Nucleic Acids in the Gas Phase

This introductory chapter sets the stage for the various methods and application that will be described in the book “Nucleic acids in the gas phase.” Using key review articles as references, nucleic acid structures are introduced, with progression from primary structure to the main secondary, tertiary, and quaternary structures of DNA and RNA. Nucleic acid function is also overviewed, from the roles of natural nucleic acids in biology to those of artificial nucleic acids in the biotechnology, biomedical, or nanotechnology fields. Importantly, the question of why studying nucleic acids in the gas phase is addressed from three different points of view. First, because isolated molecules in vacuo cannot exchange energy with their surroundings, reactivity can be studied in well-defined energetic conditions. Second, isolating molecules from their solvent and environment allow to study their intrinsic properties. Finally, the rapidly expanding field of mass spectrometry, an intrinsically gas-phase analysis method, calls for better understanding of ion structure and reactivity in vacuo.

During the past 30 years, tremendous efforts have been made to implement mass spectrometry and spectroscopy technologies for the characterization of biomolecules in the gas phase. Progresses in the study of gas phase oligonucleotides and DNA have come with the advent of different sources capable to transfer these fragile biomolecules from the condensed phase into the gas phase. These techniques have been largely employed in the spectroscopy and mass spectrometry communities and have stimulated much research with applications of mass spectrometry to structural biology and applications of spectroscopy to detailed understanding of the intra- and intermolecular interactions. The key point in all the experimental techniques is to counterpoise the extremely low volatility of nucleobases, oligonucleotides, and higher-order DNA structures while keeping these thermally fragile molecules intact. The aim of this chapter is to describe the transfer of nucleic acids to the gas phase, including the technical and experimental issues that have been successfully overcome over the last decades.

Evolution has refined nucleic acids to display well-defined three-dimensional structures that are functional under aqueous physiological conditions. While the structure of nucleic acids is well known in solution, it is unclear how nucleic acids react when transferred to a fully anhydrous environment. Simple physical chemistry considerations suggest that a heavily charged poly-anion would adopt fully extended conformations in vacuum, and that multistranded structure would dissociate, to guarantee that charged residues separate as much as possible to reduce Coulomb repulsion. However, and quite counterintuitively, a vast amount of experiments demonstrate that this is not the case and that oligomeric nucleic acids adopt quite compact structures in the gas phase, which in some cases might preserve memories of the original conformation in solution. In this chapter, we review our current understanding of nucleic acid structure in the gas phase.

This chapter deals with nucleic acid ions and their interactions with electrons and photons in the gas phase based on the many different experiments that have been performed relating to this topic within the last 10 years. The fragmentation caused by electron attachment to anions is discussed, and the role of hydration is touched upon. Photoelectron spectroscopy has established the electron binding energies of mononucleotide anions, dinucleotides and larger strands. These are significantly lower than the thresholds for electron-induced electron detachment from anions. Thresholds were measured from electron scattering experiments and product ion masses from mass spectrometry. The site of electron removal is either the base or the phosphate group, and it is likely different for photodetachment and electron detachment. Work has not been limited to anions only, but cations have also been studied. Neutral reionisation of protonated nucleobases has shed light on the lifetime of the neutral intermediate species, which was found to be significantly different to that of the temporary nucleobase anion formed in collisional electron transfer to nucleotide anions. Dissociative recombination experiments involving oligonucleotide monocations have demonstrated that there are certain electron kinetic energies where the cross section for the formation of neutral species is high (resonances), and in closely related electron-capture dissociation experiments on multiply charged cations, the actual fragmentation channels were obtained. Both for oligonucleotide anions and cations, formation of radicals by loss and capture of electrons, respectively, largely governs the dissociation patterns. This is of high relevance for sequencing. Finally, gas-phase absorption spectroscopy has revealed differences in absorption between mononucleotides, single strands, double strands and quadruplexes, which is related to the electronic coupling between two or more bases.

