Dimer Formation and Fluorescence Labeling of aPNA

The orthogonally-protected Cys moiety thus serves as a convenient handle that can be used to link aPNA modules together in order to expand its nucleic acid binding capability and/or attach molecular probes. 

As a prelude to our binding studies, the secondary structure of aPNA itself was examined using CD spectroscopy [52]. The first aPNA to be studied was the tail-to-tail bl dimer, [Ac-Cys-Gly-Ser -Asp-Ala-Glu-Ser -Ala-Ala-Lys-Ser -Ala-Ala-Glu-Ser -Ala-Aib-Ala-Ser -Lys-Gly-NH2]2- The far-UV CD spectra of this aPNA in water at 30 °C showed the double minimum at 220 nm (n-n transition) and 206 nm (n-n transition) as well as the maximum at 193 nm (n-n transition), characteristic of a peptide a-hehx. Upon increasing the temperature, the intensity of the minimum at 200 nm decreased indicating a transition from a-helix to random stracture. An isodichroic point at 202 nm was suggestive of a temperature-depen-dent a-helix to random coil transition. The helical content of this T5(bl)-dimer at 20°C in water was estimated to be 26% [40]. 

the CD spectra of the backbone 2 aPNA Ac-Cys -Lys-Ser -Ala-Ala-Lys-Ser -Ala-Ala-Lys-SerhAla-Ala-Lys-Ser -Ala-Ala-Lys-Ser -Gly-Lys-NH2, was measured as a function of pH in phosphate buffer. At pH 7, the secondary structure of this aPNA was largely random coil. However, the a-hehcity of this aPNA increased with the pH until it reached a maximum at pH 11. Analogous pH-depen-dent secondary structure has also been reported for the amphipathic KALA peptide Trp-Glu-Ala-Lys-Leu-Ala-[Lys-Ala-Leu-Ala]2-Lys-His-Leu-Ala-Lys-Ala-Leu-Ala-Lys-Ala-Leue-Lys-Ala-Cys-Glu-Ala-OH [53]. In our case however, maximum a-helicity 

These studies showed thaL in the absence of nucleic acid, the backbone 1 aPNA had significant a-hehcal content at pH 7 whereas the backbone 2 aPNA was largely in a random coil conformation at physiological pH. The latter aPNA did become a-helical at higher pHs in a manner reminiscent of the structurally related amphipathic peptides. 

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