A Receptor‐Guided Design Strategy for Ligand Identification

Abstract

Biomedical sciences require effective tools to manipulate,d etect, and study biological phenomena. Oligo-(deoxy)nucleotide ligands represent such tools,but the current strategies to generate them are restricted. Their limited availability is insufficient to address the broad range of targets related to biomedical research. Exemplified by targeting the hydrophobic molecule (À)-D 9 -tetrahydrocannabinol (THC), we report areceptor-guided design (RGD) strategy to generate chemically modified oligodeoxynucleotide libraries for the tailored selection of clickmers. Angewandte ChemieCommunications

Dynamic changes in DNA populations revealed by split–combine selection

“…These ndings are in line with previous data, in which varying interaction properties of starting libraries towards the small molecule D 9 -tetrahydrocannabinol were determined when different click-in residues with distinct aromatic and hydrophobic characteristics were employed. 16,19 Aer conducting eight split-combine SELEX cycles, we did two consecutive deconvolution cycles (selection cycles 9 and 10) followed by NGS analysis. NGS revealed a reduction of unique sequences in the DNA libraries obtained from cycles 6 and 8 (Fig.…”

Compatibility of 5-ethynyl-2′F-ANA UTP with in vitro selection for the generation of base-modified, nuclease resistant aptamers

“…The efficiency of 5-ethynyl-2'F-ANA-synthesis might be further improved by using engineered polymerases 31 and the sequencing protocol simplified by application of nanopore sequencing. 63 Work towards these aims as well as application of the click-SELEX protocol 24,64 to identify aptamers featuring high binding affinity and nuclease resistance is currently ongoing.…”

Advances and Challenges in Small‐Molecule DNA Aptamer Isolation, Characterization, and Sensor Development

“…To overcome this problem, the Mayer group utilized ab ase-modified aptamer library containing benzyl-modified deoxyuridine bases. [61] Very hydrophilic targets (e.g., carbohydrates) have also proven difficult. Stojanovic and co-workers remedied this problem by isolating aptamers against these targets bound to organometallic receptors,e ffectively increasing the number of epitopes available for aptamer binding.…”

“…[61,113,114] Alternatively,h igh concentrations of free target molecule can be added, [80,115,116] preferentially eluting library strands that have strong affinity for the free target. Quantification can be achieved by using libraries that have been previously tagged with molecules such as fluorescein [114,117] or 32 P [61] or by performing PCR with chemically labeled primers.I nr are cases where the fluorescent properties of atarget change upon binding to the library, fluorescence enhancement can be used to monitor enrichment of the pool. [118] Alternatively,o ne can perform gel electrophoresis of the unlabeled library and subsequently quantify the library by staining with aD NA-binding dye.…”

“…These affinity chromatography techniques serve as the basis of aw ide array of aptamer affinity characterization methods. [25,29,61,67,170,171] Specifically,t he ligand or aptamer is immobilized onto as olid substrate,s uch as microbeads,a nd then incubated with varying concentrations of its binding partner and washed extensively to remove unbound strands. Thenon-immobilized binding partner retained on the column is then eluted using high temperatures,c haotropic agents,o r the free form of the immobilized partner.Abinding isotherm can be made by plotting the amount of bound partner as afunction of the total amount of binding partner added to the substrate.Intheir inaugural work on aptamers,Ellington and Szostak used asmall-molecule target-immobilized column to characterize the binding affinity and specificity of their aptamers.…”