No abstract

In this study, we describe the thermal and optical properties of the thrombin binding aptamer (TBA) that has been modified at syn-G-tetrad positions with fluorescent 8-heteroaryl-2'-deoxyguanosine derivatives consisting of pyrrolyl ((Pyr)dG), furyl ((Fur)dG), thienyl ((Th)dG), benzofuryl ((Bfur)dG), indolyl ((Ind)dG) and benzothienyl ((Bth)dG). Insertion of the modified base into the syn-G5 position of TBA decreases duplex stability, but enhances stability of the antiparallel G-quadruplex (GQ) structure produced by TBA in the presence of K(+) ion and its molecular target, thrombin. The resulting modified TBA (mTBA) oligonucleotides have been employed in duplex → GQ exchange to monitor thrombin binding affinity and rates of GQ formation driven by thrombin binding. Our studies demonstrate that 8-heteroaryl-dG bases can be inserted into syn-G-tetrad positions of TBA without perturbing thrombin binding affinity and that the 8-thienyl-dG ((Th)dG) analog is particularly useful as an emissive probe for monitoring duplex → GQ exchange due to its heightened emissive sensitivity to change in DNA topology compared to the other 8-heteroaryl-dG analogs. The positional impact of a single (Th)dG probe versus multiple (Th)dG incorporation at syn-G sites of TBA highlight an advantage for di-substituted mTBA oligonucleotides for increased emission intensity and rates of duplex → GQ exchange that can be vital for diagnostics through aptamer detection strategies.

Emissive C8-aryl-2'-deoxyguanosines placed within G-tetrads of G-quadruplex structures are useful probes for distinguishing G-quadruplexes from duplex structures using fluorescence spectroscopy. Here, we report the positional impact of C8-furyl-dG ((Fur)dG) on G-quadruplex folding in the human telomere 22-mer oligonucleotide (HTelo22, (d[AG3(T2AG3)3])). The (Fur)dG probe was inserted into four different positions within the three unique G-tetrads of HTelo22, and G-quadruplex folding was monitored by UV-vis thermal denaturation, circular dichroism, and fluorescence spectroscopy. Our studies demonstrate the impacts of C8-aryl-dG adduct formation on G-quadruplex polymorphism in K(+) solution and in the presence of the additives and cosolutes, CH3CN, polyethylene glycol (PEG-600), and N-methyl mesoporphyrin IX (NMM). Our experiments predict that C8-aryl-dG derivatives can serve as useful tools for various in vitro studies aimed at understanding both the implications of DNA adduct formation within G-quadruplex structures and the unique implications imposed by various folding topologies on biological function/recognition.

DNA aptamers are single-stranded oligonucleotides that are generated by an in vitro selection method to bind targets with high affinity and specificity. Understanding molecular recognition by DNA aptamers is of fundamental importance in the development of biosensor applications. The small molecule ochratoxin A (OTA) is a fungal-derived food toxin, and OTA DNA aptamers have been established for the development of rapid detection platforms required for food safety. One such OTA aptamer (OTAA) is a guanine-rich DNA oligonucleotide that folds into an antiparallel G-quadruplex (GQ) upon OTA binding, although structural details of the GQ fold and its interaction with OTA are currently unknown. In the present study, the fluorescent nucleobase analogue, 8-thienyl-2′-deoxyguanosine (ThdG), was inserted into various G sites of OTAA to determine the probe impact on GQ folding and OTA binding affinity. Our results suggest that OTAA contains three lateral (l) loops connecting two stacked G-tetrads with an anticlockwise loop progression to afford a −(lll) GQ topology. The phenolic ring system of OTA undergoes π-stacking interactions with the G-tetrads of OTAA. Our results also demonstrate aptamer sites that can be modified with ThdG to afford a fluorescent light-up signal upon OTA binding.

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.