Detection of oligonucleotide duplex forms by ion-spray mass spectrometry

We report herein a systematic mass spectrometric study of a series of thirty-one non-selfcomplementary, matched, DNA duplexes ranging in size from 5-to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same T m yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin "charge level" to compare our results with others' and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O 6 -methylguanine causes mutations. (J Am Soc Mass Spectrom 2006, 17, 1383-1395

Section: Full-scan Mass Spectrometry Of Dna Duplexessupporting

confidence: 86%

DNA stability in the gas versus solution phases: A systematic study of thirty-one duplexes with varying length, sequence, and charge level

Pan

Sun

Lee

2006

“…The advent of soft ionization techniques and the advances of analyzers design have made this possibility a reality. Indeed, relatively short duplexes formed by complementary deoxyoligonucleotides were among the first noncovalent complexes detected by ESI without unwelcome dissociation [95][96][97]. Although this desirable outcome can be obtained also by MALDI with a judicious selection of matrix, additives, and other experimental conditions [98 -101], ESI remains the ionization technique of choice for the detection of nucleic acid noncovalent assemblies (reviewed in references [102][103][104][105]).…”

Section: Elucidating Structure-function Relationshipsmentioning

confidence: 99%

A eole for the MS analysis of nucleic acids in the post-genomics age

Fabris

2010

The advances of mass spectrometry in the analysis of nucleic acids have tracked very closely the exciting developments of instrumentation and ancillary technologies, which have taken place over the years. However, their diffusion in the broader life sciences community has been and will be linked to the ever evolving focus of biomedical research and its changing demands. Before the completion of the Human Genome Project, great emphasis was placed on sequencing technologies that could help accomplish this project of exceptional scale. After the publication of the human genome, the emphasis switched toward techniques dedicated to the exploration of sequences not coding for actual protein products, which amount to the vast majority of transcribed elements. The broad range of capabilities offered by mass spectrometry is rapidly advancing this platform to the forefront of the technologies employed for the structure-function investigation of these noncoding elements. Increasing focus on the characterization of functional assemblies and their specific interactions has prompted a re-evaluation of what has been traditionally construed as nucleic acid analysis by mass spectrometry. Inspired by the accelerating expansion of the broader field of nucleic acid research, new applications to fundamental biological studies and drug discovery will help redefine the evolving role of MS-analysis of nucleic acids in the post-genomics age. (J Am Soc Mass Spectrom 2010, 21, 1-13)

“…Duplexes can be transferred into the gas phase by soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) without dissociation of the double strands occurring [9][10][11][12]. Soon after successful ionization of duplex DNA, first tandem mass spectrometric (MS/MS) studies were published [13,14].…”

Section: Introductionmentioning

confidence: 99%

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

Nyakas

Blum

Stucki

et al. 2012

Abstract. 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.