Unlocking Pb2+ Sensing Potential in a DNA G-Quadruplex via Loop Modification with Fluorescent Chalcone Surrogates.

The ability of guanine (G)-rich DNA to bind toxic lead (Pb2+) ions within a G-quadruplex (GQ) motif is a leading DNA biosensor strategy. A major analytical hurdle for GQ detection of Pb2+ is competitive GQ templating by potassium (K+) ions. We employ the on-strand DNA synthesis of internal fluorescent chalcone surrogates within the 15-mer thrombin binding aptamer (TBA15) to address this challenge. Replacement of thymidine at the 3-position (T3) within TBA15 with an indole-4-hydroxy-indanone (Ind4HI) chalcone strongly decreases K+-GQ stability while enhancing Pb2+-GQ stability to increase Pb2+ binding specificity. The new T3-Ind4HI probe exhibits a 15-fold increase in fluorescence intensity upon binding of Pb2+ by the modified TBA15 and can detect 6.4 nM Pb2+ in the presence of 10 mM K+. Thus, replacement of the T3 residue of TBA15 with the new Ind4HI probe modulates metal ion affinity by native TBA15 to solve the analytical challenge posed by K+ in real water samples for detecting Pb2+ to meet regulatory guidelines by using a GQ biosensor.

Harnessing a 4-Formyl-Aniline Handle to Tune the Stability of a DNA Aptamer-Protein Complex via Fluorescent Surrogates.

Interactions between DNA aptamers and protein targets hold promise for the development of pharmaceuticals and diagnostics. As such, the utilization of fluorescent nucleobase surrogates in studying aptamer-protein interactions is a powerful tool due to their ability to provide site-specific information through turn-on fluorescence. Unfortunately, previously described turn-on probes serving as nucleobase replacements have only been strongly disruptive to the affinity of aptamer-protein interactions. Herein, we present a modified TBA15 aptamer for thrombin containing a fluorescent surrogate that provides site-specific turn-on emission with low nanomolar affinity. The modification, referred to as AnBtz, was substituted at position T3 and provided strong turn-on emission (Irel ≈ 4) and brightness (ε·Φ > 20 000 cm-1 M-1) with an apparent dissociation constant (Kd) of 15 nM to afford a limit of detection (LOD) of 10 nM for thrombin in 20% human serum. The probe was selected through a modular "on-strand" synthesis process that utilized a 4-formyl-aniline (4FA) handle. Using this platform, we were able to enhance the affinity of the final aptamer conjugate by ∼30-fold in comparison with the initial conjugate design. Molecular dynamics simulations provide insight into the structural basis for this phenomenon and highlight the importance of targeting hydrophobic protein binding sites with fluorescent nucleobase surrogates to create new contacts with protein targets.

A modular aldol approach for internal fluorescent molecular rotor chalcone surrogates for DNA biosensing applications.

Fluorescent molecular rotors (FMRs) are critical tools for probing nucleic acid structure and function. Many valuable FMRs have been incorporated into oligonucleotides, although the methods of doing so can be cumbersome. Development of synthetically simple, high yielding modular methods to fine-tune dye performance is crucial to expand the biotechnological applications of oligonucleotides. Herein, we report the utility of 6-hydroxy-indanone (6HI) with a glycol backbone to serve as a handle for on-strand aldehyde capture as a modular aldol approach for site-specific insertion of internal FMR chalcones. Aldol reactions with aromatic aldehydes containing N-donors proceed in high yield to create modified DNA oligonucleotides, which in the duplex match the stability of the fully paired canonical B-form with strong stacking interactions between the planar probe and the flanking base pairs, as evidenced by molecular dynamics (MD) simulations. The FMR chalcones possess remarkable quantum yields (Φfl up to 76%) in duplex DNA, coupled with large Stokes shifts (Δν up to 155 nm), light-up emissions (Irel up to 60-fold) that span the visible region (λem 518-680 nm) with brightness up to 17 480 cm-1 M-1. The library also contains a FRET pair and dual emission probes, suitable for ratiometric sensing. The ease of aldol insertion coupled with the excellent performance of the FMR chalcones permits their future wide-spread use.