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.     

Erratum to: Investigation of metal-oligonucleotide complexes by nanoelectrospray tandem mass spectrometry in the positive mode

The formation and fragmentation of multiply metal-coordinated oligonucleotides was studied by nanoelectrospray tandem mass spectrometry in the positive ion mode. Fundamental aspects of the gas-phase behavior of metal-oligonucleotide complexes are revealed. The addition of transition metal ions, such as iron(II), iron(III), and zinc(II), leads to very stable metal-oligonucleotide complexes which show heavily altered fragmentation patterns in contrast to uncomplexed oligonucleotides. The site of metal ion complexation was located by collision-induced dissociation (CID) experiments. It was found that all three metal ions investigated predominantly coordinate to the central phosphate groups of the oligonucleotides. Furthermore, it is demonstrated that the fragmentation of such complexes depends highly upon the metal ion complexed as well as on the sequence of the nucleobases in the oligonucleotide.     

Investigation of metal-oligonucleotide complexes by nanoelectrospray tandem mass spectrometry in the positive mode

The formation and fragmentation of multiply metal-coordinated oligonucleotides was studied by nanoelectrospray tandem mass spectrometry in the positive ion mode. Fundamental aspects of the gas-phase behavior of metal-oligonucleotide complexes are revealed. The addition of transition metal ions, such as iron(II), iron(III), and zinc(II), leads to very stable metal-oligonucleotide complexes which show heavily altered fragmentation patterns in contrast to uncomplexed oligonucleotides. The site of metal ion complexation was located by collision-induced dissociation (CID) experiments. It was found that all three metal ions investigated predominantly coordinate to the central phosphate groups of the oligonucleotides. Furthermore, it is demonstrated that the fragmentation of such complexes depends highly upon the metal ion complexed as well as on the sequence of the nucleobases in the oligonucleotide.     

Positive ion formation in the ultraviolet matrix-assisted laser desorption / ionization analysis of oligonucleotides by using 2,5-dihydroxybenzoic acid

The development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its demonstrated performance with large proteins has generated substantial interest in utilizing this technique as an alternative to gel electrophoresis for DNA sequence analysis. However, a lack of understanding of the desorption and ionization processes has greatly hampered advances in this field. This article explores the formation of positively charged oligonucleotides in UV (355-nm) MALDI analysis by using the matrix 2,5-dihydroxybenzoic acid. Whereas substantial fragmentation is observed in the positive-ion mode by using the short oligomer d(TAGGT), no fragmentation is evident in the negative-ion mode under identical conditions. The fragmentation products are consistent with a previously published model in which base protonation initiates base loss, which leads to subsequent cleavage of the phosphodiester backbone. Several polydeoxythymidilic acids containing modified nucleosides were used to investigate positive-ion formation. The results support the hypothesis that positive ions are formed by protonation of the nucleobases. Because base protonation initiates base loss, fragmentation is intrinsic to positive-ion formation in the MALDI analysis of oligonucleotides. This result explains the dramatic difference in fragmentation observed in positive-ion compared to negative-ion UV-MALDI mass spectra of oligonucleotides.     

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.     

Fragmentation mechanisms of oligodeoxynucleotides: Effects of replacing phosphates with methylphosphonates and thymines with other bases in T-rich sequences

This article reports another step in an ongoing effort to understand the fragmentation of T-rich oligodeoxynucleotides. We extended an earlier investigation of T-rich 4-mers to T-rich 6-mers, 8-mers and 10-mers by using four different tandem mass spectrometric methods. The methods include low-energy collisionally activated decomposition (CAD) of electrospray ionization (ESI)-produced ions, source-CAD of ESI-produced ions, post-source decay (PSD), and CAD of matrix assisted laser desorption ionization (MALDI)-generated ions. The most abundant fragment ions produced from [M - 2H]2- precursors upon low-energy CAD in an ion trap are the [a - B]- and their complementary w ions. The predominant cleavage sites for T-rich oligodeoxynucleotides are always the 3' C-O bonds adjoining a non-T nucleobase (i.e., a base with a higher proton affinity (PA) than that of T). The relative abundance of [a - B]- correlates with the PAs of the nucleobases, underscoring the importance of proton transfer to the base. The propensity to form [a - B]- ions falls in the order of G > C approximately A > T. Structural isomers up to 10-mers can be readily sequenced and distinguished with each of the four tandem mass spectrometric methods applied. The fragmentation of oligodeoxynucleotides in which various phosphates were replaced with methylphosphonate is a measure of the participation of the phosphate proton in the formation of [a - B] ions. For 4 and 5-mers, transfer of an acidic proton from the 5'-phosphate to the departing base is the initiating step in the formation of [a - B]- ions.     

