T7 RNA Polymerase Transcription with 5-Position Modified UTP Derivatives

Abstract

Seven UTP derivatives modified at the 5-position through an amide linkage were tested as substrates for T7 RNA polymerase (T7 RNAP) transcription. All UTP derivatives gave good yields of full-length transcript even from DNA templates that showed a significant number of abortive transcripts using unmodified UTP. A kinetic assay to determine the relative K(m) and V(max) for T7 RNAP transcription gave surprisingly similar values for UTP and the 5-position hydrophobic modifications phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl. The 5-position modifications imidazole and amino, which could both be positively charged, gave K(m) values significantly higher than UTP. All seven UTP derivatives gave relative V(max) values similar to UTP, indicating that insertion of these modified bases into the transcript did not impede its elongation.

Slow Off-Rate Modified Aptamer Arrays for Biomarker Discovery and Diagnostic Applications

“…Simultaneously, we realized that novel nucleotide adducts, with new functionalities at the five positions on pyrimidines, as shown in Fig. 8.1 (Vaught et al 2004(Vaught et al , 2010, would provide chemistry missing in classic aptamers and might yield SOMAmers that bound their cognate partners through extra binding interactions (Gold et al 2010a;Zichi et al 2008). The onrates are typically slower than diffusion limited, but the off-rates are also slow (halflives >30 min) (Gold et al 2010a).…”

“…The result of the SOMAscan assay is a mixture of SOMAmers that quantitatively reflects protein concentrations in the original sample. The modified nucleotides in SOMAmers maintain canonical base pairing (Vaught et al 2004(Vaught et al , 2010 and hybridize effectively to unmodified DNA oligonucleotides on the array. We currently use custom DNA microarrays manufactured by Agilent Technologies.…”

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).…”

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

“…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].…”