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

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'-P-O 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.     

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions.     

New aspects of the fragmentation mechanisms of unmodified and methylphosphonate-modified oligonucleotides

A set of pentanucleotides was investigated by electrospray tandem mass spectrometry with the focus on the fragmentation mechanism. Results reveal new aspects of the fragmentation mechanism of modified and unmodified oligonucleotides and demonstrate the influence of the nucleobases on the decomposition of oligonucleotides. Adenine-rich oligonucleotides fragment easily resulting in abundant peaks corresponding to the DNA-typical a-B- and w-ions. On the other hand, thymine was found to have a stabilizing effect, which is reflected by the preferred formation of the w(4)-ions and the relatively low abundance of shorter w-ions upon dissociation of pentanucleotides. Data from investigation of the formation of w(4)-ions support a beta-elimination mechanism. Results obtained by investigation of oligonucleotides with an abasic site confirm this mechanism, which is independent of nucleobase loss. Experiments with methylphosphonate oligonucleotides show a remarkable change in the fragmentation pattern due to the modification. It was found that charges are located on the nucleobases and initiate the fragmentation mechanism. The stability of the oligonucleotide is reduced and no a-B-fragment ions are formed wherever there is a methylphosphonate group within the backbone. This fact also demonstrates that fragmentation is locally controlled.     

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.

An experimental and theoretical study of the gas-phase decomposition of monoprotonated peptide nucleic acids

Peptide nucleic acids (PNAs) are DNA/RNA mimics which have recently generated considerable interest due to their potential use as antisense and antigene therapeutics and as diagnostic and molecular biology tools. These synthetic biomolecules were designed with improved properties over corresponding oligonucleotides such as greater binding affinity to complementary nucleic acids, enhanced cellular uptake, and greater stability in biological systems. Because of the stability and unique structure of PNAs, traditional sequence confirmation methods are not effective. Alternatively, electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry shows great potential as a tool for the characterization and structural elucidation of these oligonucleotide analogs. Extensive gas-phase fragmentation studies of a mixed nucleobase 4-mer (AACT) and a mixed nucleobase 4-mer with an acetylated N-terminus (N-acetylated AACT) have been performed. Gas-phase collision-induced dissociation of PNAs resulted in water loss, cleavage of the methylene carbonyl linker containing a nucleobase, cleavage of the peptide bond, and the loss of nucleobases. These studies show that the fragmentation behavior of PNAs resembles that of both peptides and oligonucleotides. Molecular mechanics (MM+), semiempirical (AM1), and ab initio (STO-3G) calculations were used to investigate the site of protonation and determine potential low energy conformations. Computational methods were also employed to study prospective intramolecular interactions and provide insight into potential fragmentation mechanisms.     

Fragmentation of oligoribonucleotides from gas-phase ion-electron reactions

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.     

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

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 high-resolution 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.     

