New solid support for the synthesis of 3'-oligonucleotide conjugates through glyoxylic oxime bond formation.

A novel solid support 1 was synthesized to incorporate glyoxylic aldehyde functionality at the oligonucleotide 3'-terminus. 6-mer and 11-mer oligonucleotide sequences containing 3'-glyoxylic aldehyde functionality were prepared by using this support. These modified oligonucleotides were coupled to reporters containing an aminooxy group to prepare oligonucleotide 3'-conjugates through glyoxylic oxime bond formation. The hydrolytic stability of a glyoxylic oxime linkage was also investigated. [reaction: see text].

A novel therapeutic with two SNAP-25 inactivating proteases shows long-lasting anti-hyperalgesic activity in a rat model of neuropathic pain.

A pressing need exists for long-acting, non-addictive medicines to treat chronic pain, a major societal burden. Botulinum neurotoxin type A (BoNT/A) complex - a potent, specific and prolonged inhibitor of neuro-exocytosis - gives some relief in several pain disorders, but not for all patients. Our study objective was to modify BoNT/A to overcome its inability to block transmitter release elicited by high [Ca2+]i and increase its limited analgesic effects. This was achieved by fusing a BoNT/A gene to that for the light chain (LC) of type/E. The resultant purified protein, LC/E-BoNT/A, entered cultured sensory neurons and, unlike BoNT/A, inhibited release of calcitonin gene-related peptide evoked by capsaicin. Western blotting revealed that this improvement could be due to a more extensive truncation by LC/E of synaptosomal-associated protein of Mr = 25 k, essential for neuro-exocytosis. When tested in a rat spared nerve injury (SNI) model, a single intra-plantar (IPL) injection of LC/E-BoNT/A alleviated for ∼2 weeks mechanical and cold hyper-sensitivities, in a dose-dependent manner. The highest non-paralytic dose (75 U/Kg, IPL) proved significantly more efficacious than BoNT/A (15 U/Kg, IPL) or repeated systemic pregabalin (10 mg/Kg, intraperitoneal), a clinically-used pain modulator. Effects of repeated or delayed injections of this fusion protein highlighted its analgesic potential. Attenuation of mechanical hyperalgesia was extended by a second administration when the effect of the first had diminished. When injected 5 weeks after injury, LC/E-BoNT/A also reversed fully-established mechanical and cold hyper-sensitivity. Thus, combining advantageous features of BoNT/E and/A yields an efficacious, locally-applied and long-acting anti-hyperalgesic.

A highly stable, sensitive, regenerable and rapid immunoassay for detecting aflatoxin B1 in corn incorporating covalent AFB1 immobilization and a recombinant Fab antibody.

A highly robust immunoassay applicable for the detection of aflatoxin B1 (AFB1) using a Fab antibody fragment was developed. A key factor was the use of covalently immobilized AFB1 which allowed an almost three fold increase in sensitivity, reduced assay time and regeneration with retention of binding capacity. Various factors that might affect the sensitivity of the assay such as pH, organic solvents, storage stability and wash stringency were critically evaluated. It was also demonstrated that the assay was applicable for determination of AFB1 in corn samples at concentration within the European union regulatory limits.

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged D-peptide analogue of insulin-like growth factor 1.

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.

New strategy for the synthesis of 3',5'-bifunctionalized oligonucleotide conjugates through sequential formation of chemoselective oxime bonds.

A convenient strategy for the synthesis of bifunctionalized oligonucleotide conjugates bearing two different reporters at the 3' and 5' ends of the oligonucleotide is presented. The method involves the preparation of oligonucleotides bearing an aldehyde and/or aminooxy functionality at each end, followed by reaction to form oxime bonds with appropriately functionalized reporters. The conjugation reactions are carried out under mild aqueous conditions with good reaction yield.

The oxime bond formation as an efficient chemical tool for the preparation of 3',5'-bifunctionalised oligodeoxyribonucleotides.

The simultaneous conjugation of peptides or carbohydrates at the 3'- and 5'-end of oligodeoxyribonucleotides was achieved very efficiently through chemoselective oxime bond formation. The method employs bifunctionalised oligonucleotides in single step without the need of protection strategy, under mild acidic conditions. The conjugates were obtained in high yields by reacting an oxyamine containing reporter groups (peptide, mono- and disaccharide) with an oligonucleotide carrying an aldehyde at each extremity.

Head-to-tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues.

A convenient strategy for the synthesis of the analogue of cyclic oligodeoxyribonucleotides is presented. The cyclization of the oligonucleotide was accomplished through intramolecular oxime bond formation between a 5'-oxyamine moiety and a 3'-aldehydic group.