We describe here frequency-resolved gas-phase spectroscopy of nucleic acids. Frequency resolved means that the effect of photons on the nucleic acid molecules is measured as a function of the photon frequency. The present chapter is primarily focused on experimental aspects, and intended as a compass to navigate a rather interdisciplinary field. Indeed, gas-phase spectroscopy usually combines photonics, mass spectrometry (when ions are detected), and theoretical chemistry. Although theory is of prime importance for the interpretation of the results, as it is the comparison between experimental and theoretical energies of the resonance transitions that allow the structural interpretation of the experimental spectra, extended discussion of theory levels will not be provided here, but relevant literature will be indicated along the text. We will cover rotational, vibrational, and electronic spectroscopy from isolated nucleobases to oligonucleotides and nucleic acid higher-order structures.

This chapter summarizes literature describing the gas-phase fragmentation of nucleic acid ions under a variety of reaction conditions. Specifically, the phenomenology of gas-phase dissociation of nucleic acid ions is determined by ion type, charge state, the energy deposition method, and the fragmentation reaction timescale. Various proposed mechanisms are summarized. The chapter is organized by dissociation method. For the most extensively studied collision-induced dissociation (CID), the literature is subcategorized by analyte and ion type. In many cases, no single fragmentation mechanism can account for all the reported products. This suggests that multiple dissociation mechanisms can contribute, depending on ion type, ion charge state, and reaction conditions.

Recent advances in electrospray ionization and the extension of radical ion based dissociation techniques to ribonucleic acids (RNA) were key factors for developing top-down mass spectrometry as a powerful method for the detailed characterization of posttranscriptional and synthetic modifications of RNA. This new approach identifies and localizes all mass-altering modifications without the need for labeling reactions, and can be used for characterization of RNA of unknown sequence.

The role of DNA in cells relies on its chemical structure. Unfortunately, a number of physical and chemical agents may damage DNA, leading to modification of genetic information or to cell death. Detecting DNA damage is thus a major issue in numerous studies. Analytical methods have therefore been developed in order to quantify modified DNA bases. In particular, mass spectrometry is used as a very specific and sensitive detector when combined to gas or liquid chromatography. This review will first briefly present the major classes of DNA damage and then focus on two mass spectrometry-based approaches for the quantification of modified bases following DNA hydrolysis into monomers.

Among the preeminent compounds that bind to DNA are numerous anticancer and antibacterial therapeutics. The development of new chemotherapeutics has accelerated the need for sensitive and versatile analytical techniques that are capable of characterizing DNA/ligand interactions including determination of binding stoichiometries, selectivities, and affinities. Electrospray ionization mass spectrometry (ESI-MS) has emerged as a useful technique for the analysis of complexes formed between DNA and small molecules due to its low sample consumption and fast analysis time. This chapter describes the exploration, optimization, and validation of ESI-MS methods for characterizing DNA–ligand interactions.

The vast majority of the human genome consists of sequences that do not code for proteins. Understanding their function must rely on approaches that are capable of providing more than mere sequence information. Alone or in combination with other techniques, mass spectrometry (MS) can provide an excellent platform for investigating non-coding nucleic acids (NA) at many different levels. This chapter reviews MS-based approaches developed to pursue the structural elucidation of species that are not readily amenable to the classic high-resolution techniques. Indeed, MS has recently found increasing applications as a detection platform for chemical probes used to interrogate NA structure in solution. These developments have been riding on the concomitant advances of computational approaches, which are rapidly closing the resolution gap with NMR and crystallography by taking full advantage of the sparse constraints afforded by alternative techniques. Further, the hierarchic nature of NA structure, which is characterized by the three-dimensional organization of discrete structural elements, lends itself well to the investigation by techniques that are capable of revealing the position of structure-defining interactions. For this reason, novel strategies are being developed to study secondary, tertiary, and quaternary interactions in the gas phase, which may retain memory of the solution architecture. The popularization of ion mobility spectrometry (IMS) has opened new avenues for investigating the overall topology of non-coding elements, which promise to contribute significantly to the elucidation of progressively larger NA systems.