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.     

Influence of co-matrix proton affinity on oligonucleotide ion stability in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this article we investigated the role organic base co-matrices play in reducing oligonucleotide fragmentation during analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The organic base co-matrix plays an important role in influencing the gas-phase behavior of desorbed oligonucleotides. No correlation was found between the solution pH values and the molecular ion stability of two model oligonucleotides. Instead, a direct correlation between the co-matrix proton affinity and the oligonucleotide molecular ion stability is seen. A co-matrix whose proton affinity is close to or greater than the proton affinity of the nucleobases can serve as a “proton sink. ” We propose that upon laser desorption/ionization, the co-matrix competes with the nucleobases of the oligonucleotide for additional protons from the matrix. When a co-matrix such as triethylamine is added, the co-matrix, rather than the oligonucleotide nucleobases, is the preferred site of proton transfer from the matrix. Titration of standard oligonucleotide matrices with several co-matrices of differing proton affinity demonstrates that the co-matrix mole fraction is an important factor in oligonucleotide molecular ion stability. When the mole fraction of the co-matrix approaches that of the matrix, nearly complete elimination of oligonucleotide fragmentation is seen. Control experiments utilizing pyridine, a co-matrix whose proton affinity is less than that of thymine or the phosphodiester backbone, demonstrate that the co-matrix plays an active role in oligonucleotide stabilization. Information on matrix:co-matrix interactions with these analytes should facilitate improvements in MALDI-MS of oligonucleotides.     

Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides

The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.     

Massenspektrometrische Untersuchung von Amiden–I: Energetische Betrachtungen zur el Ektronenstosin-Duzierten Fragmentierung von Piperidin- und Piperidein- Amiden

The activation energies for the formation of [RC?O]⁺-ions from different piperidine and piperideine-amides have been estimated, and a connection with the C? N-bonding energy of the amide function has been discussed. A cyclic fragmentation mechanism has also been established by means of energetic points.     

Sodium controlled selective reactivity of protonated deoxy-oligonucleotides in the gas phase

Metastable fragmentation of the positively charged, hexameric oligonucleotides 5′-d(TTXYTT) (X and Y are dC, dG, or dA) and 5′-d(CTCGTT), 5′-d(TTCGTC) and 5′-d(CTCGTC) is studied after matrix assisted laser desorption/ionization (MALDI). The influence of the degree of sodiation, i.e., when the acidic protons are one by one exchanged against sodium ions, is systematically studied for the exchange of up to seven protons against sodium ions. Exchanging the acidic protons against sodium gradually quenches the backbone cleavage through the w and a-B channels, and quantitative quenching of these channels is generally achieved with the exchange of four protons against sodium ions. At the same time, the exchange of protons against sodium ions promotes the loss of a neutral, high proton affinity base. The formation of the w and a-B fragments is found to be highly dependent on the sequence of the central bases. A single mechanism consistent with these observations is proposed. In addition to the quenching of the classical w and a-B reaction channels, a drastic and abrupt on/off-switching of new reaction channels is observed as the degree of sodiation successively increases. These channels involve selective loss of the two central bases and the excision of a phosphodiester group and a sugar unit from the center of the oligonucleotides. Synchronously, the two terminal fragments recombine to form a tetramer containing the two terminal nucleosides from each end of the hexamer. Possible mechanism explaining these remarkable channels are discussed.     

Mass spectrometric investigation of the sequence selectivity for adduction of heterocyclic aromatic amines on single-strand oligonucleotides

Heterocyclic aromatic amines (HAAs) generated during the cooking of meats are known to be genotoxic substances able to form covalent bonds with DNA bases after metabolic activation. This work aimed at the investigation of the influence of the local environment of nucleobases along the nucleotidic sequence on its modification induced by two different HAAs, namely 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), in order to identify possible sequences more susceptible to modification. A systematic study of the neighbouring base effect on the adduction was emphasized. Thus, PhIP and IQ adducts have been synthesized with various T-rich model single-strand oligonucleotides displaying different flanking bases (A, G, C or T) at the 3' or the 5' side of the targeted guanine, which allowed a comparison of the flanking base effects on adduction. Modified oligonucleotides were then analyzed by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry. The localization of the modifications induced by PhIP or IQ along the oligonucleotide sequence was achieved by tandem mass spectrometry, and modification yields of the various model sequences were compared. Results indicate a favouring sequence context effect on the G-C8-IQ adduct formation with the sequence 5'GGG3'. Although higher than IQ, modification yields observed with PhIP showed a less obvious effect of the neighbouring base on the G-C8-PhIP adduct formation, with a preferential sequence 5'GGA/G/T3'.     

