Download or read book Discrete Assembly of Synthetic Peptide-DNA Triplex Structures from Polyvalent Melamine-Thymine Bifacial Recognition written by Yingying Zeng and published by . This book was released on 2013 with total page 90 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) haven been known to direct DNA triplex assembly via Hoogsteen or reverse Hoogsteen base-pairing. However, with this conventional approach, target DNA sequences are almost solely homopurines. While the Janus-Wedge targeting concept has also been an elegant expansion of the recognition code for DNA triplex formation, it was largely restricted to preformed duplex assembly. There are fewer synthetic systems to induce triplex structures in single stranded oligonucleotides. With these goals in mind, we have designed and synthesized a 21-residue a-peptide (EM*)10G that simultaneously recognizes two oligodeoxythymidine (dT10) tracts to form triplexes with a peptide-DNA strand ratio of 1:2. The synthetic peptide has a controlled display of 10 melamine rings on lysine side chains (termed as M*), which addresses bifacial thymine-recognition interfaces along the length of the 21-residue peptide. Alternate residues are glutamic acid bearing negative charges to afford aqueous solubility and avoid nonspecific electrostatic binding with DNA. Binding stoichiometry was obtained by UV and fluorescence titration, indicating the formation of ternary peptide-[dT10]2 complex as well as heterodimeric peptide-[dT10C10T10] hairpin structure with triplex stems. Signal change on circular dichroism (CD) provided the direct evidence of DNA conformational change induced by the synthetic peptide. Clean transition trend from DNA to DNA-peptide complex bands based on native electrophoresis further supported discrete peptide-DNA assembly. Monophasic transition with cooperative melting was observed for both triplex and hairpin by UV and fluorescence denaturation. Highly exothermic assembly profiles (-28.5 kcal/mol for triplex and -31.4 kcal/mol for hairpin per peptide-DNA triplet stack) from differential scanning calorimetry (DSC) were comparable to those of DNA-DNA and melamine-cyanuric acid recognition, suggestive of similar driving forces. Binding affinity was quantified using fluorescence quenching and fluorescence anisotropy, yielding apparent dissociation constant (Kd) 4000 nM2 for the triplex and 2.7 nM for the hairpin. Recognition exhibited selectivity for thymine over other nucleobases. Further more, partial and full methylation of melamine rings on the peptide abolished all detectable binding to dT10 tract, supporting the idea that assembly depends on the hydrogen-bonding directed melamine-thymine bifacial recognition rather than nonspecific aggregation. We anticipate that this novel assembly element may present promising artificial regulator to manipulate the structure and function of thymine- or- uracil rich targets.