GG sequence of DNA and the human telomeric sequence react with cis-diammine-diaquaplatinum at comparable rates

G-quadruplex structures of telomeric sequences are of growing interest because they inhibit telomerase, an enzyme involved in the maintenance of telomere length of cancer cells. As we have shown previously, the antiparallel structure of G-quadruplexes can be cross-linked in vitro by the anti-tumour drug cisplatin. The question arises whether platination of quadruplex structures of human telomeric sequences by cisplatin could be relevant from a biological point of view. Therefore, we have compared the kinetics of reactions of the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), with the human telomeric quadruplex structure, a duplex DNA and a single-stranded DNA containing one specific platination GG site. The ratio between the platination rate constants was obtained using two intramolecular competition experiments: either a construct with a junction between duplex DNA containing a unique GG platination site and the quadruplex structure of the human telomeric sequence AG(3)(T(2)AG(3))(3), or a construct with a junction between duplex DNA and a single strand containing each a unique GG platination site. Those competition experiments allowed us to conclude that the platination of the quadruplex is favoured over that of the GG duplex by a factor of about two whereas the GG duplex is platinated three times faster than the GG single strand.     

Hybridization of complementary and homologous peptide nucleic acid oligomers to a guanine quadruplex-forming RNA

Peptide nucleic acid (PNA) oligomers targeted to guanine quadruplex-forming RNAs can be designed in two different ways. First, complementary cytosine-rich PNAs can hybridize by the formation of Watson-Crick base pairs, resulting in hybrid PNA-RNA duplexes. Second, guanine-rich homologous PNAs can hybridize by the formation of G tetrads, resulting in hybrid PNA-RNA quadruplexes. UV thermal denaturation, circular dichroism, and fluorescence spectroscopy experiments were used to compare these two recognition modes and revealed 1:1 duplex formation for the complementary PNA and 2:1 (PNA2-RNA) quadruplex formation for the homologous PNA. Both hybrids were very stable, and hybridization was observed at low nanomolar concentrations. Hybrid quadruplex formation was equally efficient regardless of the PNA strand polarity, indicating a lack of interaction between the loop nucleobases on the PNA and RNA strands. The implications of this finding on sequence specificity as well as methods to improve affinity are also discussed.     

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.     

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.     

Benzo(h)quinoline derivatives as G-quadruplex binding agents

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K_a in the range of 3 × 10⁵ M^?1 and with a 10-fold selectivity for quadruplex over duplex DNA.     

Strand invasion by mixed base PNAs and a PNA-peptide chimera

Peptide nucleic acid oligomers (PNAs) have a remarkable ability to invade duplex DNA at polypurine–polypyrimidine target sequences. Applications for PNAs in medicine and biotechnology would increase if the rules governing their hybridization to mixed base sequences were also clear. Here we describe hybridization of PNAs to mixed base sequences and demonstrate that simple chemical modifications can enhance recognition. Easily synthesized and readily soluble eight and 10 base PNAs bind to plasmid DNA at an inverted repeat that is likely to form a cruciform structure, providing convenient tags for creating PNA–plasmid complexes. PNAs also bind to mixed base sequences that cannot form cruciforms, suggesting that recognition is a general phenomenon. Rates of strand invasion are temperature dependent and can be enhanced by attaching PNAs to positively charged peptides. Our results support use of PNAs to access the information within duplex DNA and demonstrate that simple chemical modifications can make PNAs even more powerful agents for strand invasion. Simple strategies for enhancing strand invasion should facilitate the use of PNAs: (i) as biophysical probes of double-stranded DNA; (ii) to target promoters to control gene expression; and (iii) to direct sequence-specific mutagenesis.     

Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA₂–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA₂ triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA₂–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA₂–PNA triplex is significantly more stable than a DNA₂–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications.     

Selective recognition of a DNA G-quadruplex by an engineered antibody

Particular guanine rich nucleic acid sequences can fold into stable secondary structures called G-quadruplexes. These structures have been identified in various regions of the genome that include the telomeres, gene promoters and UTR regions, raising the possibility that they may be associated with biological function(s). Computational analysis has predicted that intramolecular G-quadruplex forming sequences are prevalent in the human genome, thus raising the desire to differentially recognize genomic G-quadruplexes. We have employed antibody phage display and competitive selection techniques to generate a single-chain antibody that shows >1000-fold discrimination between G-quadruplex and duplex DNA, and furthermore >100-fold discrimination between two related intramolecular parallel DNA G-quadruplexes. The amino acid sequence composition at the antigen binding site shows conservation within the light and heavy chains of the selected scFvs, suggesting sequence requirements for G-quadruplex recognition. Circular dichroism (CD) spectroscopic data showed that the scFv binds to the prefolded G-quadruplex and does not induce G-quadruplex structure formation. This study demonstrates the strongest discrimination that we are aware of between two intramolecular genomic G-quadruplexes.     

An in silico study of the differential effect of oxidation on two biologically relevant g-quadruplexes: possible implications in oncogene expression

G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential "hot-spots" for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.     

High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element

Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed.     

G-quadruplex formation in human telomeric (TTAGGG)4 sequence with complementary strand in close vicinity under molecularly crowded condition

Chromosomes in vertebrates are protected at both ends by telomere DNA composed of tandem (TTAGGG)_n repeats. DNA replication produces a blunt-ended leading strand telomere and a lagging strand telomere carrying a single-stranded G-rich overhang at its end. The G-rich strand can form G-quadruplex structure in the presence of K⁺ or Na⁺. At present, it is not clear whether quadruplex can form in the double-stranded telomere region where the two complementary strands are constrained in close vicinity and quadruplex formation, if possible, has to compete with the formation of the conventional Watson–Crick duplex. In this work, we studied quadruplex formation in oligonucleotides and double-stranded DNA containing both the G- and C-rich sequences to better mimic the in vivo situation. Under such competitive condition only duplex was observed in dilute solution containing physiological concentration of K⁺. However, quadruplex could preferentially form and dominate over duplex structure under molecular crowding condition created by PEG as a result of significant quadruplex stabilization and duplex destabilization. This observation suggests quadruplex may potentially form or be induced at the blunt end of a telomere, which may present a possible alternative form of structures at telomere ends.     

RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer

Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha-ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha-ras mRNA rests upon the stability of PNA-mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA-RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C.G*C(+) and T.A*T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA-RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA-RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA-RNA complexes using matrix-assisted laser desorption/ionization time-of -flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes.     

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.