Expanding the chemistry of DNA for in vitro selection.
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.
Vaught
Jonathan D
JD
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Bock
Chris
C
Carter
Jeff
J
Fitzwater
Tim
T
Otis
Matt
M
Schneider
Dan
D
Rolando
Justin
J
Waugh
Sheela
S
Wilcox
Sheri K
SK
Eaton
Bruce E
BE
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016133:Polymerase Chain Reaction; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D052157:Aptamers, Nucleotide; C505714:TNFRSF9 protein, human; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9