Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation.
Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.
Lundin
Karin E
KE
Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Novum PL 5, Karolinska University Hospital, Huddinge, SE-141 86 Stockholm, Sweden. karin.lundin@crc.ki.se
Hasan
Maroof
M
Moreno
Pedro M
PM
Törnquist
Elisabeth
E
Oprea
Iulian
I
Svahn
Mathias G
MG
Simonson
E Oscar
EO
Smith
C I Edvard
CI
D000818:Animals; D004278:DNA, Superhelical / Q000737:chemistry*; D015316:Genetic Therapy; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry*
D004278:DNA, Superhelical; D020135:Peptide Nucleic Acids