Lendell L. Cummins

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°u2009C 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.

Characterization of Oligonucleotide MetabolismIn Vivovia Liquid Chromatography/Electrospray Tandem Mass Spectrometry with a Quadrupole Ion Trap Mass Spectrometer

The pattern of nuclease degradation observed for an antisense phosphorothioate oligonucleotide in pig kidney was determined using liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) and LC/ESI-MS/MS with a quadrupole ion trap mass spectrometer. Metabolites were separated by length using reversed-phase high-performance liquid chromatography with aqueous hexafluoropropan-2-ol-triethylamine and a methanol gradient. The individual masses of metabolites in each LC peak were determined via deconvolution and converted into potential nucleotide compositions. The nucleotide composition was used to locate metabolites within the known oligomer sequence. The identity of metabolites was confirmed using on-line LC/MS/MS to generate fragment ions suitable for sequence verification. A limited number of shorter oligonucleotide fragments were observed, suggesting that metabolism in vivo may be sequence dependent.

Rapid diagnosis of bloodstream infections with PCR followed by mass spectrometry.

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods.

Enhanced Diagnostic Yields of Bacteremia and Candidemia in Blood Specimens by PCR-Electrospray Ionization Mass Spectrometry

ABSTRACT A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields.

Remarkable enhancement of binding affinity of Heterocycle-modified DNA to DNA and RNA. Synthesis, characterization and biophysical evaluation of N2-imidazolylpropylguanine and N2-imidazolylpropyl-2-aminoadenine modified oligonucleotides

Abstract Oligonucleotides containing novel N2-Imidazolylpropylguanine and N2-Imidazolylpropyl-2-aminoadenine moieties were synthesized and studied for their hybridization and biophysical properties. Interestingly, these heterocycle modified oligonucleotides showed a remarkable enhancement of heteroduplex binding affinity when hybridized to complementary DNA.

Rapid deconvolution of combinatorial libraries using HPLC fractionation

Abstract The rapid deconvolution of combinatorial libraries prepared via solution-phase simultaneous addition of functionalities is demonstrated using HPLC. Libraries containing 25–216 members are screened for biological activity, and active libraries are fractionated into discreet pools using reversed-phase HPLC. The entire process can be completed from a single synthesis of 15–50 mg, without the need for fixed positions, serial synthesis, or tagging. The method is demonstrated for libraries with charged and uncharged functional groups, and for a library containing substituted benzyl moieties. Biologically active combinatorial libraries prepared via solution phase simultaneous addition of functionalities are deconvoluted using HPLC fractionation.

PHOSPHOROTHIOATE OLIGONUCLEOTIDE METABOLISM: CHARACTERIZATION OF THE “N+”-MER BY CE AND HPLC-ES/MS

Abstract Unique phosphorothioate oligonucleotide metabolites of ISIS 2302 were isolated from pig kidney and characterized by capillary electrophoresis (CE) and high pressure liquid chromatography-electrospray mass spectrometry (HPLC-ES/MS) analysis. Fractionation by anion exchange HPLC followed by CE and HPLC-ES/MS analysis enabled facile identification of many metabolites, including a unique metabolite that has a slower migration time than intact ISIS 2302. The appearance of this slower migrating “N+” metabolite in CE analysis was correlated to three masses observed during HPLC-ES/MS analysis. These masses are consistent with the addition of a ribonucleotide or a phosphorothioate deoxyribonucleotide to intact ISIS 2302.

Synthesis, Biophysical and Biological Evaluations of Novel Antisense Oligonucleosides Containing Dephosphono-Internucleosidic Linkages

Abstract Efficient large-scale syntheses of methylene(methylimino) (MMI) linked mixed base nucleosidic dimers have been accomplished. These dimers were successfully incorporated into deoxyoligonucleotides by automated solid-support synthesis. The hybridization properties, nuclease stability, RNase H mediated cleavage and in vitro biological activity of novel chimeric oligomers have been studied. The biophysical and biological evaluation of these chimeric oligomers containing MMI linkages suggests that MMI is a promising chemical modification of the backbone linkage for the construction of antisense molecules useful as therapeutics.

Monitoring solution‐phase combinatorial library synthesis by capillary electrophoresis

Capillary electrophoresis has been applied to monitor model reactions in solution-phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non-aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations.