Henri Sasmor

Characterization of a Potent and Specific Class of Antisense Oligonucleotide Inhibitor of Human Protein Kinase C-α Expression

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-α expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2′ modifications have been incorporated into the protein kinase C-α targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2′-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5° C per modification) and an increase in resistance toward both 3′-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-α in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-α protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos orjunB. These results demonstrate that 2′-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-α expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.

Oligonucleotide charge states in negative ionization electrospray-mass spectrometry are a function of solution ammonium ion concentration

The charge state distribution for oligonucleotides detected using negative ionization electro-spray-mass spectrometry has been studied as a function of solution counterion concentration. In the absence of added buffer, an average charge state (Z) of −7.2 is observed for a 10 µM aqueous solution of a 14mer DNA at pH 7.0, with [M − 8H]8− the most abundant ion. As the solution concentration of ammonium acetate increases from 0.1 to 33 mM, Z shifts to −3.8 with [M − 4H]4− the most abundant charge state. The shift in most abundant charge state from [M − 7H]7− to [M − 4H]4− occurs abruptly between 1.0 and 10 mM NH4OAc. Above 100 mM NH4OAc, the value of Z plateaus at −3.1, with [M − 3H]3− the most abundant charge state. The addition of 1–50 mM glycine to the analyte solution does not alter Z, suggesting that the changes in charge state observed by using ammonium acetate result from a solution equilibration of cations around the DNA strand, rather than nonspecific gas-phase proton transfers during the electrospray process. The fraction of neutralized phosphate groups reaches a maximum of 0.79 ± 0.03 independent of length and sequence.

Pharmacokinetics in mice of a [3H]-labeled phosphorothioate oligonucleotide formulated in the presence and absence of a cationic lipid

Abstract Formulation of phosphorothioate oligonucleotides with cationic lipids enhances the pharmacological activity of the oligonucleotide in cellular based assay systems. The effect of cationic lipid formulation on the pharmacokinetic behavior of a phosphorothioate oligodeoxynucleotide in mice was investigated in the present study. In the absence of any formulation, the phosphorothioate oligonucleotide exhibited a plasma T 1 2 of 10.2 min and was broadly distributed to many peripheral tissues with liver, kidney, skeletal muscle and skin being the major organs of disposition. Formulating the oligonucleotide with a DMRIE/DOPE (50:50) formulation, such that there was a 2.5:1 positive charge excess on the lipidic structure, resulted in marked changes in the distribution of the oligonucleotide. Significant increases in the amount of oligonucleotide distributed to liver, lung and spleen were observed when the oligonucleotide was formulated with cationic lipids. These results suggest that it is possible to change the biodistribution of phosphorothioate oligodeoxynucleotides by formulating with cationic liposomes.

Synthesis, hybridization, and nuclease resistance properties of 2′-O-aminooxyethyl (2′-O-AOE) modified oligonucleotides

Abstract The novel RNA mimic 2′-O-AOE has been incorporated into antisense oligonucleotides. This 2′-O-modification significantly enhances hybridization against target RNA, and furthermore, exhibits specificity for RNA over DNA. The nuclease resistance (SVPD) of 2′-O-AOE modified phosphodiester oligonucleotides is significantly higher than the unmodified DNA and comparable to the 2′-O-MOE oligonucleotides.

Use of 2-diphenylmethylsilylethyl (DPSE) protecting group in oligonucleotide synthesis via phosphoramidite approach

Abstract 2-Diphenylmethylsilylethyl (DPSE) is a new protecting group for the internucleotidic bonds in the synthesis of deoxyribooligonucleotides by the phosphoramidite approach. This group is stable to acidic conditions and can be removed under mild conditions using aqueous ammonium hydroxide.

Non-Covalent Complexes of Oligonucleotides Observed Using Electrospray Ionization Mass Spectrometry

The development of electrospray (ESI) and matrix-assisted laser desorption (MALDI) ionization techniques for the transformation of proteins and nucleic acids into gas phase ions has reinvigorated mass spectrometry in the biological sciences. Gas-phase ions generated using electrospray ionization are believed to retain their solution structures during the desolvation process.(1) As a result, it is now possible to detect and characterize the structure and properties of molecules too large for study via solution techniques such as NMR spectroscopy. ESI and MALDI ionization provide high sensitivity and transmission efficiency for detection of fmole quantities using FTICR, quadrupole ion trap, or conventional quadrupole mass spectrometers.