DNA assembly using bis-peptide nucleic acids (bisPNAs)

DNA nanostructures are ordered oligonucleotide arrangements that have applications for DNA computers, crystallography, diagnostics and material sciences. Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization, but its potential for application in the field of DNA nanotechnology has yet to be thoroughly examined. We report the synthesis and characterization of tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson–Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. We observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate simultaneous binding of two inward projecting strands. These results suggest that bisPNA molecules can be used for self-assembling DNA nanostructures provided that the arrangement of the hybridizing DNA oligonucleotides does not interfere with simultaneous hybridization to the bisPNA molecule.     

Enhanced strand invasion by peptide nucleic acid-peptide conjugates

Efficient and selective recognition of DNA by proteins is due to sequence-specific interactions with a target site and nonselective electrostatic interactions that promote the target's rapid location. If synthetic molecules could mimic these functions, they would render a wide range of chromosome sequences accessible to rationally designed probes. Here we describe conjugates between bispeptide nucleic acids (bisPNAs) designed to specifically recognize duplex DNA and peptides that have been designed to promote rapid sequence recognition. Peptide design was based on the surface of staphylococcal nuclease, a cationic DNA binding protein with low sequence selectivity. We observe that attachment of the designed peptide increases rates of strand invasion by 100-fold relative to unmodified bisPNA. The peptide can contain D-amino acids, increasing the likelihood that it will be stable in cell extract and inside cells. Binding of the conjugate containing the D-amino acid peptide occurred over a broad range of experimental conditions and was sensitive to a single mismatch. Strand invasion was efficient at neutral to basic pH, a wide range of temperatures (0-65 degrees C), and in the presence of up to 7 mM Mg(2+) and 100 mM Na(+) or K(+). Our data suggest that attachment of peptides that mimic cationic protein surfaces to PNAs can afford conjugates that mimic the rapid and selective binding that characterizes native DNA binding proteins. Rapid strand invasion over a wide range of experimental conditions should further expand the utility of strand invasion by PNAs.     

Studies on epimerization-free methods for the preparation of aminosuccinyl peptides

Summary We have found that guanidine acetate catalyses the transformation of a -benzyl-aspartyl peptide (Boc-Asp-(OBzl)-Leu-Trp-OMe) to an aminosuccinyl peptide (Boc-Asu-Leu-Trp-OMe). The reaction was accompanied by partial epimerization. However, not even a small amount of epimerization could be detected when the aminosuccinyl peptide was synthesised from Boc-Asp-Leu-Trp-OMe with the addition of DIC, HOPfp and guanidine acetate (as a catalyst). This reaction seems to be suitable for the epimerization-free solid phase synthesis of aminosuccinyl peptides, e.g. Asu⁶-Lamprey-III-GnRH (Glp-His-Trp-Ser-His-Asu-Trp-Lys-Pro-Gly-NH₂).     

Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.     

Synthetic α-helical peptides incorporating intercalators for DNA recognition

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz ¹H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential d_NN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an α-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.     

An efficient and versatile synthesis of bisPNA-peptide conjugates based on chemoselective oxime formation

Oligomers with two identical peptide nucleic acid sequences joined by a flexible hairpin linker (bisPNA) can stably bind to specific DNA sequences without altering plasmid supercoiling, thus offering a unique opportunity to attach various functional entities to high molecular weight DNA. Current synthetic approaches, however, severely limit the possibility to link peptides or other chemical moieties (i.e., sugars, oligonucleotides, etc.) to bisPNA. Here we report a novel strategy for the synthesis of bisPNA-peptide conjugates in which chemoselective ligation of bisPNA to peptides was accomplished through oxime formation between an oxy-amine-containing peptide and a bisPNA-methyl ketone (complementary modifications can also be used). The described synthesis is highly efficient, does not require a protection strategy, and is carried out under mild aqueous conditions. Through this methodology long peptide sequences in either C to N or N to C polarity can be linked to bisPNA. In addition, this protocol makes the conjugation of cysteine-containing peptides feasible and allows disulfide bond formation to be controlled. This same approach can be exploited to link oligonucleotides, sugars, or other chemical entities to bisPNA.     

Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model

PurposeThe clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4⁺ cells, including regulatory T cells (Tregs).MethodsTo determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)_257–264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model.ResultsWe showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a⁺ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model.ConclusionThe use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy.     

