Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography

Gilár, Martin; Bouvier, Edouard S. P.

doi:10.1016/s0021-9673(00)00521-5

Cited by 65 publications

(65 citation statements)

References 12 publications

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“…Oligonucleotide synthesis typically yields the target oligonucleotide contaminated with truncated by-products, so-called failure, and mismatch sequence oligonucleotides (7,8). Using low-quality primers for PCR may result in a failure to amplify a selected sequence, as well as in generation of undesirable nonspecific amplification products.…”

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confidence: 99%

Analysis and Purification of Synthetic Oligonucleotides by Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array and Mass Spectrometry Detection

Gilár

2001

Analytical Biochemistry

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confidence: 99%

Analysis and Purification of Synthetic Oligonucleotides by Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array and Mass Spectrometry Detection

Gilár

2001

Analytical Biochemistry

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114

105

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“…Reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most important techniques for analysis of biomolecules, such as proteins, oligonucleotides, and so on [1][2][3]. Oligonucleotides are short sequences of DNA and/or RNA that have unique properties.…”

Section: Introductionmentioning

confidence: 99%

“…High performance liquid chromatography (HPLC) is the most commonly used technique for the separation and determination of unmodified and therapeutic oligonucleotides [1][2][3]8]. There are many theoretical studies concerning the interaction of oligonucleotides within the surface of stationary phase [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…There are many theoretical studies concerning the interaction of oligonucleotides within the surface of stationary phase [8][9][10]. Most of them include complex mechanisms that comprise different interactions in hydro-organic solvents [1][2][3][8][9][10]. Many different and novel approaches to the study of the retention mechanism in HPLC may be found in the literature [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

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Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase

2015

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Abstract:The main aim of the present investigation was to determine retention behavior and interactions of oligonucleotides with alkylamide stationary phase. Five oligonucleotides, differing in the sequence, were tested. Changes in the composition of the mobile phase, i.e. pH (5.0−7.0) and buffer concentration (30−75 mM) were investigated in detail. In addition, the hydrophobic and electrostatic parameters were measured for the different pH's and salt concentrations. The theoretical model of interactions between individual elements of the separation system, (e.g. solute, stationary phase, and mobile phase) based on zeta potential measurements, hydrophobic and electrostatic parameters calculation, and molecular modeling, has been considered.

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“…GTI-2040 has been shown recently to have promising response and acceptable tolerability in phase I clinical trials as a single agent or in combination with cytarabine for the treatment of advanced solid tumors and acute myeloid leukemia (AML) (Desai et al, 2005;Klisovic et al, 2008). Using a highly specific ion-pair HPLC/tandem mass spectrometry method (Griffey et al, 1997;Gilar and Bouvier, 2000;Dai et al, 2005b), we recently reported the metabolism of GTI-2040 as the sequential nucleotide deletion via the 3Ј exonuclease (Wei et al, 2006). A series of progressively 3Ј chain short-end metabolites were identified in several biological matrices including plasma from AML patients, solutions containing 3Ј exonuclease, and in human liver microsomes.…”

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confidence: 99%

Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

Wei

Dai²,

Liu³

et al. 2008

Drug Metab Dispos

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ABSTRACT:Enzyme kinetics of GTI-2040 (5-GGC TAA ATC GCT CCA CCA AG-3), a phosphorothioate ribonucleotide reductase antisense, were investigated for the first time in 3 exonuclease solution and human liver microsomes (HLMs), using the ion-pair high-performance liquid chromatogram method for quantification of the parent drug and two major 3N-1 and 3N-2 metabolites. Enzyme kinetics of GTI-2040 in 3-exonuclease solution were found to be well characterized by the Michaelis-Menten model, using the sum of formation rates of 3N-1 and 3N-2 (ϳtotal metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high-and low-affinity constants (K d s) of 0.03 and 3.8 M, respectively. Enzyme kinetics of GTI-2040 in HLMs were found to deviate from Michaelis-Menten kinetics when the total GTI-2040 substrate was used. However, after correction for the unbound fractions, the formation rate of total metabolites could be described by Michaelis-Menten kinetics. Using the free substrate fraction, the K m and V max of GTI-2040 were determined to be 6.33 ؎ 3.2 M and 16.5 ؎ 8.4 nmol/mg/h, respectively. Using these values, in vitro hepatic intrinsic clearance (CL int ) in HLM was estimated to be 2.61 ؎ 0.56 ml/h. The CL int was then used to predict GTI-2040's in vivo intrinsic clearance in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 l/h, representing 24.1% of the observed in vivo mean scaled hepatic intrinsic clearance of 758.7 l/h in patients with acute myeloid leukemia. We concluded that the saturable nonspecific binding of GTI-2040 in HLMs complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLMs only partially predicted the in vivo intrinsic clearance.Antisense oligonucleotides (ODNs) are short, single-strand DNA molecules designed to hybridize with specific mRNA strands, thereby selectively inhibiting the production of specific gene products (Stein and Cheng, 1993;Dias and Stein, 2002). This approach has been explored to target expression of genes important for malignant transformation and other pathogenetic mechanisms. For a successful therapeutic use of ODN compounds robust in vivo, stability is critical. Previously used unmodified ODNs degraded rapidly in biological fluids by nucleases, which limited their clinical use. More recently, by substituting one of the nonbridge oxygen atoms, phosphorothioate analogs have been synthesized and shown to be more resistant to exonucleases than the unmodified ODNs Aboul-Fadl, 2005). Several phosphorothioate ODNs (PS-ODNs) are currently undergoing clinical evaluation for a number of diseases, including cancer, viral infections, and inflammatory disorders Jansen and Zangemeister-Wittke, 2002;Marcucci et al., 2005). It has been reported that the in vivo pharmacodynamic effects of antisense correlate with the intracellular drug levels and clinical response Dai et al., 2005a;Marcucci et al., 2005). Moreover, it is anticipated that a well defined pharmacokinetics-pharmacodynamics cor...

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Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography

Cited by 65 publications

References 12 publications

Analysis and Purification of Synthetic Oligonucleotides by Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array and Mass Spectrometry Detection

Analysis and Purification of Synthetic Oligonucleotides by Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array and Mass Spectrometry Detection

Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase

Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

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