Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

Abstract

In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC50 values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.

Metal-containing peptide nucleic acid conjugates

“…New strategies for bioavailability such as conjugation to lipid domains 94 or oligophosphonates 95 were developed. Another novel and versatile idea is the use of metal complexes to increase cellular uptake.…”

Insights on chiral, backbone modified peptide nucleic acids: Properties and biological activity

“…For this reason, many researchers have focused their attention on developing new monomers to be used in the construction of second generation PNAs lacking the abovementioned issues. [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] Out of the above 1-3, water solubility is the least severe, with the exception of PNA's sequences rich in adenine and guanine. In addition, it should be noted that most of the cellular experiments are carried out at sub-micromolar or nanomolar.…”

“…In addition, it should be noted that most of the cellular experiments are carried out at sub-micromolar or nanomolar. 66 concentrations, at which most of PNAs are soluble. 13,65 Several cellular types, i.e., neurons and astrocytes, were shown to be permeable to PNAs without the use of a delivery system.…”

Antiviral Efficacy of a Short PNA Targeting microRNA-122 Using Galactosylated Cationic Liposome as a Carrier for the Delivery of the PNA-DNA Hybrid to Hepatocytes

“…lipophilic ligands have been suggested. 23 Use of oligonucleotide hybrids or PNA-DNA hybrids have been also attempted, 24 which introduces a negative charge to the uncharged backbone.…”