Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye

Abstract

Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25 equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.

Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids

“…3, black trace). Consistent with our previous results (Hnedzko et al 2016), photobleaching of PNA 3-HF488 under these conditions was relatively slow (Fig. 3, red trace).…”

“…The kinetics of triplex formation was studied using PNA 3 labeled at the amino terminus with HiLyte Fluor 488 dye (PNA 3-HF488, Fig. 1D) that was prepared following our recently developed methodology (Hnedzko et al 2016). The binding kinetics were studied using the stopped-flow method under pseudo-first order conditions where PNA 3-HF488 was mixed with 10 equivalents of HRP 2 labeled at the 5 ′ -end with Black Hole Quencher 1 (HRP 2-BHQ1) in phosphate buffer (pH 7.4) at 25°C.…”

“…To investigate the role of M modification in cellular internalization of PNA, we used our recently developed methodology (Hnedzko et al 2016) to prepare a set of fluorescently labeled PNA sequences shown in Figure 1D. PNA 1 was conjugated to four L-lysine residues, one lysine at the N terminus and three lysine residues at the C terminus, to evaluate PNA uptake due to lysine residues in the absence of M modifications.…”

Cellular uptake of 2‐aminopyridine‐modified peptide nucleic acids conjugated with cell‐penetrating peptides

“…PNAs were synthesized at 2 μM scale on an Expedite 8909 automated DNA synthesizer using a standard PNA synthesis protocol and were labeled at their N‐termini with HiLyte Fluor 488 fluorescent dye as previously reported by us. [ 35 ] The use of two synthesizers to separately assemble peptide and PNA portions provided best yields and resource economy. All PNAs were analyzed using LCMS (Figures S1‐S12).…”

Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs

“… 1 , 26 The desired label is then introduced at the N terminus by performing an acid–amine coupling reaction on the solid support before the final cleavage cum global deprotection step. 12 , 13 , 27 This approach has been commonly used in preparing fluorescently modified PNA probes for imaging specific nucleic acid sequences in cells. Alternatively, base-modified monomers are incorporated into PNA oligomers at the desired position during solid-phase synthesis.…”