PACE REPORT

Towards PNA chemical replicators

Experiments for characterization of PNA template directed PNA-PNA ligation reactions have been performed in aqueous medium according the general scheme Fig. 5a (see also Refs. [65, 66]). In order to allow subsequent implementation of a PNA replication system in a microfluidics environment as well as real time monitoring it was decided to establish a fluorescence readout based on FRET (fluorescence resonance energy transfer). We therefore designed a ligation-replication system as presented in Fig. 5. In order to optimize the system we first tested a system based on the fluorecein-AlexaFluor532 pair (Tables 3 & 4). In this system a weak but clear FRET signal around 550 nm was observed upon duplex formation between the two PNAs 1 & 4 (Fig. 6a), and it was also possible to obtain a fluorescence kinetics trace in a ligation reaction (Fig. 6b).

    

Figure 5: a) template directed PNA-PNA ligation (condensation) scheme b) scheme of the PNA-PNA ligation-replication cycle, starting with one duplex (top) and ending with two (bottom). The strategy is to measure the replicating turnover via FRET signal (bulb symbol) appearing only upon the formation of duplexes; hence the enhancement of the signal after each replication round. Building-block oligomers are asymmetric, kvatromers and hexamers, to ensure lower hybridization tendency of kvatromers and hence lower signal that is not due to ligation. Kvatromers as well as templates have FRET dyes so that duplexes have dyes at the opposite ends since this combination generated the highest FRET signal.

Figure 6: Chemical ligation of PNAs 2 & 3 using PNA 1 as template monitored by fluorescence (FRET) Blue: before EDC addition. Red: After EDC addition. Panel B: Time kinetics after EDC addition.

However, control experiments showed that the kinetics reflected that EDC activation of the PNA was the rate limiting step rather than the ligation per se. In view of this we decided to explore the Cy3/Cy5 FRET pair which has proven more effective in a DNA hybridization context (ref). However, for synthetic reasons we resorted to the analogous AlexaFluor pair AF555/AF647 pair, which have excitation/emission properties (Table 4, Fig. 7) almost identical to those of the Cy3/Cy5 pair. Using this pair in a PNA context we could obtain a FRET signal at 665 nm of more than 5 fold (Fig. 8). A small series of PNA pairs were synthesized having the two fluorophores at different positions in the duplex (same end, opposite ends or end and middle), and from these studies it was concluded the same end system resulted in quenching and no FRET signal whereas the end-middle system gave the highest FRET signal. As the latter unfortunately is very poorly compatible with a ligation reaction and leaves little freedom as to changes in the relative lengths of the ligation, we decide to rely on an end-end system (Table 5). Thus this entire set of PNAs were synthesized and the two individual EDC activated ligation reactions (PNAs 5, 6 & 7 and PNAs 8, 15 & 16) were studied by HPLC, MALDI-TOF mass spectrometry and fluorescence spectroscopy.

 
Tables 3, 4 and 5

Figures 7) Absorption and emission spectra of the AlexaFluor and Cy5 dyes. 8) FRET signal of a PNA duplex-AlexFluor AF555/AF647 pair. Emission of single stranded AF647-PNA (PNA5) (blue spectrum) and PNA555-PNA647 duplex (PNAs 8 & 5) (red spectrum) excited at the AF555 absorption wavelength (555nm).

A typical HPLC trace for the PNA 5, 6, 7 ligation reaction is presented in Fig. 9a. In the absence of EDC, only the template (PNA5) and the two short precursors (PNAs 6 &7) are detected (Fig. 9b). After addition of EDC a new peak appears (no 4) and the relative intensity of two of the peaks (nos 1 & 2) decreases, indicating that the ligation product, 4, has formed at the expense of two precursors (1 & 2): This interpretation was confirmed by MALDI-TOF mass spectrometric analysis (Fig. 9c) of all four peaks, identifying these as the four PNAs (7, 6, 5 & 8, respectively). Also the UV spectra of the individual peaks in terms of the attached fluorophores was consistent with this assignment. A similar analysis of the other half (PNAs 8, 15, 16 & 5) yielded analogous results (Fig. 10), thereby establishing both halves of the FRET PNA replicator.

Figure 9: HPLC and MALDI-TOF mass spectrometric analysis of chemical ligation of the PNA5-6-7 system (Table 5). Panel B: ligation mixture before addition of EDC. Panel A: after EDC addition. The identity of all peaks were verified by MALDI, e.g. panel C for the product peak no 4.

Figure 10: HPLC and MALDI-TOF mass spectrometric analysis of chemical ligation of the PNA8-15-16 system (Table 7). Panel B: ligation mixture before addition of EDC. Panel A: after EDC addition. The identity of all peaks were verified by MALDI.