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

  • Yvonne Hari
  • Branislav Dugovič
  • Alena Istrate
  • Annabel Fignolé
  • Christian J. Leumann
  • Stefan Schürch
Research Article

Abstract

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.

Graphical Abstract

Keywords

Antisense Duplex DNA Modified DNA Activation entropy Tandem mass spectrometry 

Introduction

tcDNA in Antisense Therapy

Antisense oligonucleotides are designed to modify the expression of a gene product by targeting specific RNAs. The antisense activity is either mediated via the induced degradation of the mRNA by RNase H or effected via steric blocking of the mRNA to alter or prevent splicing and translation [1]. Antisense oligonucleotides comprise chemically modified structures to enhance their resistance against endo- and exonucleases and to increase the binding affinity to complementary RNA. One group of antisense oligonucleotides is designed to restrict the steric flexibility of nucleic acids and to lock the sugar-moiety in a duplex-like conformation to improve the binding of targeted RNA. Examples of this type of antisense chemistry include locked nucleic acids and tricyclo-DNA. Tricyclo-DNA (tcDNA) is a structural analogue of DNA in which the 3'-carbon and the 5'-carbon atoms of the deoxyribose core are linked by an ethylene bridge with an annealed cyclopropane unit [2] (see Figure 1). The modification locks the sugar-moiety in the C2'-exo conformation typically observed for RNA in duplexes. This induced structural resemblance optimizes the binding to RNA targets. Thermal melting experiments confirm the remarkable stability of tcDNA:RNA heteroduplexes. A single tcDNA modification in a DNA:RNA heteroduplex with 12 base pairs can increase the melting temperature by up to +3.1 °C. For fully modified tcDNA oligonucleotides, a stabilization of +2.4 °C per modification was observed relative to the DNA:RNA hybrid [3]. The higher stability of the tcDNA-hybrids was found to arise from the decreased formation entropy of the modified duplexes attributed to the prearranged structure of the tcDNA single strand. Moreover, crystallographic data [4], CD spectroscopic analysis, and molecular dynamics simulations [3] suggest that in heteroduplexes the tricyclo sugar-moiety mimics the conformation of nucleic acids in A-form duplexes, which makes them selective for RNA over DNA.
Figure 1

The structure of tcDNA and nomenclature of typical backbone fragments

The selective binding of RNA targets encourages the application of tcDNA as antisense oligonucleotide. It is currently being tested for the treatment of Duchenne muscular dystrophy (DMD) [5]. DMD is the most severe form of muscular dystrophies and arises from a lack of the structural protein dystrophin due to random mutations or exon deletion in the dystrophin gene [6]. The goal of antisense therapy is to induce alternative splicing that leads to the expression of an internally deleted but functional protein. Goyenvalle et al. showed that tcDNA promotes significantly higher levels of dystrophin in the mdx mouse model than 2'-OMe-thiophosphate oligonucleotides and morpholino oligomers [5]. In the course of this study, first tandem mass spectrometric analysis of the antisense oligonucleotide revealed unique fragmentation characteristics that are discussed in more detail herein.

MS/MS of Oligonucleotides

Antisense oligonucleotides are designed to exhibit high in vivo stability, which renders them inaccessible for enzyme-based sequencing techniques. Today, tandem mass spectrometry (MS/MS) is the method of choice for the sequencing of structurally modified nucleic acids. Collision-induced dissociation (CID) leads to the scission of the sugar-phosphate backbone at one of the four phosphodiester bonds, giving rise to four ion pairs referred to as a-/w-, b-/x-, c-/y-, and d-/z-ions [7]. The loss of nucleobases constitutes an alternative or concomitant fragmentation pathway of oligonucleotides.

