Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

C‐5 Propynyl Modifications Enhance the Mechanical Stability of DNA

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or ‐medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base‐pairing and easy integration during chemical DNA synthesis. Through single‐molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C‐5 propynyl modifications on the mechanical stability of double‐stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non‐modified DNA duplexes.     

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

We report herein a systematic mass spectrometric study of a series of thirty-one non-self-complementary, 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 Tm 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 O6-methylguanine causes mutations.     

Interactions of mitoxantrone with duplex and triplex DNA studied by electrospray ionization mass spectrometry

We have examined interactions between mitoxantrone (MXT) and DNA duplexes or triplexes with different base compositions by using electrospray ionization mass spectrometry (ESI‐MS), respectively. MXT interacts preferentially with DNA duplexes compared to the triplexes. In the mass spectrum of the duplex–MXT mixture, the complex peaks dominated in the ratios of duplex/MXT of 1:1, 1:2 and 1:3, and the 1:2 duplex/MXT peak was the most abundant. In contrast, only 1:1 triplex–MXT complexes were observed in the mass spectrum of the triplex–MXT mixture, and the most intensive peak was a free triplex ion without MXT. Moreover, no sequence selectivity of MXT to different DNA duplexes was found while MXT showed greater affinity to the triplexes that have adjacent TAT or C⁺GC sequences. In the course of sustained off‐resonance irradiation collision‐induced dissociation (SORI‐CID), the MXT‐duplex complexes generated two separated strands, and the MXT remained on the purine strand side. UV/Vis spectra showed that MXT interacted with DNA by intercalation. Compared with emodin (a duplex intercalator) and napthylquinoline (a triplex binder), we found that the side chain of MXT might play a role in the binding of MXT to the duplexes and the triplexes. ESI‐MS shows an advantage in speed and straightforwardness for the study of drug interactions with nucleic acids. Copyright © 2008 John Wiley & Sons, Ltd.     

The structure of a mixed LNA/DNA:RNA duplex is driven by conformational coupling between LNA and deoxyribose residues as determined from 13C relaxation measurements

A study of the internal dynamics of an LNA/DNA:RNA duplex has been performed to further characterize the conformational changes associated with the incorporation of locked nucleic acid (LNA) nucleotides in a DNA:RNA duplex. In general, it was demonstrated that the LNA/DNA:RNA duplex has a very high degree of order compared to dsDNA and dsRNA duplexes. The order parameters of the aromatic carbon atoms in the LNA/DNA strand are uniformly high, whereas a sharp drop in the degree of order was seen in the RNA strand in the beginning of the AUAU stretch in the middle of the strand. This can be related to a return to normal dsRNA dynamics for the central A:U base pair. The high order of the heteroduplex is consistent with preorganization of the chimera strand for an A-form duplex conformation. These results partly explain the dramatic increase in T(m) of the chimeric heteroduplex over dsDNA and DNA:RNA hybrids of the same sequence.     

Stabilization of a Bimolecular Triplex by 3′‐S‐Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single‐stranded nucleic acids are also relevant in third‐strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson–Crick (W–C) base‐paired DNA duplexes and a Hoogsteen base‐paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA‐like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3′‐S‐phosphorothiolate (3′‐SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. ¹H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3′‐SP modifications is more compact. The 3′‐SP modification was previously shown to stabilise G‐quadruplex and i‐motif structures and therefore is now proposed as a generic solution to stabilising multi‐stranded DNA structures.     

The structure of LNA:DNA hybrids from molecular dynamics simulations: the effect of locked nucleotides

Locked nucleic acids (LNAs) exhibit a modified sugar fragment that is restrained to the C3'-endo conformation. LNA-containing duplexes are rather stable and have a more rigid structure than DNA duplexes, with a propensity for A-conformation of the double helix. To gain detailed insight into the local structure of LNA-modified DNA oligomers (as a foundation for subsequent exploration of the electron-transfer capabilities of such modified duplexes), we carried out molecular dynamics simulations on a set of LNA:DNA 9-mer duplexes and analyzed the resulting structures in terms of base step parameters and the conformations of the sugar residues. The perturbation introduced by a single locked nucleotide was found to be fairly localized, extending mostly to the first neighboring base pairs; such duplexes featured a B-type helix. With increasing degree of LNA modification the structure gradually changed; the duplex with one complete LNA strand assumed a typical A-DNA structure. The relative populations of the sugar conformations agreed very well with NMR data, lending credibility to the validity of the computational protocol.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