Macroscopic Self‐Assembly Based on Complementary Interactions between Nucleobase Pairs

We have created a selective macroscopic self‐assembly process by using polymer gels modified with complementary DNA oligonucleotides or nucleobases. The hydrogels modified with complementary DNA oligonucleotides adhered to each other by simple contact. The organogels modified with complementary nucleobases selectively formed macroscopic assemblies by agitation in nonpolar organic solvents. The adhesion strength of each gel was estimated semi‐quantitatively by stress–strain measurements. We achieved direct adhesion between macroscopic materials both in water and in organic media, based on complementary hydrogen bonds.     

Matrix-assisted laser desorption mass spectrometry of oligodeoxythymidylic acids.

Ferulic acid, sinapinic acid and 2,5-dihydroxybenzoic acid (DHBA) have been tested as matrix materials for matrix-assisted laser desorption of the pure oligonucleotide pd(T)12 and a mixture of oligonucleotides pd(T)12 through pd(T)18 using pulsed 337 nm radiation combined with reflecting time-of-flight mass spectrometry. The three matrix materials are compared with respect to obtainable mass resolution, degree of fragmentation, and adduct formation for these oligonucleotides. DHBA was found to produce the least fragmentation and adduct formation, as well as the highest mass resolution.     

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.     

Conformational change induced by metal-ion-binding to DNA containing the artificial 1,2,4-triazole nucleoside

A conformational switch can be induced upon the addition of transition-metal ions to oligonucleotides that contain a row of successive artificial nucleobases flanked by complementary sequences of natural nucleobases, provided that the artificial bases cannot undergo self-pairing via hydrogen bonding but only via the formation of metal-ion-mediated base pairs. Such oligonucleotides adopt a hairpin structure in the absence of transition-metal ions, yet they show a preference for the formation of a regular double helix if the appropriate metal ions are present. We report here our experimental data on the structure of the oligonucleotide d(A7X3T7) (A=adenine, T=thymine, X=1,2,4-triazole) in the absence and presence of silver(I). This study comprising temperature-dependent UV spectroscopy, CD spectroscopy, MALDI-TOF measurements, fluorescence spectroscopy, and dynamic light scattering opens up a new approach to the generation of a large variety of metal-ion sensors with the possibility of fine-tuning their sensing capabilities, depending on the artificial nucleoside that is used.     

Zum Mechanismus Massenspektrometrischer Fragmentierungsreaktionen—V: Substituenteneffekte auf Intramolekulare Substitutionen des Phenylrestes in den Massenspektren von N,N-Dimethyl-N′-Phenylformamidinen

The mass spectrum of N,N-dimethyl-N′-phenyl-formamidine (I) contains a large peak due to [M ? H]+-ions. As is shown by deuterium labelling, one of the ortho hydrogen atoms of the phenyl group is lost. The same result has been observed for the corresponding fragmentation of thioformanilide (V). This can be explained by the formation of benzimidazolium-ions (a). The effect of substituents at the phenyl group on the intensities and AP of these ions and on the IP of the molecular ions has been investigated. A mechanism of the cyclization reaction is proposed.     

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.     

Development of bridged nucleic acid analogues for antigene technology

In the last decade, increased efforts have been directed toward the development of oligonucleotide-based technologies for genome analyses, diagnostics, or therapeutics. Among them, an antigene strategy is one promising technology to regulate gene expression in living cells. Stable triplex formation between the triplex-forming oligonucleotide (TFO) and the target double-stranded DNA (dsDNA) is fundamental to the antigene strategy. However, there are two major drawbacks in triplex formation by a natural TFO: low stability of the triplex and limitations of the target DNA sequence. To overcome these problems, we have developed various bridged nucleic acids (BNAs), and found that the 2',4'-BNA modification of oligonucleotides strongly promotes parallel motif triplex formation under physiological conditions. Some nucleobase analogues to extend the target DNA sequence were designed, synthesized, and introduced into the 2',4'-BNA structure. The obtained 2',4'-BNA derivatives with unnatural nucleobases effectively recognized a pyrimidine-purine interruption in the target dsDNA. Some other examples of nucleic acid analogues for stable triplex formation and extension of the target DNA sequence are also summarized.