The preferred fragmentation channel is determined by the structure of the nucleic acid. A detailed account of the fragmentation pathways of ESI-generated natural and modified oligonucleotide ions can be found in a recent review [8]. In non-modified DNA, the most frequently observed fragments are a-B- and w-ions. This ion pair is formed in a two-step mechanism triggered by the loss of a nucleobase [9]. In RNA, the primary fragmentation products are c- and y-ions, which has been explained by an alternative fragmentation mechanism involving the 2'-OH group [10]. Moreover, some of the synthetic modifications introduced in antisense oligonucleotides are also known to alter the gas-phase dissociation mechanism. While thiophosphate oligonucleotides fragment in a DNA-like manner [11], no preferred dissociation channel was observed for 2'-methoxylated oligonucleotides [12], methylphosphonates [13], or locked nucleic acids [14]. In fact, the fragmentation patterns point towards the coexistence of multiple mechanisms. Recent tandem mass spectrometric studies on LNA [14] and homo-DNA [15] accentuate the impact of the sugar-moiety on the gas-phase dissociation of oligonucleotides. Consequently, understanding the fragmentation of tricyclo-DNA is a key factor for the reliable MS-based sequencing and quantification of the antisense oligonucleotides.

MS/MS of DNA Duplexes

There are two measures of the gas-phase stability of a duplex: the relative signal intensities of duplex and single strands and the activation energy required for fragmentation. The relative signal intensities of matched and mismatched duplexes correlate well with the in-solution stability of the duplex [16]. Alternatively, the stability of a duplex can be evaluated with the E50 value, which corresponds to the activation energy required to dissociate 50% of the duplex. In general, the E50 value and the melting temperature Tm measured in solution correlate for duplexes of similar GC content and identical size, which indicates that hydrogen bonding and base stacking are conserved in the gas phase [17, 18]. Although similar relationships between solution and gas-phase stability were demonstrated for DNA and RNA duplexes, there is no comprehensive study that directly compares different types of nucleic acid duplexes.

The significance of the activation energy determined in MS/MS experiments is limited, when alternative fragmentation channels, such as nucleobase loss and backbone cleavage, compete with strand separation. High charge states promote strand separation [19] and favor asymmetric charge distribution within the duplex [20]. The formation of backbone fragments, on the other hand, is promoted by high GC content [18] and generally indicates high gas-phase stability of the duplex. Double resonance experiments show that as in single strands, neutral base loss precedes backbone cleavage in the duplex [21]. Gabelica et al. [22] described product ions formed after the ejection of backbone fragments from the intact duplex, which are believed to arise from backbone cleavage in the single stranded, fraying ends of the oligonucleotides. This result suggests that the strand separation occurs by gradual unzipping of the duplex rather than a two-state transition and gives direct evidence for parallel fragmentation reactions. Whenever multiple dissociation pathways are accessible, the preferred channel is determined by two factors, the activation entropy and the activation enthalpy [23]. This reasoning was applied to duplexes [22], where changing the activation conditions was found to promote different dissociation channels. The authors argue that the rate constant for the strand dissociation increases more steeply with the internal energy of the system than the rate constant of the base loss. The entropy-favored channel can thus be accessed using fast activation regimes. Herein, we demonstrate how structural modifications can impact both the activation enthalpy and entropy of fragmentation channels and thus determine the preferred reaction pathway of nucleic acid heteroduplexes.

Experimental

Oligonucleotides, Chemicals, and Solvents

The DNA decamer 5'-AACTGTCACG-3' was purchased from Microsynth (Balgach, Switzerland) and used without further purification. Two sequence isomers containing single tcDNA modifications were synthesized according to a previously published, adapted phosphoramidite chemistry procedure [2]. The modified nucleotide in the decamer 10-2 (5'-AACTGTCACG-3') comprises an amide linker. The two tc-sugar-moieties in the decamer 10-3 (5'-AACTGTCACG-3') are fluorinated in the 2'-position. The oligonucleotides were diluted to a final concentration of 25 μM in a water:acetonitrile:TEA (49:49:2) solvent prior to analysis. Fluka HPLC water, Fluka HPLC acetonitrile, and triethylamine were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland).

