Insertion of CG repeats and 3' terminus overhangs drive B-to-Z transition: A case study with NF-κB bearing DNA...

Z-DNA, a non-canonical helical structure of DNA plays a vital role in various biological processes, including transcription and genomic stability. Though low concentration of trivalent cations is known to induce B-Z transition, the effect of short CG repeats, overhangs sequences, loop length and order of nucleotides on Z-DNA formation in larger DNA is utterly unknown. Earlier, a series of self-assembled branched DNA (bDNA) nanostructures having 5T in the loop are reported to be resistant to B-to-Z DNA transition irrespective of the overhang sequences. Since the presence of alternative purine/pyrimidine sequences and direction of oligonucleotides play a vital role during replication and transcription, we hypothesize that the insertion of a small number of CG repeats, or a change in direction of overhang sequences may influence the B-to-Z DNA transition. Here, we show that Z-DNA formation was induced by inserting CG repeats into bDNA structures that were previously resistant to B-Z transition. Moreover, B-Z transition was also observed when overhangs were introduced at the 3' terminus. The generality of the approach of B-Z transition was demonstrated in a series of bDNA structures including the bDNA having NF-kβ sequences. Different dye binding experiments suggest the formation of Z-DNA in bDNA having overhangs at the 3' terminus against the control of bDNA with 5' overhangs. Interestingly, the melting temperature (Tm) was substantially reduced to 55 °C in the Z-DNA as compared to the LaCl₃-induced condensed DNA having Tm of 77 °C. Fluorescence study also supports the presence of minor groove in Z-DNA which binds Hoechst. ITC indicates an entropy- and enthalpy-driven favorable binding between lanthanide cations and bDNA. Thus, the present study establishes a synthetic bDNA nanotechnology platform for systematically investigating how local sequence architecture, including the insertion of CG repeats, loop length, and overhang orientation influences B-to-Z conformational switching under controlled experimental conditions.