Impact of biophysical parameters on the biological assessment of peptide nucleic acids, antisense inhibitors of gene expression

Abstract

Peptide nucleic acids (PNA) are oligodeoxynucleotide (ODN) analogs in which the sugar phosphate backbone of the ODN has been replaced by one derived from units of N-ethylaminoglycine. PNAs recognize DNA and RNA in a sequence specific manner and form complexes that can be characterized by biophysical methods. The binding motif i s context dependent; homopyrimidine PNAs combine with complementary polypurine targets to form stoichiometric 2:l complexes, whereas PNAs containing both purine and pyrimidine bases afford a 1 :I heteroduplex with rnis-match sensitivity comparable to that found in dsDNA. These complexes mediate the antigene and antisense effects of PNAs via the steric blockade of enzyme complexes responsible for DNA transcription, cDNA synthesis, and RNA translation. PNAs, like ODNs, are taken up by cells via endocytosis leading to their entrapment within intracytoplasmic vesicles. Under circumstances where agent delivery i s solved by cell microinjection, PNAs can effect selective inhibition of endogenous and exogenous genes. The impact of biophysical parameters on the biological assessment of PNAs as antisense inhibitors of gene expression i s presented and discussed. Q 1995 Wiley-Liss, Inc.

Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within the gag-pol Transframe Domain

“…A clear fluorescent signal was demonstrated in the majority of cells incubated with PNA at Ն30 M compared to untreated H9 LAI cells ( Fig. 2A to C), albeit the signal was virtually confined to the cytoplasm, as has previously been reported (104) (Fig. 2D).…”

“…by Research Genetics, Inc., Huntsville, Ala. Although all these PNA oligomers were easily dissolved in water, some oligomers, including PNA GAG1 , PNA GAG2 , PNA PR1 , PNA PR2 , PNA RT1 , PNA RT5 , PNA RT8 , and PNA INT3 , formed fine precipitates when added to the culture medium at higher concentrations, as has previously been reported (104). There was no discernible relationship between the precipitate formation and effects on cell growth or HIV-1 production (see below).…”

“…First, in order to determine concentrations of PNAs required to achieve a sufficient cellular delivery in tissue culture, H9 LAI cells were incubated with fluorescein-tagged PNA at 10 to 100 M. We elected to deliver PNA by simply adding it to the culture medium, thus relying on endocytosis (42,104,131) rather than the microinjection technique commonly adopted in the previous studies (10,45,104). After an overnight incubation, H9 LAI cells were examined under a fluorescence microscope.…”

“…Peptide nucleic acid (PNA) (27,45,103,145), initially developed as a reagent for strand invasion of the duplex DNA, is a DNA mimic, consisting of a peptide backbone of N- (2-aminoethyl)glycine units in place of a deoxyribose backbone. Although unmodified PNA has a relatively poor cellular uptake compared to that of ODN (104,144), it has unique molecular characteristics which may enhance its utility as a genome-intervening tool, such as resis-tance to nucleases and proteases (24) and sequence-specific hybridization to DNA or RNA targets using Watson-Crick base pair formation (27) with much higher thermal stability than that of ODN (27,28,51).…”

PNA-Mediated Purification of PCR Amplifiable Human Genomic DNA from Whole Blood

“…The probe contains a biotin group that allows the complex between the PNA and its target nucleic acid to be captured by streptavidin-coated magnetic particles. The biotin group is separated from the first PNA segment by a Lys-Gly di-peptide spacer, the two PNA segments are linked by a hybrid spacer molecule containing two positively charged Lysresidues and two ado linkers [8-amino 3,5-dioxaoctanoic acid (20)], and a further lysine residue is placed at the NH 2 terminus to increase solubility (14). The T m of the complex between the bisPNA T 7 and its A 7 DNA target sequence was calculated from a melting curve as 63.3°C.…”