Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids

Abstract

The so-called acpcPNA system bears a peptide backbone consisting of 4'-substituted proline units with (2'R,4'R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue : yellow or blue : red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications.

Preparation and Performance Evaluation of a Pyrrolidinyl Peptide Nucleic‐Acid‐Based Displacement Probe as a DNA Sensor

“…To resolve the slow kinetics issue, brief heating was again applied and found to expectedly promote efficient strand exchange, leading to almost complete fluorescence restoration and successful discrimination between the complementary DNA ( comp–dsDNA1 ) and a single mismatched DNA ( sm–dsDNA1 ) (Figure for K4Flu1 / dSmQ1 , and Figure S16 and S17 for K1Flu1 / dSmQ1 and K4TMR1 / dSmQ1 , respectively). These results, along with our recently published study, corroborated the power of acpcPNA in undergoing duplex invasion, thus suggesting that the displacement probe should be well applicable to double stranded DNA targets.…”

Recognition of mixed-sequence DNA using double-stranded probes with interstrand zipper arrangements of O2′-triphenylene- and coronene-functionalized RNA monomers

“…Examples of major advances toward this end include triplex forming oligonucleotides (TFOs), 1 peptide nucleic acids (PNAs), 2,3 and minor groove binding polyamides, 4–6 which were followed by optimized TFOs and PNAs that can be applied across a broader range of targets. 7–16 More recently, engineered proteins such as CRISPR/Cas have gained widespread popularity, 17 despite concerns regarding inadequate binding specificity and cellular delivery. 18 Many DNA targeting applications will benefit from simpler hybridization-based probes that enable rapid, potent, and specific recognition of mixed-sequence dsDNA under physiologic conditions.…”

Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes

“…Consequentially, significant efforts have been devoted to develop alternative approaches, which has resulted in TFOs and PNAs with reduced target site limitations. 6 − 11 More recently, engineered proteins 12 , 13 such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and—in particular—CRISPR/Cas9 systems, 14 have gained a tremendous amount of attention, despite mounting concerns regarding recognition specificity and cellular delivery. 15 Another class of compounds that has emerged from these efforts are the so-called pseudocomplementary DNA and PNA (pcDNA/pcPNA), 16 − 18 in which a short DNA or PNA duplex is modified to contain pseudocomplementary base pairs between 2-thiothymine and 2,6-diaminopurine ( Figure 1 a).…”