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of ±0.7 µm or ±2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension.     

Enhancement of Stability of Double and Triple Stranded DNA by Cationic Amphiphilic α-Helix Peptide

Abstract

It was found that synthetic amphiphilic α-helix peptide could bind stabilize double or triple stranded DNA. The stabilization effect was significant for cationic α-helix peptides which indicated the importance of electrostatic interaction of positive charge of peptide and negative charge of DNA. It was also pointed out that α-helix content was increased in the presence of DNA.     

An ATPase inhibitory peptide with antibacterial and ion current effects

An 84-residue bactericidal peptide, PSK, was purified from a Chrysomya megacephala fly larvae preparation. Its amino acid sequence is similar to that of a previously reported larval peptide of the Drosophila genus (SK84) noticed for its anticancer and antimicrobial properties. The PSK sequence is also homologous to mitochondrial ATPase inhibitors from insects to humans (35–65% sequence identity), indicating an intracellular protein target and possible mechanism for PSK. It contains a cluster of six glycine residues, and has several two- and three-residue repeats. It is active against both Gram-positive and Gram-negative bacteria via a mechanism apparently involving cell membrane disintegration and inhibition of ATP hydrolysis. In addition, PSK induces an inward cationic current in pancreatic β cells. Together, the findings identify a bioactive peptide of the ATPase inhibitor family with specific effects on both prokaryotic and mammalian cells.     

Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca2+-binding EF-hand sequence

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM-40) is a metal-binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid-G₁, and that a synthetic peptide (amino acids 54–73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide-bonded region was in part responsible for the growth-suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648–2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C-terminal EF-hand sequence comprising a high-affinity Ca²⁺-binding site of SPARC and represented by a synthetic peptide (amino acids 254–273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790–25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [³H]-thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019–0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose-responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti-SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell-surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration-dependent: peptide 4.2 K, containing an Asp₂₅₈ → Lys substitution, and peptide 4.2 AA, in which the two disulfide-bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF-hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild-type 4.2 sequence, that was indicative of a compromised affinity for Ca²⁺. Moreover, ablation of the disulfide bond in peptide 4.2 AA potentially destabilized the Ca²⁺-binding loop structure, as assessed by fluorescence spectroscopy, such that the peptide competed poorly for the binding of [¹²⁵I]-peptide 4.2 to BAE cells. We conclude both that Ca²⁺-coordinating Asp at position 258 and the conformation of peptide 4.2 are necessary for the inhibition of DNA synthesis by SPARC in cultured endothelial cells.     

Monitoring the early aggregatory behaviour and size of Aβ1-42 in the absence & presence of metal ions using dynamic light scattering

Background and aimAβ₁₋₄₂ is an amyloidogenic peptide found within senile plaques extracted from those who died with a diagnosis of Alzheimer’s disease. The potent neurotoxicity of this peptide is related to its propensity to form aggregated conformations in vivo, a process that is influenced by the species and concentration of metal ions present within the local environment. This study examines the impact of different metals upon the early aggregatory behaviour and size of Aβ₁₋₄₂ under simulated physiological conditions.MethodsThe size and aggregatory behaviour of Aβ₁₋₄₂ in the presence and absence of metal ions was monitored during the initial 30 min of fibril formation in real-time using dynamic light scattering.ResultsIntensity scattering measurements showed a clear tendency towards aggregation with regards to Aβ₁₋₄₂ only solutions (10 μM). Both equimolar Al³⁺ & Cu²⁺ lowered and stabilised the dimensions of Aβ₁₋₄₂ aggregates; however, a diminutive but significant increase in size was still observed over a 30-min period. While excess Al³⁺ continued to supress the size of Aβ_1−42, a 10-fold increase in the concentration of Cu²⁺ accelerated peptide aggregation relative to that observed for equimolar metal but not compared to Aβ₁₋₄₂ alone.ConclusionThese results infer that Al³⁺ ions stabilise and aid in the maintenance of smaller, toxic intermediates while excess Cu²⁺ facilitates the formation of larger, more inert, amorphous species exceeding 1 μm in size. Furthermore, we propose that metal-induced toxicity of Aβ₁₋₄₂ is reflective of their ability to preserve smaller oligomeric species in vitro.     

Identification of cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a K_D value of 210 nM in SPR analysis and CTPS1-selective inhibition with an IC₅₀ value of 110 nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor.