The human BCL2
gene is one of the most studied oncogenes due to its high importance in apoptosis regulation. Named after B-cell lymphoma 2, in which it was first discovered, the BCL2
gene encodes an apoptosis inhibitor protein. BCL2 protein is localized on the mitochondrial membrane and maintains a delicate balance between programmed cell death and survival. Due to its crucial role in cell fate regulation the BCL2
gene is an important target in anti-cancer treatment at present. Targeting BCL2
gene expression has been approached in various ways, such as interfering with transcription or post-translational regulation. Triplex-forming oligonucleotides (38
), antisense oligonucleotides (39
), miRNAs (miR-15
induce apoptosis by targeting BCL2) inhibiting protein–protein interactions (40
) have been proposed. Some methods showed effectiveness in in vitro
experiments, but issues concerning delivery of oligonucleotides to the target, stability within an intracellular environment, and target specificity are still major obstacles. One of the promising techniques which may overcome classical issues of DNA–RNA oligonucleotide delivery is implementation of PNAs as targeting probes. Their advantages, including charge neutrality (or weak charge due to possible chemical modifications), backbone flexibility, resistance to cellular nucleases and their ability to form base pairs with DNA and RNA, make them potentially promising agents for gene targeting. It has been demonstrated that PNAs can be successfully delivered into cells via conjugation with nuclear localization and cell penetrating peptides, and inhibit expression of target genes (41–44
). Targeting based on structure recognition proposed in this study could significantly increase specificity of PNA probes and reduce off-target effects.
Another issue in gene targeting is the availability of the target sequence for a probe. This especially applies to the targets in chromosomal DNA as it is packed within the chromatin. Secondary DNA structures which ‘protrude’ from chromatin might be more vulnerable to nucleic acid probes. Furthermore, these secondary structures might even indicate or be crucial in active gene expression (45–48
). Of these types of DNA secondary structures, guanine quadruplexes are believed to play an important role in gene regulation; as mentioned earlier, guanine-rich sequences with the potential to form G-quadruplexes are frequently found in promoter regions of many genes, especially in regulatory genes (1–6
In our study, we addressed the question of the possibility of quadruplex targeting and stabilization by short PNAs. Our results showed that when added in equimolar ratio with the PNA probe, the DNA strand effectively displaces PNA from a PNA–DNA complex. This concerns all three central PNAs: cPNA1
(). This observation might explain the previously described (50
) inability of PNAs to invade relaxed plasmid DNA, and requires further studies on PNA structure to achieve more stable PNA–DNA complexes. Our further studies also supported the observation of PNA displacement by DNA strand. We incubated cPNA1
with the original plasmid with BCL2
insert and no band shifts corresponding to PNA–DNA complexes were observed (). However, in our previous studies, chemical probing showed efficient PNA invasion (22
). The possible explanation may be the displacement of PNAs by complementary DNA strand once superhelical stress is relieved after the plasmid is cut by restriction enzymes. Based on these results, we concluded that gel shift assay cannot serve as a good method to study PNA invasion efficiency in plasmid DNA, so we proceeded with chemical probing.
PNA invasion studies enabled us to answer the main question for this study—what is the role of quadruplex formation on PNA invasion? In addition to plasmid with the original BCL2 sequence, we used also a plasmid with mutant sequence so that quadruplex cannot form while the sequence of PNA binding site is preserved (). We used chemical probing to test the opened state of G-strand. It revealed that only plasmid DNA with the original BCL2 sequence allowed the PNA oligomers to invade and bind. This also provides a means of assessing the stability of the G-quadruplex. Among the tested PNAs, positively charged (cPNA1) and neutral (cPNA2) have the highest potential to sequence specifically invade a naturally supercoiled plasmid with the BCL2 sequence. cPNA2 showed higher specificity to the BCL2 sequence compared to cPNA1. cPNA2 invasion was absent completely, as judged by local melting in the plasmid containing the mutant BCL2 insert. However, in the plasmid containing the original BCL2 sequence, we observed strong evidence of local melting within the BCL2 sequence, especially within the PNA binding site. Moreover, according to DMS protection studies, G-runs within the BCL2 sequence tend to form a quadruplex in the case of a combination of cPNA2 and bis-PNA.
There are several studies performed where formation of secondary structures in DNA is related with the increased ability to bind complementary oligonucleotide probes. Similar results were shown by Zhang et al
) where another secondary structure, cruciform, facilitated PNA invasion. Nielsen et al
) showed the role of multiple t-loops to increase the ability of single-stranded DNA to invade plasmid DNA; however, telomeric protein TRF2 was required for effective invasion. Duquette et al.
) showed quadruplex formation in the G-strand of plasmid DNA during transcription while the C-strand was bound to de novo
synthesized RNA along a relatively high length of transcribed DNA (up to 500
bp). Belotserkovskii et al.
) show that Bis-PNAs hybridized to the (GAA/CTT) repeats of the frataxin gene results in varying degrees of transcription blockage, although no secondary DNA structures were necessary for effective DNA duplex invasion or PNA/DNA triplex stability. We have shown in the BCL2
sequence that G-quadruplex forming potential facilitates PNA invasion, while PNAs, once binding to the cytosine-rich complementary strand, promotes quadruplex stabilization. This provides strong evidence for PNA probes that are not only sequence specific, but also quadruplex specific. We also show that zwitterionic cPNA2
maintained binding affinity while improving the sequence and quadruplex specificity by avoiding large, non-specific aggregates. In future studies, we plan to implement these type of PNAs for in vivo
studies on the regulation of the BCL2
promoter through G-quadruplex stabilization.
In conclusion, we show G-quadruplex formation in the guanine-rich promoter region of the human BCL2 gene is a prerequisite for a stable invasion of C-strand binding PNAs. Our results demonstrate a new mode of sequence-specific targeting with short, duplex-forming PNAs as means of stabilizing G-quadruplexes through targeting the complementary C-rich strand. This approach could provide a basis for future applications of gene expression regulation through G-quadruplex stabilization.