Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Therapeutic approaches for treatment of various diseases aim at the interruption of transcription or translation. Modified oligonucleotides, such as 2′-O-methyl- and methylphosphonate-derivatives, exhibit high resistance against cellular nucleases, thus rendering application for, e.g., antigene or antisense purposes possible. Other approaches are based on administration of cross-linking agents, such as cis-diamminedichloroplatinum(II) (cisplatin, DDP), which is still the most widely used anticancer drug worldwide. Due to the formation of 1,2-intrastrand cross links at adjacent guanines, replication of the double-strand is disturbed, thus resulting in significant cytotoxicity. Evidence for the gas-phase dissociation mechanism of platinated RNA is given, based on nano-electrospray ionization high-resolution multistage tandem mass spectrometry (MS n ). Confirmation was found by investigating the fragmentation pattern of platinated and unplatinated 2′-methoxy oligoribonucleotide hexamers and their corresponding methylphosphonate derivatives. Platinated 2′-methoxy oligoribonucleotides exhibit a similar gas-phase dissociation behavior as the corresponding DNA and RNA sequences, with the 3′-C-O bond adjacent to the vicinal guanines being cleaved preferentially, leading to wx-ion formation. By examination of the corresponding platinated methylphosphonate derivatives of the 2′-methoxy oligoribonucleotides, the key role of the negatively charged phosphate oxygen atoms in direct proximity to the guanines was proven. The significant alteration of fragmentation due to platination is demonstrated by comparison of the fragment ion patterns of unplatinated and platinated 2′-O-methyl- and 2′-O-methyl methylphosphonate oligoribonucleotides, and the results obtained by H/D exchange experiment

OMA and OPA—Software-Supported Mass Spectra Analysis of Native and Modified Nucleic Acids

The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated double-stranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structure

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO-), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO- ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3 -)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3 - loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO- and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs. Graphical abstract

Gas-phase Dissociation of homo-DNA Oligonucleotides

Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS3 of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series. Figure

Mass Spectrometric Characterization of Therapeutic Nucleic Acids

The power of high-resolution mass spectrometry for the investigation of higher-order nucleic acid structures and highly modified nucleic acids is demonstrated in the present study. Experiments on selected G-rich oligodeoxynucleotides including tetra-, bi-, and monomolecular quadruplexes, provide new and detailed insight into their gas-phase dissociation. Experimental challenges are discussed which arise upon preparation and mass spectrometric analysis of DNA quadruplexes, e.g. the influence of cone voltages and the annealing conditions. Electrospray ionization tandem mass spectrometric experiments revealed the unique behavior of unplatinated and platinated quadruplexes. For the tetramolecular quadruplex, the major fragmentation channels are: i) strand separation, resulting in single-, double-, and triple-stranded ions. ii) successive decomposition of the quadruplex precursor ion by ammonia depletion followed by single or multiple release of an-B- and wm-fragments. In this process, truncated quadruplex ions (TQIs) are generated, which can undergo additional nucleobase losses. iii) the loss of nucleobases as a predominant dissociation pathway of intact tetramolecular quadruplexes, and the corresponding triplexes or TQIs. Results give evidence for the formation of the bimolecular DNA quadruplexes. Subsequent tandem mass spectrometric experiments on unplatinated bimolecular quadruplexes revealed ammonium ion loss as the initial dissociation event prior to disintegration into single strands. Incubation with cisplatin showed that the bimolecular type is partially prone to platination. Collision-induced dissociation (CID) experiments on platinated precursors revealed unplatinated and platinated single strands. The presence of these peaks indicates that cisplatin has bound to the loop region of the bimolecular quadruplex with stronger affinity to guanine, than to adenine. For the monomolecular quadruplex, ammonia depletion and subsequent disintegration into the linear strand was found to be the initial dissociation event, followed by backbone fragmentation, which results in the generation of DNA-typical fragments. In a second part, new mass spectrometric results describing sugar-modified oligonucleotides are presented. Accurate analytical tools are needed for the comprehensive structural elucidation of synthetic modified oligonucleotides and for investigation of the interaction between the drugs and native nucleic acids. Classical, enzyme-based sequencing techniques are no longer applicable for characterization of highly modified nucleic acids. ESI-MS/MS is an alternative analytical method to gain sequence and structure information. Mechanistic aspects of the gas-phase dissociation of structurally modified nucleic acids are described. Since homo-DNA, bicyclo-, and tricyclo-DNA exhibit altered sugar moieties, new gas-phase fragmentation mechanisms are expected and proposed. Moreover, the formation of higher-order assemblies such as homo-DNA duplexes, as well as bicyclo-DNA triplexes and quadruplexes is demonstrated for the first time. Finally, a novel Java-based software tool is presented, which was developed to assist mass spectra interpretation of natural and modified nucleic acids. The tool consists of two parts: (i) the oligonucleotide mass assembler (OMA), which calculates the electrospray series and fragment ions of a sequence of interest, and (ii) the oligonucleotide peak analyzer (OPA), which subsequently compares the OMA-generated peak lists with experimental data sets resulting from the MS experiments. The tool provides the possibility to implement user-defined building blocks (linkers, sugar-moieties, and nucleobases) into the OMA, thus rendering the software expandable and extremely versatile. The ease of use of the software is demonstrated on the example of the platinated DNA duplexes and quadruplexes. The results show the influence of cisplatin on the gasphase fragmentation of these higher-order structures. Additionally, software-supported interpretation of product ion spectra resulting from the analysis of tricyclo-DNA revealed its unique gas-phase dissociation characteristics

Elucidation of Nucleic Acid–Drug Interactions by Tandem Mass Spectrometry

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid–drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO–), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the MNCO– ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3–)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3– loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO– and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar modified ONs

OMA and OPA—Software-Supported Mass Spectra Analysis of Native and Modified Nucleic Acids

The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated doublestranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structures