For the characterization of duplexes, nine fully modified tcDNA oligonucleotides ranging from 10 to 15 nucleotides were synthesized. All fully modified tcDNA oligonucleotides contain thiophosphate linkers and a 5'-terminal phosphate group. Complementary DNA and RNA oligonucleotides were purchased from TriLink Biotechnologies (San Diego, CA, USA). They were used without further purification and redissolved in Fluka HPLC water to provide stock solutions of 1 mM. Equimolar amounts of complementary nucleic acids were combined in 250 mM NH4OAc to yield a final duplex concentration of 50 μM. A 7.5 M ammonium acetate solution purchased from Sigma-Aldrich was used to prepare the buffer solution for the annealing. The samples were annealed in a Eppendorf Mastercycler gradient PCR cycler (Vaudaux-Eppendorf AG, Schönenbuch, Switzerland). A temperature gradient was run from 90 °C to 18 °C over 2.5 h. For direct competition experiments, the tcDNA oligonucleotide was mixed at a 1:1:1 molar ratio with both complementary DNA and RNA strands in ammonium acetate buffer and annealed according to the same procedure. Prior to analysis, the duplex samples were diluted with an aqueous solution of 50% methanol to a final concentration of 2.5 μM. Fluka HPLC grade methanol (Sigma-Aldrich Chemie GmbH) was used to prepare the solvent.

Mass Spectrometry

All ESI-MS experiments were performed on a LTQ Orbitrap XL instrument (Thermo Fischer Scientific, Bremen, Germany) equipped with a nano-ESI source. The experiments were carried out in the negative ion mode with a spray voltage of –700 to –850 V. The capillary voltage was –45 V, the capillary temperature 150 °C, and the tube lens voltage was set to –200 V. Mass spectra were acquired in the FTMS mode from m/z 200 to 2000 with the mass resolution set to 100,000. Tandem mass spectrometric experiments were performed in the ion trap using helium as collision gas. The selection window for the precursor was defined as ±2.5 m/z. The activation time was set to 30 ms and the relative collision energies (rCE) applied for fragmentation ranged from 13 to 40. The Xcalibur Software Suite including Qualbrowser ver. 2.0.7 (ThermoFisher Scientific) was used for data processing. Data analysis was supported by the OMA and OPA software tool [24].

Melting Curves

Complementary oligonucleotides were diluted in 20 mM ammonium acetate buffer solution to a final oligonucleotide concentration of 1 μM. Thermal melting curves were recorded in a Varian Cary-100 UV/VIS photospectrometer. The samples were heated to 85 °C; after that a cooling-heating-cooling cycle was run in the temperature range of 2–85 °C and a gradient of 0.5 °C per min. Three consecutive cycles were run and all annealing and melting curves were superimposed. The Cary WinUV software (Agilent Technologies, Santa Clara, CA, USA) was used for data acquisition. Data analysis was performed using the R software environment and the melting temperature was determined as the maximum of the first derivative of the superimposed curves.

Results and Discussion

Fragmentation Mechanism of tcDNA

CID of fully modified tcDNA discerns a-B- and w-ions as the most prevalent fragmentation products, which suggests a DNA-like fragmentation mechanism. Further evidence for this hypothesis stems from the MS/MS data on the DNA decamer 5'-AACTGTCACG-3' harboring single tcDNA nucleotides. Substituting the first cytidine by a tc(5'-methyl-C)-nucleotide (10-2) leads to significant changes in the product ion spectrum, as shown in Figure 2. First, a tremendous increase of the intensity of the a3-B-ion indicates that the modified site is more prone to backbone cleavage. Second, cytosine is lost more easily from the modified sugar-moiety than from deoxyribose. For some charge states, the loss of the modified cytosine is even preferred over the loss of purines. Thus, the facile base loss correlates with the high intensity of the a3-B-ion, which implies that base loss triggers the backbone cleavage of tcDNA. Consequently, backbone cleavage must be impeded by stabilizing the N-glycosidic bond in the tc-sugar-moiety, e.g., by fluorination of the sugar-moiety in the 2'-position. This effect is revealed in the product ion spectrum of the decamer 10-3. The absence of the a3-B- and a7-B-ions (see Figure 2) demonstrates that the highly electronegative fluorine atom undermines the cleavage of the 3'-C–O bond adjacent to the modified sugar-moiety. Consistently, no base loss from the modified nucleotides is observed here. The reported MS/MS data show conclusively that the fragmentation of tcDNA oligonucleotides follows a DNA-like mechanism. Moreover, it indicates the destabilization of the N-glycosidic bond in the modified nucleotide compared to DNA.
Figure 2

Comparison of the relative signal intensities of a-B-ions of an all DNA decamer and two sequence isomers harboring tc-modified cytidines at a charge state of –3

