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

Abstract

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.

Mass Spectrometry of Nucleic Acid Noncovalent Complexes

“…The results demonstrated that the backbone nature can have strong effects on the dissociation of DNA duplexes. Similarly, a study on tricyclo-DNA conjugates, which are more rigid than the natural counterparts, shows a higher propensity for base loss, which was attributed to changes in the activation entropy (pre-exponential factor) in the rigidified duplexes . Burmistrova et al.…”

“…Similarly, a study on tricyclo-DNA conjugates, which are more rigid than the natural counterparts, shows a higher propensity for base loss, which was attributed to changes in the activation entropy (preexponential factor) in the rigidified duplexes. 963 Burmistrova et al showed by IMS that (18-bp) 7− duplex rearrangements could occur at collision energies below those required for base loss or strand dissociation. 702 The dynamics of conformational changes of DNA duplexes in the gas phase was also studied by gas-phase FRET.…”