Chapter 10
Delivery of Peptide Nucleic Acids Using an Argininocalix[4]
arene as Vector
Alessia Finotti, Jessica Gasparello, Alessandro Casnati, Roberto Corradini,
Roberto Gambari, and Francesco Sansone
Abstract
The importance of peptide nucleic acids (PNAs) for alteration of gene expression is nowadays firmly
established. PNAs are characterized by a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine
units and have been found to be excellent candidates for antisense and antigene therapies. Recently, PNAs
have been demonstrated to alter the action of microRNAs and thus can be considered very important tools
for miRNA therapeutics. In fact, the pharmacological modulation of microRNA activity appears to be a very
interesting approach in the development of new types of drugs. Among the limits of PNAs in applied
molecular biology, the delivery to target cells and tissues is of key importance. The aim of this chapter is to
describe methods for the efficient delivery of unmodified PNAs designed to target microRNAs involved in
cancer, using as model system miR-221-3p and human glioma cells as in vitro experimental cellular system.
The methods employed to deliver PNAs targeting miR-221-3p here presented are based on a macrocyclic
multivalent tetraargininocalix[4]arene used as non-covalent vector for anti-miR-221-3p PNAs. High
delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity, and easy preparation
makes this vector a candidate for a universal delivery system for this class of nucleic acid analogs.
Key words Peptide nucleic acids, Delivery, Cell transfection, Calixarenes, Non-viral vectors, Calixarene amphiphiles, MicroRNAs, miRNA therapeutics
Abbreviations
3
0 UTR
3
0 -Untranslated region
DCM
Dichloromethane
DMF
Dimethylformamide
DMSO
Dimethyl sulfoxide
FBS
Fetal bovine serum
Fl
Fluorescein
MiRNA/miR MicroRNA
PBS
Phosphate-buffered saline
PNA
Peptide nucleic acid
RT-qPCR
Reverse transcription-quantitative polymerase chain reaction
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
123
Delivery of Peptide Nucleic Acids Using an Argininocalix[4]
arene as Vector
Alessia Finotti, Jessica Gasparello, Alessandro Casnati, Roberto Corradini,
Roberto Gambari, and Francesco Sansone
Abstract
The importance of peptide nucleic acids (PNAs) for alteration of gene expression is nowadays firmly
established. PNAs are characterized by a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine
units and have been found to be excellent candidates for antisense and antigene therapies. Recently, PNAs
have been demonstrated to alter the action of microRNAs and thus can be considered very important tools
for miRNA therapeutics. In fact, the pharmacological modulation of microRNA activity appears to be a very
interesting approach in the development of new types of drugs. Among the limits of PNAs in applied
molecular biology, the delivery to target cells and tissues is of key importance. The aim of this chapter is to
describe methods for the efficient delivery of unmodified PNAs designed to target microRNAs involved in
cancer, using as model system miR-221-3p and human glioma cells as in vitro experimental cellular system.
The methods employed to deliver PNAs targeting miR-221-3p here presented are based on a macrocyclic
multivalent tetraargininocalix[4]arene used as non-covalent vector for anti-miR-221-3p PNAs. High
delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity, and easy preparation
makes this vector a candidate for a universal delivery system for this class of nucleic acid analogs.
Key words Peptide nucleic acids, Delivery, Cell transfection, Calixarenes, Non-viral vectors, Calixarene amphiphiles, MicroRNAs, miRNA therapeutics
Abbreviations
3
0 UTR
3
0 -Untranslated region
DCM
Dichloromethane
DMF
Dimethylformamide
DMSO
Dimethyl sulfoxide
FBS
Fetal bovine serum
Fl
Fluorescein
MiRNA/miR MicroRNA
PBS
Phosphate-buffered saline
PNA
Peptide nucleic acid
RT-qPCR
Reverse transcription-quantitative polymerase chain reaction
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
123