Inducing and modulating anisotropic DNA bends by pseudocomplementary peptide nucleic acids

Abstract

DNA bending is significant for various DNA functions in the cell. Here, we demonstrate that pseudocomplementary peptide nucleic acids (pcPNAs) represent a class of versatile, sequence-specific DNA-bending agents. The occurrence of anisotropic DNA bends induced by pcPNAs is shown by gel electrophoretic phasing analysis. The magnitude of DNA bending is determined by circular permutation assay and by electron microscopy, with good agreement of calculated mean values between both methods. Binding of a pair of 10-meric pcPNAs to its target DNA sequence results in moderate DNA bending with a mean value of 40 -45°, while binding of one self-pc 8-mer PNA to target DNA yields a somewhat larger average value of the induced DNA bend. Both bends are found to be in phase when the pcPNA target sites are separated by distances of half-integer numbers of helical turns of regular duplex DNA, resulting in an enhanced DNA bend with an average value in the range of 80 -90°. The occurrence of such a sharp bend within the DNA double helix is confirmed and exploited through efficient formation of 170-bp-long DNA minicircles by means of dimerization of two bent DNA fragments. The pcPNAs offer two main advantages over previously designed classes of nonnatural DNAbending agents: they have very mild sequence limitations while targeting duplex DNA and they can easily be designed for a chosen target sequence, because their binding obeys the principle of complementarity. We conclude that pcPNAs are promising tools for inducing bends in DNA at virtually any chosen site.I ntrinsic or induced DNA bending plays an important role in various biological processes such as transcription, replication, recombination, DNA packaging, and repair (1-6). It has therefore been recognized that biological functions relying on DNA bending may be influenced by nonnatural DNA-bending agents (7-11). So far, two classes of artificial DNA-bending agents have been investigated: tethered triple-helix-forming oligonucleotides (7-9) and six-zinc-finger peptides (10, 11). In both classes, two DNA-binding domains are connected by a linker and targeted to two separated sites in double-stranded DNA (dsDNA), thus inducing bending at the intervening region. Whereas tethered triplex-forming oligonucleotides can easily be designed, they suffer from severe sequence limitations because they require the presence of two Ն15-bp-long homopurine-homopyrimidine (hypr) tracts, located not far from each other in the target DNA. On the other hand, the design of artificial DNA-binding peptides such as the reported six-zinc-finger peptides is not straightforward and may require a selection process starting with a pool of peptides for any chosen DNA target sequence (12). Thus, a DNA-binding ligand capable of sequence-specific recognition and bending of DNA, which could easily be designed for a chosen DNA target sequence, would be of great interest.Certain types of peptide nucleic acids (PNAs) are known to bind complementary target sites in dsDNA sequence-specifically through invasion of the ...

Stacked–Unstacked Equilibrium at the Nick Site of DNA

“…DNA molecules with points of increased flexibility and/or locations of static bending are known to move slower in PAGE when compared to a straight DNA molecule of the same length. [34][35][36][37] Conformational exchange between the straight (stacked) and kinked (unstacked) states gives rise to a single band on the gel. The fact that the band corresponding to the nicked DNA fragment is no broader than the band corresponding to the intact fragment indicates that the exchange is fast with respect to the equilibration in the gel pores, like it takes place in other cases of fast exchange between different DNA conformations during PAGE.…”

Promotion of Double‐Duplex Invasion of Peptide Nucleic Acids through Conjugation with Nuclear Localization Signal Peptide

“…The mobility of invasion complex is generally less than that of the target DNA itself because of the bend of DNA caused by the invasion complex formation. [47] As shown in Figure S1). However NLS-pcPNAs can form an invasion complex even at low PNA concentrations.…”

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…It is noteworthy that the invasion bends the DNA strand and alters its biological and physicochemical properties at the site. 185 This is highly in contrast with other DNA binders which are mostly accommodated in the grooves of double-stranded DNA without inducing significant structural change there. In the next section (2.1.4), this structural change is used to construct artificial restriction DNA cutters for site-selective scission of double-stranded DNA.…”