Correlation of Solution and Gas-Phase Stability of DNA, RNA, and tcDNA Duplexes

The relative stabilities of six duplexes in the gas-phase and in solution were determined. The oligonucleotide 5'-AACCTCGGCTTACCT-3' (15-1) was obtained as DNA, tcDNA, and RNA (T = U in RNA) and combined with their DNA or RNA complements to yield the two homoduplexes DNA:DNA and RNA:RNA as well as four heteroduplexes (see Table 1). The stability of the duplexes in solution increases in the following order: RNA:DNA ~ DNA:DNA < DNA:RNA < RNA:RNA ~ tcDNA:DNA << tcDNA:RNA. This result confirms earlier thermal melting studies on tcDNA that demonstrate remarkable stability and selectivity of tcDNA for RNA complements [3]. The difference between the DNA:RNA and RNA:DNA heteroduplexes arises from the changing pyrimidine content of the DNA strand. In such duplex pairs, the hybrid with the higher deoxypyrimidine content is usually more stable in solution [25]. The relative stabilities of all duplexes in the gas-phase were assessed by collision-induced dissociation. Because the charge state of the precursor affects the fragmentation, the MS/MS results of all duplexes with z = –6, which were accessible for all six duplexes, are compared herein. The normalized collision energy was incremented in steps of one and the values listed in Table 1 indicate the experimental conditions at which strand separation emerged. For all duplexes, the onset of strand separation coincided with the formation of backbone fragments and the loss of nucleobases from the single strands. The energy transferred to the precursor is converted in multiple parallel or successive reaction steps, and strand separation is clearly not a two-state transition. Nonetheless, the collision energy at the onset of dissociation correlates well with the melting temperatures for nonmodified duplexes. Base loss is most pronounced from tcDNA oligonucleotides, intermediate from DNA, and scarcely observed from RNA. Thus, the stability of the N-glycosidic bond increases in the following order: tcDNA < DNA < RNA.
Table 1

Fragmentation Pathways of Sequence Identical Duplexes at a Charge State of –6. Parallel fragmentation channels are abbreviated as follows: D for strand separation, B for base loss and backbone cleavage, and T for the formation of truncated duplex ions. The backbone cleavage leading to truncated duplexes are indicated with red lines

Previous studies showed that the formation of backbone fragments is promoted by a high GC content in the DNA duplex, and therefore, it is considered indicative of high gas-phase stability [18]. Comparison of the four nonmodified duplexes in Table 1 reveals that this does not hold true for RNA duplexes. The RNA:RNA duplex requires the highest activation energy for dissociation but exhibits least backbone fragments. As shown in Figure 3, the extent of backbone cleavage is highest in the DNA:DNA duplex and intermediate in the DNA:RNA and RNA:DNA hybrids. In these hybrids, the DNA single strand is cleaved preferably, whereas the additional 2'-hydroxy group stabilizes the RNA backbone. Deprotonation of the 2'-OH group, which would trigger backbone cleavage, evidently requires higher activation energy than strand separation. The presence of backbone fragments is, therefore, no appropriate measure of the duplex stability in general but depends on the gas-phase stability of the single stranded nucleic acids.
Figure 3

Product ion spectra of three sequence-isomeric duplexes with charge state z = –6. (a) RNA:RNA, (b) DNA:RNA (c) DNA:DNA

Formation of Truncated Duplexes

For the two modified heteroduplexes, the collision energy was smaller than expected based on the stability determined by thermal melting. Moreover, the tcDNA duplexes stand out from the set of sequence-isomeric duplexes because an additional fragmentation channel was observed here. The loss of one or more nucleobases, mainly purines, from the intact duplex was detected. Nucleobase loss can induce the cleavage of the backbone to give raise to truncated duplex ions. For both heteroduplexes, D-tca2 and D-tcw3-AH ions were detected, which indicate backbone cleavage in the tcDNA strand after the 2nd or the 12th nucleotide, respectively. The formation of truncated duplexes was first reported for DNA duplexes in 2002 [22]. The authors stated that truncated duplex ions can occur in precursors with relatively high gas-phase stability and are formed after partial unzipping of the duplex in the gas-phase. Our data demonstrate that the unwinding of the duplex takes place from both termini.

