Project description:We describe herein a new conformationally constrained analog of PNA carrying an alternating ?/? amino acid backbone consisting of (2'R,4'R)-nucleobase-subtituted proline and (1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). The acpcPNA has been synthesized and evaluated for DNA, RNA and self-pairing properties by thermal denaturation experiments. It can form antiparallel hybrids with complementary DNA with high affinity and sequence specificity. Unlike other PNA systems, the thermal stability of acpcPNA·DNA hybrid is largely independent of G+C contents, and is generally higher than that of acpcPNA·RNA hybrid with the same sequence. Thermodynamic parameters analysis suggest that the A·T base pairs in the acpcPNA·DNA hybrids are enthalpically stabilized over G·C pairs. The acpcPNA also shows a hitherto unreported behavior, namely the inability to form self-pairing hybrids. These unusual properties should make the new acpcPNA a potentially useful candidate for various applications including microarray probes and antigene agents.
Project description:Conjugation of peptides with oligonucleotides offers opportunities for combining the strengths of both biopolymer classes. Herein, we show that the combination of a peptide-based module with an antisense oligonucleotide module provides for enhancements of potency and a widened scope of cell delivery options. The peptide unit comprises a Smac mimetic compound (SMCs) which antagonizes the action of inhibitor of apoptosis proteins (IAPs) frequently overexpressed in cancer cells. To counteract SMC resistance, the antisense module downregulates the cellular FLICE-like protein (c-FLIP), a master regulator of the extrinsic apoptosis pathway. We compared c-FLIP antisense units based on oligophosphorothioate (PSO) and peptide nucleic acid (PNA) architectures. Owing to the ease of synthesis, PNA conjugates combined with a cell penetrating peptide (CPP) offer a seemingly ideal solution for cell delivery of dual activity agents. However, our investigations revealed that such congeners have to be handled with care to avoid off-target effects. By contrast, PSO conjugates provided a more robust and specific activity for inducing death of SMC-resistant A549 cells due to a simultaneous activation of caspases and c-FLIP knockdown. We show that lipofection is a convenient approach for delivery of peptide-PSO conjugates into cells. The results highlight that the combination of the peptide and the DNA world confers properties inaccessible by the unconjugated components.
Project description:A highly efficient chemical ligation was developed for quantitative conjugation of PNA with DNA (PNA or peptide) using the copper-catalyzed azide-alkyne cycloaddition reaction. While PNAs with an alkyne at the C-terminus and an azide at the N-terminus have been used, an efficient click-click reaction occurs. The PNA click ligation is sequence-specific and capable of single nucleotide discrimination.
Project description:It is becoming increasingly clear that the transfer of an electron across a protein-protein interface is coupled to the dynamics of conformational conversion between and within ensembles of interface conformations. Electron transfer (ET) reactions in conformationally mobile systems provide a "clock" against which the rapidity of a dynamic process may be measured, and we here report a simple kinetic (master equation) model that self-consistently incorporates conformational dynamics into an ET photocycle comprised of a photoinitiated "forward" step and thermal return to ground. This kinetic/dynamic (KD) model assumes an ET complex exists as multiple interconverting conformations which partition into an ET-optimized (reactive; R) population and a less-reactive population ( S). We take the members of each population to be equivalent by constraining them to have the same conformational energy, the same average rate constant for conversion to members of the other population, and the same rate constants for forward and back ET. The result is a mapping of a complicated energy surface onto the simple "gating", two-well surface, but with rate constants that are defined microscopically. This model successfully describes the changes in the ET photocycle within the "predocked" mixed-metal hemoglobin (Hb) hybrid, [alpha(Zn), beta(Fe3+N 3 (-))], as conformational kinetics are modulated by variations in viscosity (eta = 1-15 cP; 20 degrees C). The description reveals how the conformational "routes" by which a hybrid progresses through a photocycle differ in different dynamic regimes. Even at eta = 1 cP, the populations are not in fast exchange, and ET involves a complex interplay between conformational and ET processes; at intermediate viscosities the hybrid exhibits "differential dynamics" in which the forward and back ET processes involve different initial ensembles of configurational substates; by eta = 15 cP, the slow-exchange limit is approached. Even at low viscosity, the ET-coupled motions are fairly slow, with rate constants of <10 (3) s (-1). Current ideas about Hb function lead to the testable hypothesis that ET in the hybrid may be coupled to allosteric fluctuations of the two [alpha 1, beta 2] dimers of Hb.
Project description:Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. ? PNAs, PNA derivatives having a substituent in the ? position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the ? position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.
Project description:DNA has shown great promise as a building material for self-assembling nanoscale structures. To further develop the potential of this technology, more methods are needed for functionalizing DNA-based nanostructures to increase their chemical diversity. Peptide nucleic acid (PNA) holds great promise for realizing this goal, as it conveniently allows for inclusion of both amino acids and peptides in nucleic acid-based structures. In this work, we explored incorporation of a positively charged PNA within DNA nanostructures. We investigated the efficiency of annealing a lysine-containing PNA probe with complementary, single-stranded DNA sequences within nanostructures, as well as the efficiency of duplex invasion and its dependence on salt concentration. Our results show that PNA allows for toehold-free strand displacement and that incorporation yield depends critically on binding site geometry. These results provide guidance for the design of PNA binding sites on nucleic acid nanostructures with an eye towards optimizing fabrication yield.
Project description:Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Together with single-guide RNAs, Cas9 also functions as a powerful genome engineering tool in plants and animals, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition and RNA-DNA base-pairing that serves as a final specificity checkpoint before DNA double-strand break formation.
Project description:PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.
Project description:Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.
Project description:Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209), comprised of a glucagon-like peptide-1 receptor (GLP1-R) agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors in vitro, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic Lep(ob)/Lep (ob) mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (HbA1c) equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide in vivo proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.