Fast atom bombardment combined with tandem mass spectrometry for determining structures of small oligonucleotides

To reveal the gas-phase chemistry of RNA and DNA fragmentation during MALDI mass spectrometry in positive ion mode, we performed hydrogen/deuterium exchange on a series of RNA and DNA tetranucleotides and studied their fragmentation patterns on a highresolution MALDI TOF-TOF instrument. We were specifically interested in elucidating the remarkably different fragmentation behavior of RNA and DNA, i.e., the characteristic and abundant production of c-and y-ions from RNA versus a dominating generation of (a-B)-and w-ions from DNA analytes. The analysis yielded important information on all significant backbone cleavages as well as nucleobase losses. Based on this, we suggest common fragmentation mechanisms for RNA and DNA as well as an important RNA-specific reaction requiring a 2=-hydroxyl group, leading to c-and y-ions. The data is viewed and discussed in the context of previously published data to obtain a coherent picture of the fragmentation of singly protonated nucleic acids. (J Am Soc Mass Spectrom 2006, 17, 1353-1368

Section: C-and Y-ionsmentioning

confidence: 99%

RNA fragmentation in MALDI mass spectrometry studied by H/D-exchange: Mechanisms of general applicability to nucleic acids

Andersen

Kirpekar

Haselmann

2006

“…The few reports found indicate that dissociation of RNA clearly differs from the dissociation of DNA. The preferred cleavage site of RNA is the 5=-POO bond, resulting in the formation of c-and y-type ions [24][25][26]. It has been shown that oligoribonucleotides possess higher gas-phase stability than oligodeoxyribonucleotides because of stabilization of the N-glycosidic bond by the 2=-OH substituent [10,27].…”

mentioning

confidence: 99%

Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry

Tromp

Schürch

2005

Oligoribonucleotides (RNA) and modified oligonucleotides were subjected to low-energy collision-induced dissociation in a hybrid quadrupole time-of-flight mass spectrometer to investigate their fragmentation pathways. Only very restricted data are available on gas-phase dissociation of oligoribonucleotides and their analogs and the fundamental mechanistic aspects still need to be defined to develop mass spectrometry-based protocols for sequence identification. Such methods are needed, because chemically modified oligonucleotides can not be submitted to standard sequencing protocols.In contrast to the dissociation of DNA, dissociation of RNA was found to be independent of nucleobase loss and it is characterized by cleavage of the 5=-POO bond, resulting in the formation of c-and their complementary y-type ions. To evaluate the influence of different 2=-substituents, several modified tetraribonucleotides were analyzed. Oligoribonucleotides incorporating a 2=-methoxy-ribose or a 2=-fluoro-ribose show fragmentation that does not exhibit any preferred dissociation pathway because all different types of fragment ions are generated with comparable abundance. To analyze the role of the nucleobases in the fragmentation of the phosphodiester backbone, an oligonucleotide lacking the nucleobase at one position has been studied. Experiments indicated that the dissociation mechanism of RNA is not influenced by the nucleobase, thus, supporting a mechanism where dissociation is initiated by formation of an intramolecular cyclic transition state with the 2=-hydroxyl proton bridged to the 5=-phosphate oxygen. A ntisense oligonucleotides are nucleic acids of about 12 to 20 nucleobases, which are designed to hybridize to a complementary messenger RNA (mRNA) sequence, thus, inhibiting gene expression [1][2][3]. Therefore, they are of great interest in human cancer therapy and for diagnostic applications. Besides the high binding specificity of antisense oligonucleotides to their target mRNA, affinity, bioavailability, and biostability are of foremost importance. However, a major drawback of the application of unmodified phosphodiester DNA or RNA oligonucleotides is that these structures are subjected to rapid nuclease degradation under physiological conditions. To improve these factors, oligonucleotide analogs, exhibiting chemical modifications of the phosphodiester backbone, of the ribose, and, to a limited extent, also of the nucleobases, are evaluated.Mechanisms of inhibition of gene expression by antisense oligonucleotides involve mRNA degrading enzymes such as RNase H, blockade of translation initiation, or modulation of splicing [4][5][6][7]. These mechanisms allow the application of new chemical modifications to increase the binding affinity and the nuclease resistance. Possible positions of the modifications are the phosphodiester backbone, the sugar unit and the nucleobases. Introduction of a phosphorothioate backbone increases nuclease resistance of antisense oligonucleotides and simultaneously serves as a very efficient su...

“…Tandem mass spectrometry has been extensively used to identify the sequence of oligonucleotide samples [42][43][44][45][46] and characterize their covalent modifications [47][48][49][50][51] based on characteristic ions series produced by fragmentation of the nucleic acid backbone. In contrast, gas-phase activation of small ligand-nucleic acid complexes has been more actively pursued to investigate their stability rather than to map the actual binding sites [52][53][54][55][56].…”

mentioning

confidence: 99%

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry

Turner

Hagan

Kohlway

et al. 2006

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (⌿-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the ⌿-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC·SL3 and NC·SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC·SL2 complex. . The relatively low mutation frequency of this ϳ120 nt stretch of genomic RNA makes the packaging signal a very promising target for the development of new therapeutic strategies to contend with the rapid emergence of drug resistant strains [7,8]. For this reason, steps have been made toward the identification of possible inhibitors that may bind specific ⌿-RNA structures and disrupt their normal functions [9 -11]. In a systematic investigation of classic nucleic acid ligands, we have recently shown that aminoglycosidic antibiotics are not only capable of binding individual domains of ⌿-RNA, but can also inhibit their interactions with NC in a structure-specific fashion [12]. Initial tests to locate the actual binding sites on the different RNA substrates, which is crucial to explain the mechanism of preferential inhibition, were based on the analysis of mutant-ligand complexes performed by electrospray ionization (ESI) [13,14] and Fourier transform mass spectrometry (FTMS) [15,16].The favorable energetics characteristic of ESI enable the analysis of relatively labile noncovalent complex...