To better understand the formation of truncated duplex ions, a set of nine tcDNA:DNA heteroduplexes listed in Table 2 were investigated. Multiple base losses and the ejection of backbone fragments were observed for all duplexes with 14 or 15 base pairs and charge state z = –6. For shorter oligonucleotides with 10-11 base pairs, precursors with lower charge states were chosen to attain the same ratio of protonated and deprotonated phosphate groups in every duplex. However, no truncated duplexes were detected for the shorter oligonucleotides. Hence the formation of truncated duplexes is not a sterically preferred dissociation pathway of tcDNA in general but requires a minimum stability of the precursor in the gas phase. Noticeably, backbone cleavage in the heteroduplexes was only ever observed within the tcDNA strand, preferably at the 3'-side of adenosines. The adenine is typically ejected in addition to a w-ion to form D-tcw-AH-ions. In agreement with the trend of base loss, alternative backbone cleavage can occur at tcG nucleosides, but the loss of pyrimidine and the backbone cleavage at tcC or tcT nucleosides is disfavored.
Table 2

Truncated Duplex Ions Formed for tcDNA:DNA Heteroduplexes. The charge state was –6 for 14 and 15, and –5 for the shorter duplexes. The top sequence corresponds to the sugar-modified strand (C* = 5-methyl-cytosine), the complementary DNA strands are listed below. The listed percentage of the RNA heteroduplex was determined in direct competition experiments to assess the selectivity of tcDNA for the RNA versus DNA complements

For all tcDNA hybrids with 14 or 15 base pairs, the observed fragmentation channels include the release of nucleobases and backbone fragments as well as strand separation, hereafter referred to as base loss and dissociation, respectively. After dissociation of the hybrids, the intact DNA or RNA complements are detected, while the modified single strand further undergoes base loss and backbone cleavage. Backbone cleavage can occur before, during, or after full strand separation, which thwarts quantitative comparison of base loss and dissociation from the product ion spectra. Nevertheless, the release of up to four nucleobases from the duplex was observed in addition to the ejection of backbone fragments, which suggests that dissociation is only a minor fragmentation pathway of the tcDNA:RNA and tcDNA:DNA duplexes.

Parallel fragmentation channels in tcDNA:DNA heteroduplexes

The coincidence of two parallel reactions in tcDNA hybrids indicates similar values for the free activation enthalpies, ∆G, of both reactions. In the DNA:DNA duplex, by contrast, there is a significant difference in the free activation enthalpies for base loss and dissociation since only the latter is observed in CID experiments. We propose that the difference between the homo- and the heteroduplex is determined by two factors: First, the activation enthalpy for the base loss is reduced in the modified duplex. Second, the activation entropy for the dissociation is lowered because of the steric constraints of the tcDNA. Consequently, moving from the DNA:DNA duplex to the tcDNA hybrid, ∆G of the base loss decreases and ∆G of the dissociation increases, i.e., the free activation enthalpies of the two reactions converge. The low activation enthalpy for the base loss results from the destabilization of the N-glycosidic bond in the tc-sugar-moiety. As discussed for the singly modified DNA decamer 10-2, base loss and backbone cleavage are preferred at the modified site. The fact that backbone cleavage within the hybrid duplex only occurs in the tcDNA strand further evidences the weakness of the N-glycosidic bond in the modified nucleoside. As to the second effect, previously published thermodynamic data obtained from UV melting experiments [3] demonstrate that the formation entropy of tcDNA duplexes in solution is reduced relative to non-modified structures. Therefore, entropic stabilization of the hybrid duplexes should be assumed in the gas-phase.

As suggested by Gabelica et al. [22], the entropy-favored fragmentation pathway can be promoted by fast activation conditions. For the tcDNA hybrids, however, changing the activation time did not change the fragmentation pattern. Alternatively, the activation entropy can be studied indirectly when the activation enthalpy for the dissociation is altered. One approach is to compare different charge states since the Coulomb repulsion increases with the number of deprotonated phosphate groups and destabilizes the duplex. The tcDNA hybrids with z = –6 preferentially fragment by base loss. If the charge state of the precursor is increased to –7, strand separation becomes more pronounced, while base loss is reduced to the minor fragmentation pathway. A similar shift between competing reactions can be observed for the nonmodified DNA:RNA heteroduplex 15-1. Here, the formation of truncated duplex ions was observed for z = –5, whereas only dissociation is observed at higher charge states. While comparing charge states allows no fine-tuning of the activation enthalpy, it confirms that both reaction pathways, dissociation as well as base loss, are in principle accessible in modified and nonmodified duplexes.