The Detection and Stability of DNA Duplexes Probed by MALDI Mass Spectrometry

Matrix‐assisted laser desorption/ionization (MALDI) with nonacidic matrices is shown to generate intact gas‐phase ions of double‐stranded DNA. Control experiments show that specific DNA duplexes are detected by this method, while nonspecific adducts form only when high monomer concentrations are present in the MALDI sample. The relative intensity of the duplex‐ion signal is found to reflect the solution‐phase stability of the double‐stranded DNA.     

Structural motifs of DNA complexes in the gas phase

DNA duplexes are known to be quite stable in the condensed phase but recent mass spectrometry results have shown that DNA complexes are also stable (at least for a limited time) in the gas phase. However, very little is known about the overall shape of the complexes in a solvent-free environment and what factors influence that shape. In this article, we present recent ion mobility and molecular modeling results that address some issues concerning the gas-phase conformations of DNA duplexes. Examples include the effect of metal ions on Watson–Crick base pairing, investigating the onset of helicity in duplexes as a function of strand length, comparison of the stability of C·G and A·T base pairs, and examining the formation of quadruplex structures.     

Fragmentation studies of sartans by electrospray ionization mass spectrometry

Sartans and related analogues with 5‐oxo‐l, 2, 4‐oxadiazole ring and tetrazole ring are investigated in detail using collision‐induced dissociation (CID) method in positive ion mode by electrospray ionization tandem mass spectrometry (ESI‐MSⁿ). It is found that the protonated sartans and related analogues tend to form the N‐substituted‐3‐substituted phenanthridin‐6‐amine ion which has a large conjugative structure. The possible fragmentation pathways were proposed for the first time, and the key structure of product ions was confirmed by high resolution tandem mass spectrometry and theoretical calculation. It is very helpful for understanding the intriguing roles of sartans analogues in fragmentation reactions and enriching the knowledge of the gas‐phase chemistry of the oxadiazole and tetrazole ring. Copyright © 2017 John Wiley & Sons, Ltd.     

Asymmetric charge partitioning upon dissociation of DNA duplexes

Upon collisional activation, a series of DNA duplexes exhibited a significant degree of asymmetric dissociation with respect to charge partitioning among the single strands. That is, the charge states of the single strand product ions did not equal q/2 for even precursor charge states or (q + 1)/2 and (q − 1)/2 for odd precursor charge states (where q is the charge of the precursor). The factors that affect this asymmetric charge partitioning were assessed. The smaller, lower charged duplexes resulted in more symmetric dissociation compared with larger duplexes in higher charge states, which displayed a high degree of asymmetry upon dissociation. The composition of the duplexes influenced charge partitioning, with those containing a greater number of A/T base pairs showing more symmetric dissociation relative to the more G/C rich duplexes. The use of higher collisional energies resulted in significantly more asymmetric dissociation. Comparisons were made with the dissociation behavior previously studied for protein noncovalent complexes and past studies of the gas-phase conformations and dissociation of DNA complexes.     

Use of ion mobility mass spectrometry and a collision cross‐section algorithm to study an organometallic ruthenium anticancer complex and its adducts with a DNA oligonucleotide

We report the development of an enhanced algorithm for the calculation of collision cross‐sections in combination with Travelling‐Wave ion mobility mass spectrometry technology and its optimisation and evaluation through the analysis of an organoruthenium anticancer complex [(η⁶‐biphenyl)Ru^II(en)Cl]⁺. Excellent agreement was obtained between the experimentally determined and theoretically determined collision cross‐sections of the complex and its major product ion formed via collision‐induced dissociation. Collision cross‐sections were also experimentally determined for adducts of this ruthenium complex with the single‐stranded oligonucleotide hexamer d(CACGTG). Ion mobility tandem mass spectrometry measurements have allowed the binding sites for ruthenium on the oligonucleotide to be determined. Copyright © 2009 John Wiley & Sons, Ltd.     

Negative charge induced dissociation: fragmentation of deprotonated N‐benzylidene‐2‐hydroxylanilines in electrospray ionization mass spectrometry

Unimolecular reactivities of different N‐benzylidene‐2‐hydroxylaniline anions were investigated in gas phase by electrospray ionization tandem mass spectrometry. All the collision‐induced dissociation spectra of N‐benzylidene‐2‐hydroxylaniline anions show similar ions at phenyl anions, neutral loss of benzonitrile and benzoxazole anions, respectively. The possible fragmentation pathway was probed through deuterium labeling and various group substituents experiments. Computational results were applied to shed light on the mechanism of fragmentation patterns. The proton in the CH=N is reactive in the formation of the concerned ions. Its direct transfer to the oxygen results in 2‐hydroxyphenyl anion. Proton abstraction between benzoxazole and phenyl anion leads to the formation of benzene and benzoxazole anion. Copyright © 2014 John Wiley & Sons, Ltd.     

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.     

Characterization of multiple fragmentation pathways initiated by collision‐induced dissociation of multifunctional anions formed by deprotonation of 2‐nitrobenzenesulfonylglycine

The correlation of anion structure with the fragmentation behavior of deprotonated nitrobenzenesulfonylamino acids was investigated using tandem mass spectrometry, isotopic labeling and computational methods. Four distinct fragmentation pathways resulting from the collision‐induced dissociation (CID) of deprotonated 2‐nitrobenzenesulfonylglycine (NsGly) were characterized. The unusual loss of the aryl nitro substituent as HONO was the lowest energy process. Subsequent successive losses of CO, HCN and SO₂ indicated that an ortho cyclization reaction had accompanied loss of HONO. Other pathways involving rearrangement of the ionized sulfonamide group, dual bond cleavage and intramolecular nucleophilic displacement were proposed to account for the formation of phenoxide, arylsulfinate and arylsulfonamide product ions at higher collision energies. The four distinct fragmentation pathways were consistent with precursor–product relationships established by CID experiments, isotopic labeling results and the formation of analogous product ions from 2,4‐dinitrobenzenesulfonylglycine and the Ns derivatives of alanine and 2‐aminoisobutyric acid. The computations confirmed a low barrier for ortho cyclization with loss of HONO and feasible energetics for each reaction step in the four pathways. Computations also indicated that three of the fragmentation pathways started from NsGly ionized at the carboxyl group. Overall, the pathways identified for the fragmentation of the NsGly anion differed from processes reported for anions containing a single functional group, demonstrating the importance of functional group interactions in the fragmentation pathways of multifunctional anions. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of in‐source collision‐induced dissociation and beam‐type collision‐induced dissociation of emerging synthetic drugs using a high‐resolution quadrupole time‐of‐flight mass spectrometer

In‐source collision‐induced dissociation (CID) is commonly used with single‐stage high‐resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in‐source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in‐source CID and beam‐type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in‐source CID. Electrospray ionization–quadrupole time‐of‐flight (ESI‐Q‐TOF) mass spectrometry was used to generate product ion spectra from in‐source CID and beam‐type CID for a series of well‐characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in‐source CID and a range of collision energies for beam‐type CID. The results indicate that large fragmentor potentials for in‐source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam‐type CID. Although conditions can be found in which in‐source CID and beam‐type CID provide similar overall spectra, the in‐source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam‐type CID.     

Electrospray‐Ionization Mass Spectrometry for Screening the Specificity and Stability of Single‐Stranded‐DNA Templated Self‐Assemblies

Supramolecular complexes consisting of a single‐stranded oligothymine ( dTn ) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen‐bonding unit have been studied with electrospray‐ionization mass spectrometry (ESI‐MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn . By changing the hydrogen‐bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single‐stranded‐DNA (ssDNA)–guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20‐component self‐assembled objects. Gas‐phase breakdown experiments on single‐ and multiple‐guest–DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI‐MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.     

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

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