Molecular models for intrastrand DNA G-quadruplexes

<p>Abstract</p> <p>Background</p> <p>Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G₃X_<it>n</it>1G3X_<it>n</it>2G₃X_<it>n</it>3G₃motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.</p> <p>Results</p> <p>We have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion.</p> <p>Conclusion</p> <p>Molecular models have been generated for a large number of G₃X_<it>n</it>1G₃X_<it>n</it>2G3X_<it>n</it>3G₃sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.</p

Conjugated PDT drug: photosensitizing activity and tissue distribution of PEGylated pheophorbide a.

The design of new photosensitizers with enhanced phototoxicity and pharmacokinetic properties remains a central challenge for cancer photodynamic therapy (PDT). In this study, Pheophorbide a (Pba) has been pegylated to methoxypolyethylene glycol (mPE G-Pba) to produce a soluble photosensitizer that exhibits a higher tissue distribution than free Pba. In vitro studies have shown that mPE G-Pba promotes a fairly strong photosensitizing effect in cancer cells, as previously observed for the unpegylated molecule. mPE G-Pba targets the mitochondria where, following photoactivation, ROS are produced which cause a cellular injury by lipid peroxidation. The effect of pegylation on the photosensitizer biodistribution has been examined in different selected organs of female mice, at different time points after intraperitoneal administration of the drug (50 μmol/Kg body weight). Other than free Pba, which showed a low tissue accumulation, mPE G-Pba has been detected in significant amounts (8 to 16 μg/ml) in liver, spleen, duodenum and kidney and, 3-5 hours after intraperitoneal injection, in moderate amounts (3 to 8 μg/ml) in brain and lung. In vivo optical imaging performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Our results indicate that although pegylation somewhat decreases the phototoxicity, it significantly increases the drug solubility and tissue distribution and tumor uptake of mPE G-Pba, making the conjugate an interesting photosensitizer for PDT

Photosensitization of pancreatic cancer cells by cationic alkyl-porphyrins in free form or engrafted into POPC liposomes: The relationship between delivery mode and mechanism of cell death

Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode

Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation

Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA–TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA–TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA–TFOs were found to abrogate the formation of a DNA–protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA–TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome

Protein hnRNP A1 and its derivative Up1 unfold quadruplex DNA in the human KRAS promoter: implications for transcription

The promoter of the human KRAS proto-oncogene contains a structurally polymorphic nuclease hypersensitive element (NHE) whose purine strand forms a parallel G-quadruplex structure (called 32R). In a previous work we reported that quadruplex 32R is recognized by three nuclear proteins: PARP-1, Ku70 and hnRNP A1. In this study we describe the interaction of recombinant hnRNP A1 (A1) and its derivative Up1 with the KRAS G-quadruplex. Mobility-shift experiments show that A1/Up1 binds specifically, and also with a high affinity, to quadruplex 32R, while CD demonstrates that the proteins strongly reduce the intensity of the 260 nm-ellipticity—the hallmark for parallel G4-DNA—and unfold the G-quadruplex. Fluorescence resonance energy transfer melting experiments reveal that A1/Up1 completely abrogates the cooperative quadruplex-to-ssDNA transition that characterizes the KRAS quadruplex and facilitates the association between quadruplex 32R and its complementary polypyrimidine strand. When quadruplex 32R is stabilized by TMPyP4, A1/Up1 brings about only a partial destabilization of the G4-DNA structure. The possible role played by hnRNP A1 in the mechanism of KRAS transcription is discussed

Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins

The human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with Tm of ∼55°C (Q1) and ∼72°C (Q2). DMS-footprinting and CD suggest that Q1 can be a parallel and Q2 a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA–protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS

Characterization of the DNA triplex formed by d(TGGGTGGGTGGTTGGGTGGG) and a critical R · Y sequence located in the promoter of the murine Ki-ras proto-oncogene

AbstractThe binding of the G-rich oligonucleotide d(TGGGTGGGTGGTTGGGTGGG) to a critical homopurine-homopyrimidine sequence located in the promoter of the murine Ki-ras proto-oncogene has been investigated. The duplex and the oligonucleotide form a triple helix as evidenced by band-shift electrophoresis, hydroxyapatite (HA) chromatography, UV-melting and circular dichroism (CD) experiments. Upon thermal denaturation in 50 mM Tris-acetate, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 0.1 mM spermine the triplex exhibits two cooperative transitions: one of these is attributed to the triplex-to-duplex transformation, the other to the duplex-to-coil transformation. The thermodynamic parameters of triplex formation have been determined by a van't Hoff analysis of the UV-melting curves which provided values of ΔH = 79 ± 8 kcal/mol, δS = 224 22 e.u., ΔG298 = 12.2 ± 1.2 kcal/mol. These data are compared with those reported for the YRY triplex motif

G4 DNA in ras genes and its potential in cancer therapy

It is now well established that in the human genome the canonical double helix coexists with folded G-quadruplex structures that are known to have important biological functions. In this review we summarize the current knowledge on quadruplex formation in the promoters of the ras genes that are mutated in about 30% of all human cancers. We describe the nuclear proteins that recognize these unusual DNA structures and discuss their function in transcription. We also examine the formation of G-quadruplexes in the 5'-untranslated region of the ras transcripts and conclude this review by reporting strategies that use either ras G-quadruplexes or proteins recognizing the ras G-quadruplexes as targets of anticancer small molecules

G4 DNA in ras genes and its potential in cancer therapy

It is now well established that in the human genome the canonical double helix coexists with folded G-quadruplex structures that are known to have important biological functions. In this review we summarize the current knowledge on quadruplex formation in the promoters of the ras genes that are mutated in about 30% of all human cancers. We describe the nuclear proteins that recognize these unusual DNA structures and discuss their function in transcription. We also examine the formation of G-quadruplexes in the 5'-untranslated region of the ras transcripts and conclude this review by reporting strategies that use either ras G-quadruplexes or proteins recognizing the ras G-quadruplexes as targets of anticancer small molecules