Dodecamer d-AGATCTAGATCT and a Homologous Hairpin form Triplex in the Presence of Peptide REWERReport as inadecuate

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We have designed a dodecamer d-AGATCTAGATCT RY12 with alternate oligopurines and oligopyrimidines tracts and its homologous 28 bp hairpin oligomer RY28 that forms a triple helix only in the presence of a pentapeptide REWER. An intermolecular triplex is formed by the single strand invasion of the RY28 duplex by RY12 in the presence of REWER. 5′- oligopurine end of RY12 binds to oligopurine sequence of RY28 in a parallel orientation and its oligopyrimidine stretch then changes strand and adopts an antiparallel orientation with the other strand of the duplex. Evidence for the formation of the triplex come from our studies of the UV melting curves, UV mixing curves, gel retardation assay, and chemical sequencing of 1∶1 mixture of dodecamer and hairpin oligonucleotides in the presence and absence of the peptide REWER. RY12 exists as a duplex that melts at 35°C. The hairpin RY28 melts at 68°C. 1∶1 mixture of RY12 and RY28 in the absence of REWER gives a biphasic transition curve with thermodynamic properties corresponding to those of the melting of the duplex of RY12 and the hairpin RY28. However, the melting curve of this mixture is triphasic in the presence of the REWER; the thermodynamic parameters associated with the first phase melting of the duplex of RY12, second phase melting of the triplex and the third phase melting of the hairpin show dependence on the molar ratio of peptide to oligonucleotides. Under appropriate conditions, gel retardation assay showed a shifted band that corresponds to a possible triplex. Chemical sequencing of KMnO4 and DEPC treated mixture of RY12, RY28 and REWER revealed the footprint of triplex.

Author: Amrita Das, Tapas Saha, Faizan Ahmad, Kunal B. Roy, Vikas Rishi



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