Manipulation of a DNA aptamer–protein binding site through arylation of internal guanine residues

Abstract

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

A 5′-BODIPY End-label for Monitoring DNA Duplex-Quadruplex Exchange

“…The modifications decrease the T m of the GQ and increase K d . However, it is clear that the 5′-BODIPY-CN probe developed in this work is far less perturbing than the commercially available 5′-FAM label that strongly decreases GQ stability (Δ T m = −9.5 °C) and thrombin binding affinity ( K d = 4.9 μM) 27 . These changes cannot be ascribed to the nature of the linker, as both dyes are attached to the 5′-end of TBA using a six-carbon tether.…”

“…Formation of the antiparallel GQ by the BODIPY-CN-TBA sample was supported by CD spectral analysis and UV thermal melting at 295 nm. Unmodified TBA displays a characteristic antiparallel GQ CD spectrum with positive peaks at 290 and 240 nm with a negative peak at 260 nm with a thermal melting temperature ( T m ) of ~53.5 °C 27 . The BODIPY-CN-TBA sample afforded a T m of 51.0 °C and provided the characteristic antiparallel GQ CD spectrum (Fig.…”

“…The impact of the BODIPY-CN chromophore on GQ stability and thrombin binding affinity is summarized in Table 1 . For direct comparison to the 5′-BODIPY-CN probe, previous data determined for native TBA 28 , 5′-FAM-labeled TBA 27 and mTBA containing C8-(4-cyanophenyl-vinyl)-dG ( CN dG) at G 6 within the G-tetrad 27 are included. The binding data for native TBA was determined using isothermal titration calorimetry (ITC) and generated an apparent binding constant K b = 3 × 10 6 M −1 , for a dissociation constant K d = 0.33 μM 28 .…”

“…Our results demonstrate the utility of 5′-BODIPY containing cyanophenyl substituents for monitoring duplex-GQ exchange in a quencher-free format. Unlike 5′-FAM 27 , the BODIPY label exhibits minimal impact on GQ stability compared to native TBA 28 and demonstrates equivalent thrombin binding affinity to that determined for a less-bulky, minimally perturbing, internal FBA 27 . Furthermore, the BODIPY end-label is considerably brighter than internal FBAs used previously by our laboratory 15 , 27 and undergoes excitation in the visible region with a reasonably large Stokes shift for aptasensor applications.…”

“…Furthermore, BODIPYs possess very high quantum yields, are often more photostable than fluorescein (FAM) and rhodamine analogs 25 and can readily be functionalized at desired positions to improve photophysical properties 26 and perhaps aptamer performance. This prompted our laboratory to synthesize BODIPY probes to test performance at the 5′-end of TBA in order to make direct comparison to internal FBAs and 5′-FAM 27 . Our results demonstrate the utility of 5′-BODIPY containing cyanophenyl substituents for monitoring duplex-GQ exchange in a quencher-free format.…”