Molecular biology is now a routine tool in almost all biological research fields. With the exponential growth in the number of molecular biological techniques, there is a recognizable need for sensitive, accurate and precise quantitation of nucleic acids. We present here two complementary instruments designed for the quantitation of nucleic acids, the GeneQuant II and the DyNA Quant 200 Fluorometer. The GeneQuant II can rapidly determine the UV absorbance of a solution and display the calculated DNA, RNA or protein concentration. In addition, the GeneQuant can display calculated melting temperatures for a given DNA oligonucleotide base sequence, a useful feature for primer design. The DyNA Quant 200 quantitates DNA on the basis of the fluorescent Hoechst 33258 dye/double-stranded (ds)DNA assay. Upon binding to dsDNA, the spectral properties of the dye change such that it becomes highly fluorescent at 460 nm when excited at 365 nm. The assay has proven to be a specific and sensitive alternative method for DNA quantitation, particularly for unpurified DNA samples. Together, the GeneQuant II and the DyNA Quant 200 are a cost-effective and convenient solution to the routine protein and nucleic acid quantification needs of the molecular biologist.
We describe the attachment of a psoralen derivative (site specific psoralen, SSP) to the 5' end of a DNA oligonucleotide and the hybridization and the photoreaction of this reagent with a complementary target site on an RNA molecule. SSP was coupled to a variety of DNA oligonucleotides to investigate the structural requirements for addition to the RNA. Efficient SSP photoadducts were made on specific uridines by designing an intercalation site at an unpaired nucleotide in the RNA strand within the heteroduplex region. The optimal location for this site was five nucleotides from the oligonucleotide 5' end and just 5' to the target uridine residue. Because the attachment of the SSP to the oligonucleotide is through a disulfide bond, the DNA oligonucleotide can be removed with reduction to leave SSP attached to the RNA strand. The SSP adduct made in this way will be useful for subsequent biochemical and biophysical experiments.
BackgroundBAY 81–8973 (Kovaltry) is an unmodified full-length recombinant factor VIII (rFVIII) for treatment of hemophilia A. The BAY 81–8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns of BAY 81–8973 and two other rFVIII (sucrose-formulated rFVIII [rFVIII-FS; Kogenate FS]) and antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM; Advate) and their pharmacokinetic (PK) characteristics.Materials and methodsN-linked glycans or terminal carbohydrates were enzymatically removed from immobilized BAY 81–8973, rFVIII-FS, and rAHF-PFM proteins and analyzed using high-performance liquid chromatography to determine the percentage of individual N-linked glycan structures and degree of sialylation of each structure. PK data were available from two separate phase 1 crossover studies in which the PK profile of BAY 81–8973 was compared with that of rFVIII-FS (n=26) and rAHF-PFM (n=18) in patients with severe hemophilia A who received a single 50 IU/kg dose of each product.ResultsBAY 81–8973 and rFVIII-FS had increased N-linked glycan branching with higher levels of sialylation compared with rAHF-PFM. Levels of trisialylated glycans were 29.0% for BAY 81–8973 vs 11.5% for rFVIII-FS and 4.8%–5.5% for rAHF-PFM; tetrasialylated glycans were 12.0% vs 2.8% and 0.6%, respectively. Degree of sialylation was 96% for BAY 81–8973, 94% for rFVIII-FS, and 78%–81% for rAHF-PFM. Based on chromogenic assay results from the single-dose phase 1 PK studies, BAY 81–8973 half-life was 15% longer than that for rFVIII-FS and 16% longer than rAHF-PFM.ConclusionIncreased N-glycan branching and sialylation were seen for BAY 81–8973 vs rFVIII-FS and rAHF-PFM. Improved PK for BAY 81–8973 relative to rFVIII-FS and rAHF-PFM as seen in single-dose crossover PK studies might be related to this greater level of branching and sialylation, which can prolong the time BAY 81–8973 remains in the circulation.
Previous studies of the Drosophila melanogaster hsp26 gene promoter have demonstrated the importance of a homopurine*homopyrimidine segment [primarily (CT)n*(GA)n] for chromatin structure formation and gene activation. (CT)n regions are known to bind GAGA factor, a dominant enhancer of PEV thought to play a role in generating an accessible chromatin structure. The (CT)n region can also form an H-DNA structure in vitro under acidic pH and negative supercoiling; a detailed map of that structure is reported here. To test whether the (CT)n sequence can function through H-DNA in vivo, we have analyzed a series of hsp26-lacZ transgenes with altered sequences in this region. The results indicate that a 25 bp mirror repeat within the homopurine.homopyrimidine region, while adequate for H-DNA formation, is neither necessary nor sufficient for positive regulation of hsp26 when GAGA factor-binding sites have been eliminated. The ability to form H-DNA cannot substitute for GAGA factor binding to the (CT)n sequence.
Background Traumatic injury and the associated acute bleeding are leading causes of death in people aged 1 to 44 years. Acute bleeding in pathological and surgical settings also represents a significant burden to the society. Yet there are no approved hemostatic drugs currently available. While clinically proven as an effective pro‐coagulant, activated factor VII (FVIIa) use in acute bleeding has been hampered by unwanted thromboembolic events. Enhancing the ability of FVIIa to quickly stop a bleed and clear rapidly from circulation may yield an ideal molecule suitable for use in patients with acute bleeding. Objectives To address this need and the current liability of FVIIa, we produced a novel FVIIa molecule (CT‐001) with enhanced potency and shortened plasma residence time by cell line engineering and FVIIa protein engineering for superior efficacy for acute bleeding and safety. Methods To address safety, CT‐001, a FVIIa protein with 4 desialylated N‐glycans was generated to promote active recognition and clearance via the asialoglycoprotein receptor. To enhance potency, the gamma‐carboxylated domain was modified with P10Q and K32E, which enhanced membrane binding. Results Together, these changes significantly enhanced potency and clearance while retaining the ability to interact with the key hemostatic checkpoint proteins antithrombin and tissue factor pathway inhibitor. Conclusions These results demonstrate that a FVIIa molecule engineered to combine supra‐physiological activity and shorter duration of action has the potential to overcome the current limitations of recombinant FVIIa to be a safe and effective approach to the treatment of acute bleeding.
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