Selection of a self-cleaving ribozyme activated in a chemically and thermally denaturing environment.

A new self-cleaving ribozyme was obtained from in vitro selection, displaying site-specific cleavage activity under denaturing conditions, such as high temperatures up to 95 °C, and denaturing solvents (20 M formamide). Adding salt such as Mg2+ also inhibited its activity. The conserved ribozyme sequence was found in the genome of several extremophiles, suggesting its potential biological relevance. This study provides an example of a ribozyme working under exotic conditions, which may expand the application of ribozymes in non-biological environments.

Sequence specific hydrogen bond of DNA with denaturants affects its stability: Spectroscopic and simulation studies.

BACKGROUND: Several different small molecules have been used to target the DNA helix in order to treat the diseases caused by its mutation. Guanidinium(Gdm+) and urea based drugs have been used for the diseases related to central nervous system, also as the anti-inflammatory and chemotherapeutic agent. However, the role of Gdm+ and urea in the stabilization/destabilization of DNA is not well understood. METHODS: Spectroscopic techniques along with molecular dynamics (MD) simulation have been performed on different sequences of DNA in the presence of guanidinium chloride (GdmCl) and urea to decode the binding of denaturants with DNA and the role of hydrogen bond with the different regions of DNA in its stability/destability. RESULTS AND CONCLUSION: Our study reveals that, Gdm+ of GdmCl and urea both intrudes into the groove region of DNA along with the interaction with its phosphate backbone. However, interaction of Gdm+ and urea with the nucleobases in the groove region is different. Gdm+ forms the intra-strand hydrogen bond with the central region of the both sequences of DNA whereas inter-strand hydrogen bond along with water assisted hydrogen bond takes place in the case of urea. The intra-strand hydrogen bond formation capability of Gdm+ with the nucleobases in the minor groove of DNA decreases its groove width which probably causes the stabilization of B-DNA in GdmCl. In contrast, the propensity of the formation of inter-strand hydrogen bond of urea with the nucleobases in the groove region of DNA without affecting the groove width destabilizes B-DNA as compared to GdmCl. This study depicts that the opposite effect of GdmCl and urea on the stability is a general property of B-DNA. However, the extent of stabilization/destabilization of DNA in Gdm+ and urea depend on its sequence probably due to the difference in the intra/inter-strand hydrogen bonding with different bases present in both the sequences of DNA. GENERAL SIGNIFICANCE: The information obtained from this study will be useful for the designing of Gdm+ based drug molecule which can target the DNA more specifically and selectively.

The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.

Conformational rearrangements of G-quadruplex topology promoted by Cu(II) 12-MCCu(II)PyrAcHA-4 metallacrown.

Cu(II) 12-MCCu(II)PyrAcHA-4 metallacrown was studied by several spectroscopic techniques as an interacting ligand with G-quadruplex DNA structures. Investigations were performed on oligonucleotides bearing human telomeric and protooncogenic c-myc sequences in buffered solution mimicking ionic conditions in cellular environment. The planar square-based Cu(II) 12-MC-4 metallacrown interacts with GQ via an end-stacking mode with 1:1 stoichiometry. Circular dichroism (CD) titration revealed capability of this metallacrown to induce transformation of the GQ hybrid topology into the parallel form. Thermal melting experiment indicated higher thermal stability of both antiparallel (ΔTm = +15 °C) and parallel (ΔTm = ≥27 °C) G-quadruplexes in the presence of Cu (II) 12-MC-4. Indirect GQ FID assay let to determine high binding affinity of the Cu(II) 12-MC-4 to antiparallel 22Htel/Na+ GQ (KMC = 3.9 (±0.4) x 106 M-1). Comparing with lower binding constants previously reported for Ln (III) 15-MC-5 and Sm (III) 12-MC-4, one can conclude that the square planar geometry and the positive charge of metallacrown play an important role in MC/GQ interactions.

Room temperature synthesis of NIR emitting Ag2S nanoparticles through aqueous route and its influence on structural modulation of DNA.

Near infra-red (NIR) light emitting nanomaterials had shown great promise in clinical imaging in view of negligible absorption by skin or tissue of mammalian. Thus, it demands for synthesizing stable NIR emitting nanomaterials in water environment. The present work presents synthesis of biologically acceptable luminescent near-IR emitting silver sulfide nanoparticles through an aqueous route using 2-mercaptoethanol. The prepared as-synthesized Ag2S nanoparticles exhibited bright photoluminescence with quantum yield of ca. 4%. X-ray diffraction (XRD) analysis indicated that the products were monoclinic α-Ag2S. Fourier transform infrared spectral analysis revealed that the stretching vibration at 2560 cm-1 responsible for S-H bond of thiol group disappeared suggesting the conjugation of 2-mercaptoethanol with Ag2S nanoparticles. In view of investigating any possible effect on genetic materials, interactions of the synthesized particles with calf thymus DNA was investigated employing Ethidium bromide (EB) as structural probe. To understand the binding mechanism, the UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopic, as well as DNA melting studies measurements were carried out. The observed results confirm that nanoparticles interact with DNA through groove binding.

