Pyrene-modified PNAs: Stacking interactions and selective excimer emission in PNA2DNA triplexes

Abstract

Pyrene derivatives can be incorporated into nucleic acid analogs in order to obtain switchable probes or supramolecular architectures. In this paper, peptide nucleic acids (PNAs) containing 1 to 3 1-pyreneacetic acid units (PNA1-6) with a sequence with prevalence of pyrimidine bases, complementary to cystic fibrosis W1282X point mutation were synthesized. These compounds showed sequence-selective switch-on of pyrene excimer emission in the presence of target DNA, due to PNA2DNA triplex formation, with stability depending on the number and positioning of the pyrene units along the chain. An increase in triplex stability and a very high mismatch-selectivity, derived from combined stacking and base-pairing interactions, were found for PNA2, bearing two distant pyrene units.

Keywords: PNA; SNP recognition; modified nucleobase; nucleic acids; pyrene excimer; triplex stabilization.

Figure 1

(a) TAT triplet structure showing Watson–Crick and Hoogsteen base pairing; the binding can be reinforced by the concurrent interaction between two groups protruding from C5 position of thymine into the major groove; (b) pyrene-modified uracil derivative used in PNA monomer in the present study; (c) sequences of PNA and DNA used. T indicates pyrene modified nucleobases; bold letters indicate the position of W1282X point mutation.

Scheme 1

Synthesis of the PNA monomer 1: i) 1. PPh₃, H₂O, THF; 2. TFA, 71%; ii) 1-pyreneacetic acid, HBTU, DIPEA, DMF, 66%; iii) NaOH, H₂O/MeOH (2:1), 91%; iv) EDC·HCl, DIPEA, DhBtOH, DMF, 68%; v) TFA, DCM, 86%.

Figure 2

Fluorescence spectra at 347 nm excitation, recorded at 20 °C of: (a) PNA1, (b) PNA2, (c) PNA3, (d) PNA4, (e) PNA5, (f) PNA6. All measurements were done in PBS buffer, pH 7; concentration of each strand was 1 μM. Full lines are for ssPNA solutions, dotted lines are for PNA:DNA1 solutions and broken lines are for PNA:DNA2 solutions.

Figure 3

(a) Increase in fluorescence intensity of the excimer band for PNA2 upon addition of complementary DNA1 (black diamonds) or mismatched DNA2 (grey squares) at 25 °C); (b) model of interaction showing both base recognition through hydrogen bonding and stacking interactions. X = A for full match, G for mismatch.

Figure 4

Ratio of the intensities of the pyrene excimer (F₄₇₄) and monomer emission (F₃₇₉) for the PNA probes in the absence of DNA (white bars), in the presence of DNA1 (full match, grey bars), and DNA2 (mismatch, black bars). Experiments were done at 20 °C in PBS at 1 μM PNA concentration (0.5 μM DNA concentration).