In an experimentally more elaborate approach to assess the role of the activation entropy of dissociation, mismatched base pairs were introduced into the duplex series based on sequence 15-1 (see Table 3). Two C·T mismatches were inserted at terminal and central positions into the tcDNA:DNA and DNA:DNA duplexes. The reduced number of hydrogen bonds in the mismatched duplexes decreases the activation enthalpy of dissociation and thereby promotes strand separation. The effect of the mismatches was first assessed in solution, where similar destabilization of the homo- and heteroduplex was observed. In both cases, the central mismatch reduces the melting temperatures by approximately 30 °C while the terminal mismatch results in a decrease of only 5 °C. The central mismatch disrupts the base stacking within the duplex and most likely increases the flexibility of the duplex to a greater extent than the terminal mismatches, hence the larger reduction of the melting temperature by the central mismatches. In the gas phase, the destabilization of the modified and nonmodified duplexes differs tremendously. The DNA:DNA duplex with neither the terminal nor the central mismatch was detected intact after electrospray ionization, while both mismatched tcDNA:DNA heteroduplexes were detected and subjected to CID. As in matched hybrids, base loss and dissociation coincide in the mismatched heteroduplexes. Despite the two unpaired thymines and cytosines, no increased loss of pyrimidine bases was observed. Consequently, base loss from the modified strand is not a result of steric accessibility for the protonation of the nucleobase. As illustrated in Figure 4, the same fragmentation pattern was observed for the two mismatched tcDNA:DNA species and the matched tcDNA:RNA and tcDNA:DNA duplexes. Experimental data for tcDNA-hybrids confirm that the collisional energy at the onset of dissociation and base loss is not affected by the mismatched base pairs, a finding that contrasts strongly with the destabilization observed in solution. The melting temperatures determined for the four duplexes in Table 3 span a wide range from 27 °C to 80 °C. The temperature span of 53 °C between the least and the most stable duplex clearly precludes any direct comparison of the duplex stabilities in solution and in the gas-phase. This discrepancy arises because UV melting experiments determine the thermodynamic stability of the duplex, whereas CID probes their kinetic stability in the gas-phase. Unlike the melting temperature, the kinetic stability does not directly refer to strand separation but to the ratio of the reaction rates of parallel processes. The reaction rate of each process is determined by its activation enthalpy and entropy. Apparently, the contribution of the two missing Watson-Crick base pairs to the activation energy of the dissociation of tcDNA duplexes is negligible. In the DNA:DNA duplex, by contrast, the remaining 13 base pairs were insufficient to preserve the assembled structure during the electrospray process. Since sequence-identical DNA:DNA and tcDNA:DNA duplexes were studied, the different behavior in the gas-phase can be attributed solely to the activation entropy. The modified sugar-moiety in tcDNA reduces the activation entropy for the dissociation of the hybrids compared to DNA duplexes. Hence, the free activation enthalpy increases and the tcDNA hybrid exhibits the higher stability in the gas-phase. The presented data illustrate the impact of the activation entropy on the gas-phase fragmentation of nucleic acid duplexes. These findings demonstrate that the relative stability of different types of nucleic acid duplexes is not assessed reliably in tandem mass spectrometric experiments.
Table 3

Fragmentation Pathways of Mismatched tcDNA:DNA and DNA:DNA Duplexes at a Charge State of –6

Figure 4

Product ion spectra two tcDNA heteroduplexes with charge state z = –6 containing the same tcDNA 15mer. (a) Matched tcDNA:RNA, (b) tcDNA:DNA harboring two central C·T mismatches