A DNA G-quadruplex/i-motif hybrid.

DNA can form many structures beyond the canonical Watson-Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C-C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.

Molecular recognition of 3+1 hybrid human telomeric G-quadruplex DNA d-[AGGG(TTAGGG)3] by anticancer drugs epirubicin and adriamycin leads to thermal stabilization.

Recent reports suggest influence of anti-cancer anthracyclines on telomere dysfunction and their possible interaction with G-quadruplex (G4) DNA as an alternate pathway to apoptosis. We have investigated interaction of epirubicin and adriamycin with G4 DNA [d-AGGG(TTAGGG)3] comprising human telomeric DNA sequence by surface plasmon resonance, absorption, fluorescence, circular dichroism and thermal denaturation. Epirubicin and adriamycin bind with affinity, Kb, = 2.5×105 and 5.2×105M-1, respectively in monomeric form leading to decrease in absorbance, fluorescence quenching and ellipticity changes without any significant shift in absorption emission maxima with corresponding induced thermal stabilization by 13.0 and 11.6°C in K+ rich solution. Na+ ions did not induce any thermal stabilization. Molecular docking confirmed external binding at grooves and loops of G4 DNA involving 4OCH3 of ring D, 9COCH2OH of ring A, 4'OH/H and 3'NH3+ of daunosamine sugar. Thermal stabilization induced by specific interactions is likely to hamper telomere association with telomerase enzyme and contribute to drug-induced apoptosis in cancer cell lines besides causing damage to duplex DNA. The findings pave the way for drug designing in view of immense possibilities of altering substituent groups on anthracyclines for enhancement of efficacy, reduced cell toxicity as well as specificity towards G-quadruplex DNA.

Effects of caffeine on the structure and conformation of DNA: A force spectroscopy study.

Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.

Synthesis of new ultrasonic-assisted palladium oxide nanoparticles: an in vitro evaluation on cytotoxicity and DNA/BSA binding properties.

Better solubility and improved toxicity of palladium complexes compared with cisplatin were major reasons for synthesis of novel Pd(II) complex, [Pd(8Q)(bpy)]NO3 (8Q=8-hydroxyquinolinate, bpy=2,2'-bipyridine). Interaction between the [Pd(8Q)(bpy)]NO3 complex and calf thymus DNA in aqueous solution has been investigated by circular dichroism (CD), UV-Visible absorption and fluorescence spectroscopic techniques. These experiments showed that prepared Pd(II) complex can effectively intercalate into CT-DNA and weakly bind to BSA in which the bovine serum albumin molecule was unfolded slightly. The cytotoxicity of the prepared complex has been evaluated on the MCF-7 and DU145 cell lines by MTT and TUNEL assay. The MTT results were showed that in DU145, the CC50 values of [Pd(8Q)(bpy)]NO3 and cisplatin are very close together (10.4 and 8.3 µM, respectively), unlike MCF-7. Accordingly, TUNEL assay was performed on DU145 and apoptosis was clearly obvious by 43% DNA fragmentation in the treated cell lines. So, we can suggest the [Pd(8Q)(bpy)]NO3 as alternative drug for cisplatin in the future which has great potential in DNA denaturation and apoptosis specially on prostate cancer. PdO nanoparticles were successfully prepared without supported any surfactants via sonochemical approach. The synthesized PdONPs were characterized using UV-Vis and FTIR spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Communicated by Ramaswamy H. Sarma.

The effect of neonicotinoid insecticide thiacloprid on the structure and stability of DNA.

The application of pesticides and chemical fertilizers constitutes a potential risk to human and animals due to the presence of their residues in the food. Thiacloprid belongs to a group of neonicotinoid insecticides. It shows a cytotoxic/cytostatic effect in human peripheral blood lymphocytes probably due to DNA damage. The use of thiacloprid is increasingly widespread worldwide, therefore is very important the assessment of its possible genotoxic and cytotoxic effects on a living organism. That is the reason why we studied the thiacloprid influence on the structure and stability of DNA in presented work. We have been studied the thiacloprid interaction with calf thymus DNA. Association constant was determined by fluorescence spectroscopy using equilibrium receptor-ligand binding analysis. The thermal denaturation of DNA was used to identify the mode of interaction. Viscosity changes were recorded to confirm/disconfirm the intercalation mode of interaction. Given the results, we can conclude that neonicotinoid pesticide thiacloprid destabilizes DNA. It changes the structure and stability of DNA through binding into the minor groove by hydrophobic or hydrogen interactions.

