HYBRIDIZATION RESPONSIVE FLUORESCENCE-LABELED PYRROLIDINYL PEPTIDE NUCLEIC ACID PROBES

Abstract

In this study, we developed fluorescence peptide nucleic acid (PNA) probes that can change the fluorescence intensity and emission wavelength upon hybridization with correct DNA targets. Such probes should be useful for DNA sequence analyses. The PNA employed in this work is the pyrrolidinyl PNA with an alternating D-proline/2-aminocyclopentane carboxylic acid backbone (acpcPNA) which shows superior stability, as well as binding affinity and specificity than DNA and commercial PNA. In the first part, internally dual-labeled acpcPNA were synthesized using a pair of orthogonally-protected (Tfa and o-Nosyl) 3-aminopyrrolidine-4-carboxylic acid (APC) that was incorporated into the acpcPNA backbone. It was proposed that the fluorescence of internally dual-labeled acpcPNA should change due to different interactions (quenching or FRET) between the two dyes in the single stranded and DNA-hybridized probes. Unfortunately, the fluorescence change was not very high, and was not in accordance with the design due to the quenching between the FRET pairs in single stranded probes. In the second part, dual end-labeled acpcPNA probes bearing a fluorophore and a quencher with end-stacking ability (anthraquinone) were developed. These single-stranded end-labeled probes exhibited very low fluorescence in single stranded state. In the presence of complementary DNA, the fluorescence of the doubly-end labeled acpcPNA was greatly enhanced (up to 18 folds). The probes exhibit good specificity in discriminating between complementary and both internally and terminally mismatched DNA, which could be improved further by a strand displacement reaction or by increasing the temperature. In the third part, the solvatrochromic dye Nile red which can change its fluorescence according to its environment was incorporated into the internal positions of acpcPNA backbone. The fluorescence of the Nile red-modified acpcPNA was enhanced when hybridized with complementary DNA and DNA with inserted base that can form a bulge. Based on spectroscopic data, the Nile red was proposed to interact with the groove in complementary PNA-DNA hybrid, or the hydrophobic pocket formed by the bulged duplex.