Modified Nucleoside Triphosphates for In-vitro Selection Techniques

Abstract

The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

Circular Bivalent Aptamers Enable in Vivo Stability and Recognition

“…Chemical modifications, including postselection modification and SELEX with modified oligonucleotide libraries (modified SELEX), are the conventional methods used to generate nuclease-resistant aptamers. 14–18 Macugen, an approved aptamer drug, is a vivid example of nuclease-resistant aptamers generated with modified SELEX. 19 However, modified nucleotides must be compatible with polymerases in SELEX, and such postselection modifications can affect aptamer specificity and affinity.…”

Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics

“…Furthermore, the majority of reports to date have involved selections with partially modified libraries (modSELEX), with some DNA or RNA components retained during the selection step. Despite this, modSELEX has proven to be a powerful approach for selecting aptamers with improved properties for in vivo applications (Dellafiore et al 2016;Lipi et al 2016); although phosphorothioate (αS)-and boranophosphate (αBH 3 )-linked RNA backbones have been explored, modified sugars have become a preferred chemistry, such as 2 ′ -amino-and 2 ′ -fluoro-pyrimidine analogs (2 ′ F-dYTPs) in particular, because of the ease of chemical synthesis of both triphosphates and oligonucleotides. DNA aptamer selection using the analogous purines (2 ′ F-dRTPs) has recently been shown (Thirunavukarasu et al 2017), and polymerases capable of using all four 2 ′ F analogs (2 ′ F-dNTPs) have been developed (Cozens et al 2012), thus aptamer selections at full-2 ′ F substitution should now be feasible.…”

“…Other modifications that have been explored in mod-SELEX include 4 ′ -thioribose pyrimidines (4 ′ S-YTPs), which conferred a 50-fold increase in resistance to RNase A degradation. Locked or bridged nucleic acids (LNA/ 2 ′ 4 ′ BNA), in which the 2 ′ O and 4 ′ C of the deoxyribose ring are linked by a (oxy)methylene bridge, are known to be even more resistant to nuclease degradation, and confer exceptional duplex stability (Dellafiore et al 2016;Lipi et al 2016). So far, only one instance has been reported in which an LNA TTP replaced dTTP in the randomized region of a DNA library for aptamer selection (Kasahara et al 2013).…”

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

“…The enzymatic synthesis of chemically modified nucleic acids plays an increasingly important role in life science and biotechnology. Polymerase-assisted incorporation of modified nucleotides has attracted substantial attention over the last decade 1 – 3 . As a result, many non-natural nucleotides have been comprehensively studied as polymerase substrates.…”