thus less stable basepairing. The chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP is reported in detail.

A Short DNA Aptamer That Recognizes TNFα and Blocks Its Activity in Vitro

“…Recently, some of the most common modifications of aptamers investigated have been their conjugation to polyethylene glycol (PEG) groups for increased stability (41,42). In addition, to increase the in vivo circulating half-life of DNA aptamers, modified nucleotides could be introduced into the libraries during synthesis such as 2' OH group with 2'fluoro or 2' amino, aromatic or alkyl moieties or introduction of Locked Nucleic Acid (LNA) (43,44). Also, aptamer libraries with longer variable regions may identify sequences with a higher G-rich content which in turn could adopt different G-quadruplex structures and yield improved inhibitors to TNFα (45).…”

Directed polymerase evolution

“…Mutant RT521 (TgoT: E429G, I521L, K726R) was shown to have moderate RT activity for HNA, ANA, and FANA, and RT521K (RT521: A385V, F445L, E664K) for CeNA and LNA. Using these polymerases and a modification of SELEX similar to that originally used by Klussmann [79] and later by Eaton and co‐workers [80], pools of DNA oligomers were first transcribed into XNAs, subjected to selection for binding to various targets, and then reverse transcribed back into DNA for amplification. This work demonstrates that family B DNAPs may be evolved to have altered substrate repertoires, and that modified oligonucleotides may be used for the selection phase of evolution (amplification was still performed with DNA).…”

Blocking the attachment of cancer cells in vivo with DNA aptamers displaying anti‐adhesive properties against the carcinoembryonic antigen

“…2′ OH group with 2′‐amino, 2′‐fluoro 2′‐O‐methyl), the phosphate (eg. Phosphorothioate) or of the base (2′‐thiopyrimidine, methyl or trifluoromethyl) (Healy et al., 2004; Latham et al., 1994; Mayer, 2009; Schoetzau et al., 2003; Willis et al., 1998). Also, Locked Nucleic acids (LNA) have been introduced within their structures to increase stability (Barciszewski et al., 2009).…”