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.

Decomposing hierarchical alignment: Co-arguments as conditions on alignment and the limits of referential hierarchies as explanations in verb agreement

Apart from common cases of differential argument marking, referential hierarchies affect argument marking in two ways: (a) through hierarchical marking, where markers compete for a slot and the competition is resolved by a hierarchy, and (b) through co-argument sensitivity, where the marking of one argument depends on the properties of its co-argument. Here we show that while co-argument sensitivity cannot be analyzed in terms of hierarchical marking, hierarchical marking can be analyzed in terms of co-argument sensitivity. Once hierarchical effects on marking are analyzed in terms of co-argument sensitivity, it becomes possible to examine alignment patterns relative to referential categories in exactly the same way as one can examine alignment patterns relative to referential categories in cases of differential argument marking and indeed any other condition on alignment (such as tense or clause type). As a result, instances of hierarchical marking of any kind turn out not to present a special case in the typology of alignment, and there is no need for positing an additional non-basic alignment type such as “hierarchical alignment”. While hierarchies are not needed for descriptive and comparative purposes, we also cast doubt on their relevance in diachrony: examining two families for which hierarchical agreement has been postulated, Algonquian and Kiranti, we find only weak and very limited statistical evidence for agreement paradigms to have been shaped by a principled ranking of person categories.