Expanding the Chemistry of DNA for in Vitro Selection

Abstract

Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.

High-content affinity-based proteomics: unlocking protein biomarker discovery

“…As expected, the contact between the protein and SOMAmer is extensive and contains elements never seen before in either classic aptamer-protein structures [80,81] or structures between natural RNAs and their binding partners [82]. The SOMAmer was identified from a singlestranded DNA library, where 5-benzyl-dUMP was substituted for every 'T' [79]. We found interactions between benzyl groups and amino acids, benzyl groups stacking on other nucleotides, and a remarkable compact hydrophobic 'turn' with benzyl groups at its core.…”

“…Dissociation rates for most aptamers with plasma proteins are fast, with half-lives of a few seconds -we reasoned that we simply had to select slow dissociation rates in the SELEX High-content affinity-based proteomics process itself. Simultaneously, we realized that novel nucleotide adducts, with new functionalities at the 5-positions on pyrimidines [78,79], would provide chemistry missing in classic aptamers and might yield SOMAmers that held on to their cognate partners through extra binding interactions [66]. The on-rates are slower than diffusion-limited rates, but the off-rates are also slow (half-lives of 30 min to several hours) [66,79].…”

Aptamers and the RNA World, Past and Present

“…Modified pyrimidines have played an enormous role in the successful enhancement of aptamers. Bruce Eaton's original five-position modifications (Dewey et al 1995) have been expanded to include both amino acid side-chain adducts (Vaught et al 2004;Vaught et al 2010) and more recently adducts that resemble "fragment-based pharmacophores" from the drug industry (Congreve et al 2008;J. Rohloff, personal communication).…”

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

“…In addition to their role in these fundamental biological transformations, their modified counterparts have advanced as a versatile and mild platform for the introduction of functional groups into oligonucleotides, a methodology that nicely complements the automated solid-phase synthesis that is usually applied 1,2 . Indeed, provided the (d)NTPs can act as substrates for RNA and DNA polymerases 3 , a wealth of functional groups including amino acids [4][5][6][7][8][9][10][11][12][13] , boronic acids 14,15 , nornbornene 16 , diamondoid-like residues 17 , side-chains for organocatalysis 18 , bile acids 19 , and even oligonucleotides 20 can be introduced into oligonucleotides.…”

“…Beyond representing a convenient vector for the functionalization of nucleic acids, modified dNTPs can be engaged in SELEX and other related combinatorial methods of in vitro selection for the generation of modified catalytic nucleic acids [21][22][23][24][25][26][27][28][29][30] and aptamers for various practical applications 10,[31][32][33][34][35][36] . The additional side-chains that are introduced by the polymerization of the modified dNTPs are thought to increase the chemical space that can be explored during a selection experiment and supplement the rather poor functional arsenal of nucleic acids 37 .…”