Detailed Characterization of Antisense DNA Oligonucleotides

Abstract

We have developed methods for verification of the structures of novel, chemically synthesized oligonucleotides having alternating methylphosphonate/phosphodiester internucleotide linkages. Matrix-assisted laser desorption ionization mass spectrometry was used to measure the molecular masses of full-length oligonucleotides, failure synthesis products, and degradation products formed by enzymatic and chemical means. These measurements provide detailed structural information, including molecular mass, length, base composition, complete nucleotide sequence, and confirmation of the sugar moieties and internucleotide linkages.

Sequencing of Production-Scale Synthetic Oligonucleotides by Enriching for Coupling Failures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…Analyzing the failure species from crude synthetic material, Keough et al (52,53) and Juhasz et al (49) verified sequence of a methylphosphonate 18-mer and an unmodified 31-mer by MALDI-TOF. The advantage of this technique is that sequencing information can be determined for modified and structural oligonucleotides resilient to enzymatic hydrolysis, Sanger termination reactions, or chemical cleavage.…”

“…A fourth, less documented methodology involves the detection of failure sequences from a crude synthesis (49,52,53). By utilizing the inefficiency of nucleotide coupling during the synthesis process, failure species (N-1, N-2, N-3, etc.)…”

“…Sequence information is generated by a series of failure ions, each of which varies by the molecular mass of the specific nucleotide not coupled. Keough et al verified the majority of the sequence of a methylphosphonate 18-mer using this technique (52,53). Juhasz et al obtained sequence information up to the 3Ј trinucleotide of a crude 31-mer (49).…”

Frontiers of Mass Spectrometry in Nucleic Acids Analysis

“…Basic understanding and evaluation of the pharmacokinetic properties of antisense reagents is foundational to their appropriate design and application. Although MALDI-MS has been applied to metabolic profiling of this class of therapeutics, [92][93][94][95][96] ESI-MS usually in combination with liquid chromatography (Lc) is the more commonly applied technique. [97][98][99][100][101][102][103][104][105][106] obtained tandem (MS/MS) mass spectrometric information is used to assess stability, modes of degradation, tissue distribution and the extent of cellular uptake of antisense reagents.…”

Shotgun Sequencing Small Oligonucleotides by Nozzle-Skimmer Dissociation and Electrospray Ionization Mass Spectrometry

“…Mass spectrometry-based approaches for obtaining sequence information from nucleic acids have been summarized previously. [1][2][3][4][5][6][7] As illustrated by Keough and co-workers, 8,9 among others, sequence characterization of oligonucleotides by tandem mass spectrometry (MS/MS) requires precursor ion isolation followed by activation through collisions with a target gas. [10][11][12][13][14][15][16][17] A drawback to such conventional MS/MS approaches is the limitation in throughput.…”