Rigorous aptamer selection sans amplification, solid phase synthesis of oligomeric thioureas, and determining hydrolytic rates of nuclease mimics

A novel recursive in vitro selection method has successfully culled ATP-binding DNA sequences from a pool of about {dollar}7times10sp{lcub}10{rcub}{dollar} different single-stranded DNA molecules. The initial pool, containing 18 random sites per DNA strand, has on average over 17,800 copies of each possible unique sequence. After six rounds of selection using an ATP-agarose column, only one amplification step is required prior to the final selection for the best binding aptamers (DNA or RNA that bind specific targets). Three of these aptamers bind ATP with micromolar dissociation constants, similar to the best ATP binding aptamers reported in the literature. The ATP-binding motif is the same in all three cases and is composed of a putative, two stacked G-quartets with the consensus sequence: AGGGGGAnnnnnnnGGAG. The new strategy can also denote ligands that will be problematic for normal aptamer selection schemes. Two such ligands identified are glucose and 3-keto-2-deoxyoctonate.; Thiourea groups form stronger hydrogen bonds than amide groups. Thus, oligomeric thioureas may have a greater tendency to aggregate and form secondary structures than oligomers built from amides, such as peptides. Herein, both solution and solid phase synthetic procedures that yield oligomeric thioureas are described. Both rely on the coupling of an isothiocyanate with an amine to produce the thiourea linkage. The monomers are derived from simple diamines. Higher yields are achieved using the solid phase method.; Radioactive end labeling can be used to determine the hydrolytic rates of nuclease mimics on moderate to long lengths of RNA or DNA. However, the reliability of end labeling as an assay can vary depending on how well the unincorporated label is removed. Therefore, gel filtration, acid precipitation, membrane diafiltration, and paper chromatography were tested to determine which was the most effective at such separation. The results in order of decreasing contamination by ({dollar}gamma{lcub}-{rcub}sp{lcub}32{rcub}{dollar}) ATP were gel filtration (40%), acid precipitation (5%), diafiltration (2%), and paper chromatography (1%); and, in order of decreasing loss of RNA, were acid precipitation (30%), diafiltration (11%), gel filtration (10%), and paper chromatography (1%).