Effect of preorganization on the affinity of synthetic DNA binding motifs for nucleotide ligands

Abstract

Triplexes with a gap in the purine strand have been shown to bind adenosine or guanosine derivatives through a combination of Watson-Crick and Hoogsteen base pairing. Rigidifying the binding site should be advantageous for affinity. Here we report that clamps delimiting the binding site have a modest effect on affinity, while bridging the gap of the purine strand can strongly increase affinity for ATP, cAMP, and FAD. The lowest dissociation constants were measured for two-strand triple helical motifs with a propylene bridge or an abasic nucleoside analog, with Kd values as low as 30 nM for cAMP in the latter case. Taken together, our data suggest that improving preorganization through covalent bridges increases the affinity for nucleotide ligands. But, a bulky bridge may also block one of two alternative binding modes for the adenine base. The results may help to design new receptors, switches, or storage motifs for purine-containing ligands.

Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures

“…We also wished to create larger structures with several binding motifs for complexing adenine‐containing ligands in designed binding pockets, by a combination of Watson–Crick and Hoogsteen base pairing . In the event, we wished to create binding motifs with a high affinity for cofactors as ligands, by employing oligopurine chains as the middle strand of the triplex that feature an abasic site analog at the position where the adenine was to be bound by base pairing . The large assemblies to be created can bind several cofactors simultaneously and can more readily be retained between porous bar‐ riers of devices than individual triplex motifs.…”

“…[25][26][27] In the event, we wished to create binding motifs with ah igh affinity for cofactors as ligands, by employing oligopurine chains as the middle strand of the triplex that feature an abasic site analog at the position where the adeninew as to be bound by base pairing. [28,29] The large assemblies to be created can bind several cofactors simultaneously and can more readily be retained between porous barriers of devices than individual triplex motifs.…”

DNA Triplexes That Bind Several Cofactor Molecules

“…Among them, f is the more rigid building block, which wasexpectedt of avor tight binding. [16] Strands 1p and 1f featured three of the shorter linkers in ar ow to yield enlarged binding sites. The corresponding strand with ah exaethylene glycol linker 1h had been studied previously in adifferent context [24] and wasincluded in the current assays, but it was not synthesized again for the current work.…”

“…But, as long as the number of single nucleotide binding sites is reasonable, high affinity for each of Figure 6. Apparentdissociation constants( K dapparent )for the complexes of FADa nd intramolecular tripleh elicalbinding motifs differing in the length of the inner purine segment (toehold) between two hexaethyleneg lycol-bridged binding sites (11)(12)(13)(14)(15)(16), as well as amotif with ad uplex-stabilizing stilbene diether bridge (17). Conditions:5mM bindingm otif,30mMF AD in phosphate buffer (10 mM,pH= 6), 1 M NaCl; centrifugation at 4 8Ct hrough filter units with am olecularw eight cut-off of 3kDa.…”

“…We wished to find out how many gaps in ag iven triplex will be toleratedb efore the triple helical structure breaks down,t hus exploring the limits of nucleotide binding in triplexes. We chose to do so by synthesizingo ligonucleotides with bridgesb etween purine segments [16] and measuring their ability to bind ATP, cAMP,o rF AD as representative examples of nucleotide-based biomolecules that act as energy source, [32][33][34] second messenger, [35] or redoxc ofactors. [36] Here we report the resultso fasystematic studyo nt he ability of oligonucleotide triplexes to accommodate severale quivalents of purine ligandsi nd esigned bindings ites.…”