Dynamic changes in DNA populations revealed by split–combine selection

Abstract

Clickmers are chemical modified aptamers representing an innovative reagent class for developing binders for biomolecules with great impact on therapeutic and diagnostic applications. To establish a novel layer for screening...

Dependence of click-SELEX performance on the nature and average number of modified nucleotides

“…S1) and were thus not further characterized. This finding is in accordance with recently published results from split-combine selection procedures, 14 in which also In-dU, Bn-dU, and BF-dU as well as ethylamine (Ea-dU) and 4-chloro benzyl (ClBn-dU) were used, leading to clickmers only when using In-dU, BF-dU and Bn-dU. The enriched libraries revealed click-dependent binding ( Fig.…”

“…These data are in line with results obtained from click-SELEX procedures targeting C3-GFP. With exception of clickmer 7EdU(1) these clickmers share a sequence motif (CTTTGAATATGTAG) with the previously identified clickmer I10, 14 indicating a robust and reproducible enrichment process.…”

“…Previously, we employed several azides for CuAAC to modify DNA libraries and subjected these to click-SELEX procedures. Among them, we used indole, benzyl, and other aromatic residues and successfully identified clickmers binding to cycle 3 Green Fluorescent Protein (C3-GFP), 5 streptavidin, 14 CXCL9, 15 and Δ 9 -tetrahydrocannabinol. 16 To more systematically investigate the suitability of individual azide moieties for the click-SELEX procedure, we performed click-SELEX targeting C3-GFP using eight different representative and available azides ( Fig.…”

“…As our study is limited to C3-GFP using other target proteins might reveal a different set of modifications which indeed lead to clickmers. ClBn-dU has been used in a split-combine click-SELEX approach, 14 in which 5 different chemical entities were used simultaneously. In this process, by-product formation was not observed, probably due to the presence of other entities, i.e.…”

“…However, currently, the methodology is limited to one modification per DNA strand but has been shown to be adaptable to multiplex formats, thereby assaying several chemical entities simultaneous. 14,15 Here we describe the suitability of different chemical entities for being used in the click-SELEX procedure, the impact of modifications on PCR performance, and the relation of selection output on the statistical number of modifications per DNA strand in the starting library. Our experiments reveal that the PCR performance is more reliable when using less modifications per DNA sequence.…”