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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010PNA-Peptide Conjugates for Regulation of DNA andRNA G-Quadruplex Structures Depending on aParticular Protease ConcentrationKenji Usui 1,2 , Keita Kobayashi 1 , and Naoki Sugimoto 1,21 Faculty of Frontiers of Innovative Research in Science and Technology (FIRST),Konan University; 2 Frontier Institute for Biomolecular Engineering Research (FIBER),Konan University, Kobe, 6500047, JapanIntroductionIt has been elucidated in the last few decades that some DNA and RNA secondarystructures modulate a variety of cellular events. It is recently suggested that a G-quadruplexstructure, which is one of the secondary structures, also regulates cellular events such astranscription, translation, pre-RNA splicing and telomerase elongation, and these events arerelated to serious diseases and aging [1,2]. Therefore, systems that could control DNA andRNA G-quadruplex structures when needed, would be able to modulate various cellularevents, and as a result the systems could provide biological effects. Although manyG-quadruplex-targeting ligands including phthalocyanine derivatives [3] were proposed sofar, next generation ligands should have more specificity to a particular G-quadruplex andmore functionality including switching, cellular penetrating and organelle-targeting. Fromthis point of view, we attempted to construct a system using alternative small molecules toregulate G-quadruplex structures with an on-off switching module depending on cellularenvironments.Results and DiscussionFirst of all, the small molecule (Lmyc) was designed (Figure 1). The molecules consisted oftwo parts. One part was composed of guanine PNA-rich sequences to induce G-richDNA/RNA sequences to form G-quadruplex structures with the PNAs. PNAs have someadvantages not only that PNAs have enzyme resistance but also that functions such ascell-penetrating or switching can be introduced. In this study, a switching moduledepending on a particular cellular environment was adopted into the other part and weselected a particular protease concentration as the cellular environment. Therefore, thissystem would induce DNA/RNA to form G-quadruplex structure when the protease wouldnot be expressed in cells, and once expressing the protease, these conjugates would bedigested and simultaneously loose the induction ability resulting in collapse of aDNA-PNA quadruplex structure (Figure 2). Calpain I, which is related with seriousdiseases such as Dystrophia and Alzheimer’s disease, was chosen as a model particularprotease. Thus a calpain I substrate sequence was put into the center of the small moleculeas the switching module.After synthesis of the peptides by Fmoc chemistry [4], we checked the G-quadruplexstructure induction by far-UV CD spectroscopy using a model DNA sequence halfTG(Figure 1), which was a part of c-Myc promoter. The DNA with the PNA peptide showed asimilar spectrum including a positive maximum in ellipticities at 260 nm to otherPNA-DNA parallel G-quadruplexes as previously described [5]. CD melting curves of thetarget DNA with/without the conjugate showed that Tm of the PNA-DNA structure at 260nm was ca. 10 ºC higher than that of the DNA alone. This implied that a G-rich DNA couldbe induced to form PNA-DNA G-quadruplex by the PNA peptide.Also we attempted to demonstrate the switch function depending on the proteaseFig. 1. Sequences of the PNA peptide and the target DNA.608