Relative Stability of tcDNA:DNA and tcDNA:RNA Duplexes

This conclusion is accentuated by the comparison of the tcDNA:DNA duplexes listed in Table 2 with their tcDNA:RNA analogues. Although the latter duplexes consistently exhibit higher melting temperatures than their tcDNA:DNA counterparts, no significant differences between the fragmentation patterns of the two sets of duplexes were found. Thus, no correlation between the relative kinetic stabilities and the stability in solution was found for tcDNA:DNA and tcDNA:RNA duplexes. An alternative way to determine the stability of higher-order structures is to consider the relative signal intensities of the single strands and the assembled structures. This comparison is most revealing in direct competition experiments, a method that constitutes a fast means of determining the selectivity of an antisense oligonucleotide for its target. When incubated with their DNA and RNA complements at equimolar concentrations, tcDNA oligonucleotides preferentially hybridize with the RNA strand (compare Figure 5). The intensity of the tcDNA:RNA signals accounts for at least 66% of the total intensity of all duplex ions. For the shorter duplexes with only 10 base pairs, this fraction rises to 100%, while the tcDNA:DNA heteroduplexes were not detected at all. Similarly, the stabilities of homo- and heteroduplexes can be compared. When the DNA 15mer 15-1 is annealed with the complementary DNA and tcDNA oligonucleotides, the tcDNA:DNA heteroduplex is formed preferentially, while the DNA:DNA homoduplex is not observed in the mass spectrum. The preferred formation of the tcDNA:RNA over the RNA:RNA duplex was confirmed in the same way. In all of these experiments, the relative signal intensities only truthfully reflect the abundance in solution if equal ionization efficiencies can be assumed for all species. This assumption is justified because infusion of the mixtures prior to the annealing shows equal signal intensities for all three single strands. In conclusion, direct competition experiments confirm the distinctive stability of the tcDNA heteroduplexes and indicate high specificity of tcDNA for the RNA target over complementary DNA.
Figure 5

Overview spectrum of a 1:1:1 mixture of the tcDNA 15mer 15-6 and its DNA and RNA complement. The double peaks arise from the loss of the 5'-terminal phosphate group from the tcDNA oligonucleotide. The uppercase letters identify the peaks as duplex (D) and monomer (M), and the lower case indices specify the type of the oligonucleotide (tcDNA, DNA, and RNA). The diagram shows the percentage of the tcDNA:RNA (green) and tcDNA:DNA (red) heteroduplexes listed in Table 2

Conclusion

The correlation between the collision energy and the melting temperature repeatedly reported within sets of similar duplexes does not extend to the comparison of different types of nucleic acid duplexes. In solution, the relative stabilities of six sequence-identical duplexes was found to increase in the order RNA:DNA ~ DNA:DNA < DNA:RNA < RNA:RNA ~ tcDNA:DNA << tcDNA:RNA. This trend does not correspond to the relative collision energies determined in MS/MS experiments. Moreover, dissociation, the favored fragmentation channel of nonmodified duplexes, constitutes only a minor pathway in tcDNA hybrids, whereas the formation of truncated duplexes is preferred. The free activation enthalpies of base loss and dissociation converge in tcDNA hybrids because the modified sugar-moiety decreases the activation enthalpy of the base loss and reduces the activation entropy of the strand dissociation. The decreased enthalpy for base loss is evidenced by the fact that bond scission preceding the ejection of backbone fragments from the intact duplex solely occurs from the modified strand owing to the weak N-glycosidic bond in tcDNA nucleosides. The activation entropy for the dissociation was studied by comparison of matched and mismatched duplexes. Herein, we report identical fragmentation patterns for two heteroduplexes based on the same tcDNA 15mer: the fully matched tcDNA:RNA hybrid and the tcDNA:DNA hybrid harboring two central C·T mismatches. This strongly contrasts with the difference in the melting temperature of 53 °C determined for the same two duplexes and illustrates the role of the activation entropy for strand separation in the gas phase. Consequently, tandem mass spectrometric experiments do not reflect the relative stability of duplexes when different types of nucleic acids are compared. For this reason, the stability of sequence identical tcDNA:RNA and tcDNA:DNA heteroduplexes is best assessed by direct competition experiments rather than CID. For the seven tested sequences, the tcDNA:RNA heteroduplex accounts for 66%–100% of the total signal intensity of all duplex species in equimolar mixtures of tcDNA and both DNA and RNA complements.

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Copyright information

© American Society for Mass Spectrometry 2016

Authors and Affiliations

  • Yvonne Hari
    • 1
  • Branislav Dugovič
    • 1
  • Alena Istrate
    • 1
  • Annabel Fignolé
    • 1
  • Christian J. Leumann
    • 1
  • Stefan Schürch
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of BernBernSwitzerland

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