Structural Aspects of Nucleic Acid Analogs and Antisense Oligonucleotides

Hybridization of complementary oligonucleotides is essential to highly valuable research tools in many fields including genetics, molecular biology, and cell biology. For example, an antisense molecule for a particular segment of sense messenger RNA allows gene expression to be selectively turned off, and the polymerase chain reaction requires complementary primers in order to proceed. It is hoped that the antisense approach may lead to therapeutics for treatment of various diseases including cancer. Areas of active research in the antisense field focus on the mechanisms of cellular uptake of antisense molecules and their delivery to specific cell sites, an improved understanding of how these molecules inhibit the production of proteins, as well as the optimization of the chemical stability of antisense molecules and the thermodynamic stability of the duplexes they form with the mRNA targets. The last two issues in particular have prompted chemists to launch an extensive search for oligonucleotide analogs with improved binding properties for hybridization with RNA and higher resistance toward nuclease degradation. During the last years this research has resulted in a flurry of new chemical analogs of DNA and RNA with modifications in the sugar–phosphate backbone as well as in the nucleobase sites. However, to date little effort has been directed toward uncovering the exact origins of the gain or loss in stability when nucleic acid analogs bind to RNA. Although large amounts of thermodynamic data have been collected, the structural perturbations induced by the modifications in hybrid duplexes are only poorly understood. For many modified oligonucleotides the compatibility of protection, coupling, and deprotection chemistry with standard DNA and RNA synthesis protocols makes it now possible to generate modified nucleic acid fragments or mixed oligonucleotides containing modifications at selected sites in quantities suitable for three-dimensional structure investigations. Such studies should reveal the structural origins of the observed changes in affinity and specificity of binding for particular modifications and may guide the development of second-and third-generation antisense molecules. In addition, the availability of a previously unimaginable variety of modified building blocks and the investigation of their structures provides the basis for a deeper understanding of the native DNA and RNA structures. This contribution will summarize the results of X-ray crystallographic structure determinations of modified nucleic acid fragments conducted in our laboratory during the last three years and the insights gained from them.     

Ion trap collision-induced dissociation of locked nucleic acids

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3′C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.     

Chemical basis of DNA sugar-phosphate cleavage by low-energy electrons

DNA damage by low-energy electrons (LEE) was examined using a novel system in which thin solid films of oligonucleotide tetramers (CGTA and GCAT) were irradiated with monoenergetic electrons (10 eV) under ultrahigh vacuum. The products of irradiation were examined by HPLC. These analyses permitted the quantitation of 16 nonmodified nucleobase, nucleoside, and nucleotide fragments of each tetramer resulting from the cleavage of phosphodiester and N-glycosidic bonds. The distribution of nonmodified products suggests a mechanism of damage involving initial electron attachment to nucleobase moieties, followed by electron transfer to the sugar-phosphate backbone, and subsequent dissociation of the phosphodiester bond. Moreover, virtually all the nonmodified fragments contained a terminal phosphate group at the site of cleavage. These results demonstrate that the phosphodiester bond breaks by a distinct pathway in which the negative charge localizes on the phosphodiester bond giving rise to nonmodified fragments with an intact phosphate group. Conversely, the radical must localize on the sugar moiety to give as yet unidentified modifications. In summary, the reaction of LEE with simple tetramers involved dissociative electron attachment leading to phosphodiester bond cleavage and the formation of nonmodified fragments.     

Synthesis and properties of novel l-isonucleoside modified oligonucleotides and siRNAs

Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this report, a novel isonucleoside containing a 5'-CH(2)-extended chain at the sugar moiety was synthesized, thus isoadenosine and isothymidine were incorporated into a DNA single strand and siRNA. It was found that isonucleoside modified oligonucleotides can form stable double helical structures with their complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified with isonucleoside () at 3' or 5' terminal can retain the silencing activity and minimize the passenger strand specific off-target effect.     

Caprin-1 Promotes Cellular Uptake of Nucleic Acids with Backbone and Sequence Discrimination

The cellular delivery of oligonucleotides has been a major obstacle in the development of therapeutic antisense agents. PNAs (Peptide Nucleic Acid) are unique in providing a modular peptidic backbone that is amenable to structural and charge modulation. While cationic PNAs have been shown to be taken up by cells more efficiently than neutral PNAs, the generality of uptake across different nucleobase sequences has never been tested. Herein, we quantified the relative uptake of PNAs across a library of 10 000 sequences for two different PNA backbones (cationic and neutral) and identified sequences with high uptake and low uptake. We used the high uptake sequence as a bait for target identification, leading to the discovery that a protein, caprin-1, binds to PNA with backbone and sequence discrimination. We further showed that purified caprin-1 added to cell cultures enhanced the cellular uptake of PNA as well as DNA and RNA.     

