Mass Spectrometric Characterization of Therapeutic Nucleic Acids

Abstract

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.