Oligonucleotides are an emerging class of drugs that are manufactured by solid‐phase synthesis. As a chemical class, they have unique product‐related impurities and degradants, characterization of which is an essential step in drug development. The synthesis cycle, impurities produced during the synthesis and degradation products are presented and discussed. The use of liquid chromatography combined with mass spectrometry for characterization and quantification of product‐related impurities and degradants is reviewed. In addition, sequence determination of oligonucleotides by gas‐phase fragmentation and indirect mass spectrometric methods is discussed. © 2019 John Wiley & Sons Ltd. Mass Spec Rev

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage l, with a 4-thiouridine (4 S -U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 59-AUG is required for stable binding. Furthermore, the addition of a 59-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.

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A method has been developed to identify oligonucleotide-peptide heteroconjugates by accurate mass measurements using mass spectrometry. The fractional mass (the decimal fraction mass value following the monoisotopic nominal mass) for peptides and oligonucleotides is different due to their differing molecular compositions. This property has been used to develop the general conditions necessary to differentiate peptides and oligonucleotides from oligonucleotide-peptide heteroconjugates. Peptides and oligonucleotides generated by the theoretical digestion of various proteins and nucleic acids were plotted as nominal mass versus fractional mass. Such plots reveal that three nucleotides cross-linked to a peptide produce enough change in the fractional mass to be recognized from non-crosslinked peptides at the same nominal mass. Experimentally, a cytochrome c digest was spiked with an oligonucleotide-peptide heteroconjugate and conditions for analyzing the sample using liquid chromatography-mass spectrometry were optimized. Upon analysis of this mixture, all detected masses were plotted on a fractional mass plot and the heteroconjugate could be readily distinguished from non-crosslinked peptides. The method developed here can be incorporated into a general proteomics-like scheme for identifying proteinnucleic acid cross-links, and this method is equally applicable to characterizing cross-links generated from protein-DNA and protein-RNA complexes. KeywordsFractional mass; mass excess; mass defect; cross-linking; oligonucleotide-peptide heteroconjugate Nucleic acid and protein complexes play crucial roles in cell functions such as DNA replication, packaging, repair and transcription and RNA maturation, transport and translation. 1-3 Biophysical methods such as X-ray crystallography, nuclear magnetic resonance (NMR) and cryo-electron microscopy are typically used to gain insight into the structure and function of these complexes. 4-8 While X-ray crystallography and NMR can provide high resolution information, not all nucleic acid-protein complexes are readily characterized by these methods. Cross-linking combined with mass spectrometry (MS) is one alternative approach for structurally characterizing nucleic acid-protein complexes, although sample preparation, analysis and data interpretation are not trivial. 9Cross-links, once identified, impose a distance constraint on the complex and allow one to draw conclusions on the three-dimensional structure of the complex by molecular modeling. 10 The combination of cross-linking, MS characterization, and molecular modeling has been called mass spectrometry in three dimensions (MS3D), due to its ability to generate three-