Binding of anticancer drug daunomycin to parallel G-quadruplex DNA [d-(TTGGGGT)]4 leads to thermal stabilization: A multispectroscopic investigation.

Guanine rich DNA sequences form four stranded G-quadruplex structures found in telomeric DNA and oncogene promoters. Small molecules binding and stabilizing the G-quadruplex disrupt telomere maintenance and gene regulation, thereby limiting the proliferation of cancer cells. The anti-cancer drug daunomycin binds to both G-quadruplex DNA and duplex DNA. We have undertaken a study of interaction of daunomycin to [d-(TTGGGGT)]4 comprising telomeric DNA sequence from Tetrahymena thermophilia to understand the mechanisms of its action. Absorbance, fluorescence and circular dicroism spectra show significant change on interaction with no change in wavelength maxima. The daunomycin dimers present in free state in solution are disrupted on binding. Presence of all sequential short inter proton distance contacts in nuclear magnetic resonance spectra confirm external binding. The observed inter molecular nuclear Overhauser enhancements, changes in chemical shift and molecular docking studies establish well defined binding of daunomycin at two different sites of G-quadruplex DNA. Thermal stabilization of [d-(TTGGGGT)]4 by 10-15 °C upon daunomycin binding is expected to reduce access of telomerase to its functional site at telomeres. The present studies on mode of action pave the way for alternate derivatives/analogues by chemical modification of anthracyclines to arrive at a more potent telomerase inhibitor.

Further aspects of Toxoplasma gondii elimination in the presence of metals.

Toxoplasma gondii, the etiological agent of toxoplasmosis, infects nucleated cells and then resides and multiplies within a parasitophorous vacuole. For this purpose, the parasite secretes many virulence factors for the purpose of invading and subverting the host microbicidal defenses in order to facilitate its survival in the intracellular milieu. Essential metals are structural components of proteins and enzymes or cofactors of enzymatic reactions responsible for these parasitic survival mechanisms. However, an excess of non-essential or essential metals can lead to parasite death. Thus, infected host cells were incubated with 20 µM ZnCl2 in conjunction with 3 µM CdCl2 or HgCl2 for 12 h in order to investigate cellular events and organelle damage related to intracellular parasite death and elimination. In the presence of these metals, the tachyzoites undergo lipid uptake and transport impairment, functional and structural mitochondrial disorders, DNA condensation, and acidification of the parasitophorous vacuole, thus leading to parasite death. Additional research has suggested that lysosome-vacuole fusion was involved in parasite elimination since acid phosphatases were found inside the parasitophorous vacuole, and vacuoles containing parasites were also positive for autophagy. In conclusion, low concentrations of CdCl2, HgCl2, and ZnCl2 can cause damage to Toxoplasma gondii organelles, leading to loss of viability, organelle death, and elimination without causing toxic effects to host cells.

Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA.

BACKGROUND: Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in >80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence. METHODS: UV-Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics. RESULTS: TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na+ or K+. T-Jump kinetic experiments show that the rates of formation and dissociation of these complexes in the ms time scale differ by one order of magnitude. MD simulations reveal that, in K+ buffer, "hybrid 1" conformation yields kinetic constants on interaction with TMPyP4 one order lower than "hybrid 2". The binding involves π-π stacking with external loop bases. CONCLUSIONS: For the first time we show that for a particular buffer TMPyP4 interacts in a kinetically different way with the two Tel22 conformations even if the complexes formed are thermodynamically indistinguishable. GENERAL SIGNIFICANCE: G-quadruplexes, endowed with technological applications and potential impact on regulation mechanisms, define a new research field. The possibility of building different conformations from same sequence is a complex issue that confers G-quadruplexes very interesting features. The obtaining of reliable kinetic data constitutes an efficient tool to determine reaction mechanisms between conformations and small molecules.

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements.

BACKGROUND: When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. RESULTS: The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. CONCLUSIONS: This study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs.

Ionic effects on the temperature-force phase diagram of DNA.

Double-stranded DNA (dsDNA) undergoes a structural transition to single-stranded DNA (ssDNA) in many biologically important processes such as replication and transcription. This strand separation arises in response either to thermal fluctuations or to external forces. The roles of ions are twofold, shortening the range of the interstrand potential and renormalizing the DNA elastic modulus. The dsDNA-to-ssDNA transition is studied on the basis that dsDNA is regarded as a bound state while ssDNA is regarded as an unbound state. The ground state energy of DNA is obtained by mapping the statistical mechanics problem to the imaginary time quantum mechanics problem. In the temperature-force phase diagram the critical force F c (T) increases logarithmically with the Na+ concentration in the range from 32 to 110 mM. Discussing this logarithmic dependence of F c (T) within the framework of polyelectrolyte theory, it inevitably suggests a constraint on the difference between the interstrand separation and the length per unit charge during the dsDNA-to-ssDNA transition.

Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study.

Pirenzepine is an anti-ulcer agent which belongs to the anti-cholinergic group of gastrointestinal disorder drugs and functions as an M1 receptor selective antagonist. Drug-DNA interaction studies are of great significance as it helps in the development of new therapeutic drugs. It provides a deeper understanding into the mechanism through which therapeutic drugs control gene expression. Interaction of pirenzepine with calf-thymus DNA (Ct-DNA) was determined via a series of biophysical techniques. UV-visible absorption and fluorescence spectroscopy confirmed the formation of pirenzepine-Ct-DNA complex. The values of binding constant from various experiments were calculated to be in the order of 103 M-1 which is consistent with the groove binding mode. Various spectrofluorimetric experiments like competitive displacement of well known dyes with drug, iodide quenching experiments and the effect of Ct-DNA denaturation in presence of drug confirmed the binding of pirenzepine to the groove of Ct-DNA. The binding mode was further established by viscometric, circular dichroic and molecular modelling studies. Thermodynamic parameters obtained from isothermal titration calorimetric studies suggest that the interaction of pirenzepine with Ct-DNA is enthalpically driven. The value of TΔS and ΔH calculated from calorimetric studies were found to be 4.3 kcal mol-1 and -2.54 kcal mol-1 respectively, indicating that pirenzepine-Ct-DNA complex is mainly stabilized by hydrophobic interaction and hydrogen bonding. The binding energy calculated was -7.5 kcal mol-1 from modelling studies which was approximately similar to that obtained by isothermal titration calorimetric studies. Moreover, the role of electrostatic interaction in the binding of pirenzepine to Ct-DNA cannot be precluded.

Selective and Robust Stabilization of Triplex DNA Structures Using Cationic Comb-type Copolymers.

DNA sequences capable of forming triplexes induce DNA double-strand breaks that have attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN). Therefore, novel functional tools that stabilize triplex DNA structures must be further investigated to spark renewed interest. In this study, we investigated the unique character of cationic comb-type copolymers for the selective stabilization of triplex DNA. The melting temperature (Tm) of triplex DNA increased from 24.5 to 73.0 °C (ΔTm = 48.5 °C) by the addition of poly(allylamine)-graft-dextran (PAA-g-Dex) under physiological conditions (at pH 7.0), while PAA-g-Dex did not stabilize but rather destabilized the DNA duplex. On the other hand, poly(l-lysine)-graft-dextran (PLL-g-Dex) stabilized both the duplex and triplex structures at pH 7.0. Thermodynamic parameters evaluated by isothermal titration calorimetry (ITC) revealed that the binding constant (Ka) for the intermolecular triplex formation in the presence of PAA-g-Dex was 1.1 × 109 M-1 at 25 °C which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 107 M-1). Stabilizing activity and selectivity of cationic copolymers toward DNA assemblies were successfully controlled by selecting appropriate backbone structures of the copolymer. Various functional molecules that stabilize DNA duplexes have been developed and used in biological research. However, there are few cationic polymers that stabilize triplex DNA selectively. This study indicates that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.

Refolding and unfolding of CT-DNA by newly designed Pd(II) complexes. Their synthesis, characterization and antitumor effects.

New antitumor Pd(II) compounds derived from oxygen donor ligands salicylate (SA) (1) and sulfosalicylate (SSA) (2) dianions and nitrogen donor heterocyclic ligand 2,2'-bipyridine (bpy) were synthesized and characterized by elemental analysis, UV-Vis, FT-IR, 1H NMR and conductivity measurements. The complexes evaluated for their cytotoxicity effects towards cancer cell line of K562 using MTT assay. They are more cytotoxic than cisplatin. The dependence of their interaction modes with CT-DNA on concentration of the compounds has been discovered in this work. CT-DNA binding studies of these complexes have been investigated by UV-Vis absorption, ethidium bromide (EB) displacement, fluorescence, circular dichroism and gel electrophoresis techniques. The apparent binding constants (Kapp) has been obtained 3.9 and 10.9×104M-1 at lower concentration range and 1.03 and 1.59×104M-1 at higher concentration range for complexes (1) and (2), respectively. These complexes effectively interact with CT-DNA in the order of (2)>(1). Fluorescence studies exhibited that the complexes quench CT-DNA-EB by simultaneous static and dynamic quenching processes. The calculated binding (Kapp, kq, KSV, n) and thermodynamic (ΔG°, ΔH°, ΔS°) parameters revealed that hydrophobic, van der Waals forces and hydrogen binding holds the Pd(II) complexes in the CT-DNA grooves. Gel electrophoresis supports the spectroscopic experiments.

CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression.

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.