Peptide Nucleic Acid-Functionalized Adenoviral Vectors Targeting G-Quadruplexes in the P1 Promoter of Bcl-2 Proto-Oncogene: A New Tool for Gene Modulation in Anticancer Therapy

Abstract

The B-cell lymphoma 2 (Bcl-2) gene encodes for an antiapoptotic protein associated with the onset of many human tumors. Several oligonucleotides (ONs) and ON analogues are under study as potential tools to counteract the Bcl-2 expression. Among these are Peptide Nucleic Acids (PNAs). The absence of charges on PNA backbones allows the formation of PNA/DNA complexes provided with higher stability than the corresponding natural DNA/DNA counterparts. To date, the use of PNAs in antigene or antisense strategies is strongly limited by their inability to efficiently cross the cellular membranes. With the aim of downregulating the expression of Bcl-2, we propose here a novel antigene approach which uses oncolytic adenoviral vectors (OAds) as a new cancer cell-targeted PNA delivery system. The ability of oncolytic Ad5D24 vectors to selectively infect and kill cancer cells was exploited to transfect with high efficiency and selectivity a short cytosine-rich PNA complementary to the longest loop of the main G-quadruplex formed by the 23-base-long bcl2midG4 sequence located 52–30 bp upstream of the P1 promoter of Bcl-2 gene. Physico-chemical and biological investigations confirmed the ability of the PNA-conjugated Ad5D24 vectors to load and transfect their PNA cargo into human A549 and MDA-MB-436 cancer cell lines, as well as the synergistic (OAd+PNA) cytotoxic effect against the same cell lines. This approach holds promise for safer chemotherapy because of reduced toxicity to healthy tissues and organs.

PNA-based Graphene Oxide/porous Silicon Hybrid Biosensor: Towards a Label-free Optical Assay for Brugada Syndrome

“…CD profile of both the DNA-FAM/PNA and DNA/PNA complexes (Figure 2, blue and green line, respectively) showed the typical spectrum of antiparallel heteroduplexes characterized by two CD maxima around 260 nm and 220 nm and two minima around 240 nm and 200 nm, thus confirming the formation of the heteroduplexes [3,5,6,45]. Moreover, a slight reduction of the positive dichroic signals at 269 nm observed for the DNA-FAM/PNA in comparison with DNA/PNA profile could be ascribed to the presence of the FAM chromophore.…”

“…PNA sequence ( Table 1) was synthesized using the 9-fluorenylmethoxycarbonyl (Fmoc) solidphase strategy, following the protocol reported elsewhere [6]. The raw product was purified by semipreparative HPLC analyses by Jasco (Easton, MD, USA) Plus pump equipped with a Jasco UV-2075 Plus UV detector using a 10 × 250 mm C-18 reverse-phase column (particle size 5 µm)(Merck Millipore Billerica, MA, USA) eluted with a linear gradient of CH3CN containing 0.1% (v/v) trifluoroacetic acid (TFA) in H2O containing 0.1% (v/v) TFA (from 0 to 100% in 45 min, flow 1.2 mL/min).…”

“…The neutral charge nature of the amide backbone also enables the PNA hybridization to the target sequence on exposure to a low-salt environment. In this case, the salt positive ions are not necessary for neutralizing the inter-strand repulsion that hampers the duplex formation between two negatively charged ONs [4][5][6][7][8]. Moreover, the PNA is chemically and thermally stable in those environments in which the nucleic acids would undergo a complete degradation [9].…”

Evaluation of an Analogue of the Marine ε-PLL Peptide as a Ligand of G-quadruplex DNA Structures

“…On the other side, linear α,ε-PLLs, easily obtainable by standard solidphase peptide synthesis procedures [15,18,19] and endowed with structural specific nucleic acids binding abilities, do not show any significant degradation after 24 h of incubation in human serum at 37 °C [14]. Among the DNA secondary structures, the G-quadruplex (G4) family is one of the most intriguing and deeply investigated [20][21][22][23]. It has been demonstrated that G4 DNA plays a crucial role in many physiological and disease-related biological mechanisms [24].…”

“…Apart from the ubiquitous potassium or sodium cations, positively-charged polyamines and triethylene tetraamine may contribute to the G4 stability and induce biologically-relevant effects [25,26]. In this context, also the polycationic PLL was evaluated for its impact on the formation of G4 structures by the human telomere in cation-deficient media and showed the interesting ability to convert the telomeric G4 Among the DNA secondary structures, the G-quadruplex (G4) family is one of the most intriguing and deeply investigated [20][21][22][23]. It has been demonstrated that G4 DNA plays a crucial role in many physiological and disease-related biological mechanisms [24].…”