Pseudo-complementary PNA Research Articles

PNA monomers fully compatible with standard Fmoc-based solid-phase synthesis of pseudocomplementary PNA

Here we report the synthesis of new PNA monomers for pseudocomplementary PNA (pcPNA) that are fully compatible with standard Fmoc chemistry. The thiocarbonyl group of the 2-thiouracil (sU) monomer was protected with the 4-methoxy-2-methybenzyl group (MMPM), while the exocyclic amino groups of diaminopurine (D) were protected with Boc groups. The newly synthesized monomers were incorporated into a 10-mer PNA oligomer using standard Fmoc chemistry for solid-phase synthesis. Oligomerization proceeded smoothly and the HPLC and MALDI-TOF MS analyses indicated that there was no remaining MMPM on the sU nucleobase. The new PNA monomers reported here would facilitate a wide range of applications, such as antigene PNAs and DNA nanotechnologies.

Affinity Isolation of Desired Restriction Fragment from Human Genome Using Double-duplex Invasion of Biotin-bound Pseudo-complementary PNA

A predetermined fragment in the human genome was successfully isolated by affinity separation using biotin–streptavidin interaction. The whole genome from human cells was first digested by restriction enzymes and then treated with a pair of pseudo-complementary peptide nucleic acids (pcPNAs). A biotin was covalently bound to either of the pcPNAs. With the use of streptavidin-coupled resin, the targeted fragment involving the invasion site of the pcPNAs was selectively picked up from the genomic digests in high yields.

A novel pseudo-complementary PNA G-C base pair

Pseudo-complementary oligonucleotide analogues and mimics provide novel opportunities for targeting duplex structures in RNA and DNA. Previously, a pseudo-complementary A-T base pair has been introduced. Towards sequence unrestricted targeting, a pseudo-complementary G-C base pair consisting of the unnatural nucleobases n6-methoxy-2,6-diaminopurine (previously described in a DNA context) and N4-benzoylcytosine is now presented for design of pseudo-complementary PNA oligomers (pcPNAs).

Mismatch recognition in PNA double-duplex invasion

Double-duplex invasion of pseudo-complementary PNA (pcPNA) to double-stranded DNA is promising for recognition of a specific sequence in double-stranded DNA. In order to apply this process for various purposes such as gene suppression, one base-pair change at the target site in DNA must be strictly distinguished by the pcPNA additives. In this study, mismatch-recognizing activity of double-duplex invasion was investigated under various salt conditions. It has been found that the mismatch-recognition of the invasion is strict enough to distinguish one base-pair alternation, as long as the invasion is achieved in the media of appropriate ionic strength (e.g., [NaCl] = 20 mM).

Promotion of PNA double-duplex invasion by glutaric dialdehyde

Invasion of two pseudo-complementary PNA additives (pcPNAs) to target site in double-stranded DNA (double-duplex invasion) was greatly promoted by the addition of glutaric dialdehyde.

Activation of Double-stranded DNA by One pcPNA Strand for Its Site-selective Scission with CeIV/EDTA

Abstract In the presence of only one strand of pseudo-complementary PNA (pcPNA), double-stranded DNA was selectively hydrolyzed by CeIV/EDTA (EDTA = ethylenediamine-N,N,N′,N′-tetraacetate) at target site. Compared with previously reported artificial restriction DNA cutters using two pcPNA strands, the present cutters are simple and promising for various applications.

Efficient Sequence-Directed Psoralen Targeting Using Pseudocomplementary Peptide Nucleic Acids

A pair of decameric pseudocomplementary PNAs which bind to their mixed purine-pyrimidine sequence target in duplex DNA by double duplex invasion has been synthesized with a derivative of 8-methoxypsoralen conjugated to one of the PNAs. It is shown that this pair of psoralen-conjugated pseudocomplementary PNA oligomers, which target a site in the pBluescriptKS+ vector, upon irradiation with long-wavelength UV light (UVA) with high efficiency and specificity form photoadducts to an adjacent 5'-TA site, and more than 50% of these adducts are DNA interstrand cross-links. Transcription elongation by T7 or T3 RNA polymerase is specifically arrested at the psoralen cross-linking site, yielding more than 90% arrested product. These results emphasize the potential of pseudocomplementary PNA oligomers for highly specific gene targeting, in particular, with respect to sequence-directed psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.

Site-specific Scission of Lambda Phage Genomic DNA by Ce(IV)/EDTA-based Artificial Restriction DNA Cutter

Abstract Lambda phage genomic DNA (48502 base pairs) was site-selectively hydrolyzed by artificial restriction DNA cutter (ARCUT), which we have developed by combining Ce(IV)/EDTA and pseudo-complementary PNA. PNA satisfactorily invaded lambda DNA and activated the target site for the scission by Ce(IV)/EDTA.

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. -Comparison with the site-selective hydrolysis using Ce(IV)/EDTAY-

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

Manipulation of double-stranded DNA by artificial restriction enzyme composed of Ce(IV)/EDTA and PNA

Through the invasion of pseudo-complementary PNA (pePNA) to double-stranded DNA, gap-like structures were formed at predetermined sites in both strands of PBR322 plasmid DNA. These gap-like sites were selectively hydrolyzed by Ce(IV)/EDTA complex, and two designed fragments were obtained. Furthermore, the scission fragment by this artificial restriction enzyme was successfully ligated with foreign DNA.

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.