Zn(II) Complexes and their Recognition of Non-Canonical Thymine and Application Towards DNA Switches
Sander, Stephanie A.
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Binding of DNA secondary structures containing non-canonical thymine to Zn(II) complexes was studied. Metal complexes produce unique binding modes for nucleic acid recognition. Specifically, the Zn 2+ metal ion center provides selective recognition to the Watson-Crick face of the thymine nucleobase, along with the pendent group attached to the 1,4,7,10-tetraazacyclododecane (cyclen) macrocycle which promotes a tight binding interaction, typically through favorable stacking interactions. The dynamic nature of DNA and all of the secondary structures it is capable of forming add a level of difficulty when designing and studying metal complex binding agents. The non-canonical context of the nucleobase of interest can account for the propensity to change conformation. Here we examine interactions with non-canonical thymine in a T-bulge, G-quadruplex and hairpin loop. Each of these structures provides a different number of unpaired thymine nucleobases whose spatial arrangements vary. The T-bulge provides a single extrahelical thymine which is located in the stem of a hairpin loop. A quadruplex from the human telomere (H-telo) was studied and has three non-canonical loops each with two thymine bases present. Finally, the hairpin loop has four unpaired thymine nucleobases which comprise the entirety of the loop portion of the hairpin. Three structurally different Zn(II) complexes have been studied as DNA recognition agents. It has been found that a single ring pendent group attached to a macrocycle does not markedly increase the binding strength of the complex to thymine containing DNA. In contrast, a three ring pendent group provides additional non-specific interactions but loses the specificity of the metal center for the unpaired thymine. For these reasons, our work is carried out using two ring pendents whose linkers to the cyclen macrocycle vary. The Zn(SL) complex contains a non-planar dansyl pendent group appended through a sulfone linker. This complex has shown a preference for the more spatially available non-canonical thymine bases present in G-quadruplex DNA. The Zn(ML) and Zn(DL) complexes are planar and demonstrated similar binding to all three non-canonical secondary structures. The methylene linker of Zn(ML) creates a highly flexible metal complex which does not bind as tightly to unpaired thymines as the rigid, direct linked pendent of Zn(DL). The compact and rigid nature of the pendent group of Zn(DL) allow it to bind tightly to a variety of secondary structures it is able to recognize. The Zn(II) complexes were also studied as promoters of a nucleic acid switch, inducing a duplex to hairpin structural change. The Zn(DL) complex promoted the conformational switch at the lowest ratio of Zn(II) complex to DNA. The switch was found to be reversible and to be selective for the Zn 2+ metal ion over other biologically relevant metal ions such as, Fe 2+ and Cu 2+ . Interestingly, the switch did not work with duplexes whose single strands were unable to form a stable secondary structure (D-4*). The lack of structure in the single-stranded components of DNA allowed for a more favorable hybridization into its duplex form, as shown through ITC studies. The more favorable hybridization of the D-4* duplex creates a more stable and stronger duplex than one containing the same A-T sequence and G-C content whose single strands are able to form hairpin structures (D-4). The switch properties were studied using an additional duplex as a comparison. The new duplex (D-7) has the same stability as measured by its thermal melt temperature (T m ), as D-4* but has single strands which are capable of forming hairpin structures, similar to D-4. Some destabilization occurred with the D-7 duplex, confirming that the ability of the product to form stable hairpins of the switch is of great importance.