Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

Abstract

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site-containing DNA duplex aptamer (AP aptamer) in combination with an abasic site-binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23-mer AP aptamer (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/5'-GTG TGC GTT GCC CTG GAC GCA GA-3'; X: the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM. Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.

Selective Stabilization of Abasic Site‐Containing DNA by Insertion of Sterically Demanding Biaryl Ligands

“…[5] At the same time, selective ligands that carry a spin label [6] or that change their optical properties when bound to the AP site [7] may be used for the sensitive detection of these lesions. Along these lines, several ligands have been shown to bind to AP sites, for example, naphthyridine derivatives, [8] nucleic-base intercalator conjugates, [9,10] aminoalkyl-substituted DNA intercalators, [11,12] DNA intercalators such as proflavine, [13] annelated quinolizinium derivatives, [14] or berberine, [15] cyclic bisacridines, [16] or metalloinsertors with sterically expansive ligands.…”

DNA Triplexes That Bind Several Cofactor Molecules

“…Thisd esign gives ah igh theoretical storaged ensity and requires less synthetic effort than composite linkers. At otal of seven different www.chemeurj.org motifs were studied (11)(12)(13)(14)(15)(16)(17), ranging from 11 that lacks ap urine toehold between the h-bridged binding regionsa nd 16 and 17 with five residues in the centralp urine segment between the two bridged binding sites ( Figure 6). Compound 17 containsastilbened iether bridge [41][42] between the two Watson-Crick pairing segments.…”

“…Dissociation constants for complexes between nucleoside monophosphates and two-or three-strand double helicesp resenting as ite for base pairing ( Figure 1a) are in the millimolar range. [7] When the two neighboring segments that offer stacking interactions to the ligand are connected by linkers, binding pockets result that readily accommodate large ring systems [8][9][10][11][12] or nucleobases, [13] but not nucleotides as guests. However, when as econd base pairing interaction is allowed to takep lace, by leaving ag ap as binding site in the center of at riple helix, dissociation constants for complexesw ithn ucleotidesr each the nanomolar range.…”

Recent Progress in Abasic Site-binding Small Molecules for Detecting Single-base Mutations in DNA

“…The ratiometric signal was indeed effective, and the strategy would be sufficiently flexible for further design of this class of probes to detect all possible mutations. Also, since the naphthyridine (ATMND) moiety can bind to the other non-Watson-Crick base pairing sites in the DNA duplex as demonstrated in our previous work, [63][64][65][66][67][68][69][70][71][72] ATMND-DBD would have a potential for further applications such as gap site binding in a binary fluorometric method, 63,64 detecting mismatches in trinucleotide repeats, 65 and affinity-labeling in aptamer assays or molecular beacons.…”