In vitro Selection of Chemically Modified DNAzymes

DNAzymes are in vitro selected DNA oligonucleotides with catalytic activities. RNA cleavage is one of the most extensively studied DNAzyme reactions. To expand the chemical functionality of DNA, various chemical modifications have been made during and after selection. In this review, we summarize examples of RNA-cleaving DNAzymes and focus on those modifications introduced during in vitro selection. By incorporating various modified nucleotides via polymerase chain reaction (PCR) or primer extension, a few DNAzymes were obtained that can be specifically activated by metal ions such as Zn²⁺ and Hg²⁺. In addition, some modifications were introduced to mimic RNase A that can cleave RNA substrates in the absence of divalent metal ions. In addition, single modifications at the fixed regions of DNA libraries, especially at the cleavage junctions, have been tested, and examples of DNAzymes with phosphorothioate and histidine-glycine modified tertiary amine were successfully obtained specific for Cu²⁺, Cd²⁺, Zn²⁺, and Ni²⁺. Labeling fluorophore/quencher pair right next to the cleavage junction was also used to obtain signaling DNAzymes for detecting various metal ions and cells. Furthermore, we reviewed work on the cleavage of 2′-5′ linked RNA and L-RNA substrates. Finally, applications of these modified DNAzymes as biosensors, RNases, and biochemical probes are briefly described with a few future research opportunities outlined at the end.     

Ion trap collision-induced dissociation of multiply deprotonated RNA: c/y-ions versus (a-B)/w-ions

The dissociation of model RNA anions has been studied as a function of anion charge state and excitation amplitude using ion trap collisional activation. Similar to DNA anions, the precursor ion charge state of an RNA anion plays an important role in directing the preferred dissociation channels. Generally, the complementary c/y-ions from 5′ P-O bond cleavage dominate at low to intermediate charge states, while other backbone cleavages appear to a limited extent but increase in number and relative abundance at higher excitation energies. The competition between base loss, either as a neutral or as an anion, as well as the preference for the identity of the lost base are also observed to be charge-state dependent. To gain further insight into the partitioning of the dissociation products among the various possible channels, model dinucleotide anions have been subjected to a systematic study. In comparison to DNA, the 2′-OH group on RNA significantly facilitates the dissociation of the 5′ P-O bond. However, the degree of excitation required for a 5′ base loss and the subsequent 3′ C-O bond cleavage are similar for the analogous RNA and DNA dinucleotides. Data collected for protonated dinucleotides, however, suggest that the 2′-OH group in RNA can stabilize the glycosidic bond of a protonated base. Therefore, base loss from low charge state oligonucleotide anions, in which protonation of one or more bases via intramolecular proton transfer can occur, may also be stabilized in RNA anions relative to corresponding DNA anions.     

Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)ⁿ⁺ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.     

PNA: Synthetic Polyamide Nucleic Acids with Unusual Binding Properties

The astonishing discovery that peptide nucleic acids (PNAs, B=nucleobase), in spite of their drastic structural difference to natural DNA, are better nucleic acid mimetics than many other oligonucleotides has resulted in an explosion of research into this class of compounds. The synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA are summarized here.     

Electron detachment dissociation for top-down mass spectrometry of acidic proteins

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.     

Electron Transfer Dissociation Reveals Changes in the Cleavage Frequencies of Backbone Bonds Distant to Amide-to-Ester Substitutions in Polypeptides

Interrogation of electron transfer dissociation (ETD) mass spectra of peptide amide-to-ester backbone bond substituted analogues (depsipeptides) reveals substantial differences in the entire backbone cleavage frequencies. It is suggested that the point permutation of backbone bonds leads to changes in the predominant ion structures by removal/weakening of specific hydrogen bonding. ETD responds to these changes by redistributing the cleavage frequencies of the peptide backbone bonds. In comparison, no distinction between depsi-/peptide was observed using collision-activated dissociation, which is consistent with a general unfolding and elimination of structural information of these ions. These results should encourage further exploration of depsipeptides for gas-phase structural characterization.     

Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.