Abstract
The constitution, configuration, and flexibility of the core sugars of DNA molecules alter their function in diverse roles. Conformational itineraries of the ribofuranosides (fs) have long been known to finely determine rates of processing, yet we also know that, strikingly, semifunctional DNAs containing pyranosides (ps) or other configurations can be created, suggesting sufficient but incompletely understood plasticity. The multiple conformers involved in such processes are necessarily influenced by context and environment: solvent, hosts, ligands. Notably, however, to date the unbiased, “naked” conformers have not been experimentally determined. Here, the inherent conformational biases of DNA scaffold deoxyribosides in unsolvated and solvated forms have now been defined using gas-phase microwave and solution-phase NMR spectroscopies coupled with computational analyses and exploitation of critical differences between natural-abundance isotopologues. Serial determination of precise, individual spectra for conformers of these 25 isotopologues in alpha (α-d) and beta (β-d); pyrano (p) and furano (f) methyl 2-deoxy-d-ribosides gave not only unprecedented atomic-level resolution structures of associated conformers but also their quantitative populations. Together these experiments revealed that typical 2E and 3E conformations of the sugar found in complex DNA structures are not inherently populated. Moreover, while both OH-5′ and OH-3′ are constrained by intramolecular hydrogen bonding in the unnatural αf scaffold, OH-3′ is “born free” in the “naked” lowest lying energy conformer of natural βf. Consequently, upon solvation, unnatural αf is strikingly less perturbable (retaining 2T1 conformation in vacuo and water) than natural βf. Unnatural αp and βp ribosides also display low conformational perturbability. These first experimental data on inherent, unbiased conformers therefore suggest that it is the background of conformational flexibility of βf that may have led to its emergence out of multiple possibilities as the sugar scaffold for “life’s code” and suggest a mechanism by which the resulting freedom of OH-3′ (and hence accessibility as a nucleophile) in βf may drive preferential processing and complex structure formation, such as replicative propagation of DNA from 5′-to-3′.
Highlights
Structural variability and flexibility of ribonucleic acids are apparent and immense in scope and intimately linked to both the existence1−3 and emergence4,5 of biological function
Ever-expanding interest in the design and use of both natural and unnatural ribonucleotides in diagnostic and therapeutic applications continues to highlight a key role for an understanding of the fundamentals that generate associated structural populations.6−8 For example, while chemical modifications at phosphate9 or nucleobase10 can usefully increase in vivo stability, it is the correct manipulation of the conformations of the core sugar scaffold that has proven key to optimal functional activity
We present a strategy for the complete structural analysis of the core sugar scaffold of DNA, 2-deoxy-D-riboside (Figure 1a,b), that exploits custom-made, high-resolution microwave spectrometers combined with complementary vaporization and sampling techniques in the gas phase
Summary
Structural variability and flexibility of ribonucleic acids are apparent and immense in scope and intimately linked to both the existence− and emergence of biological function. As the pioneering work of Eschenmoser highlighted, there is no necessity for ribosidic or even furanosidic structures, and alternative polynucleotides can be constructed based on, for example, Lthreo-furanosides or even configurationally varied pyranosides.. As the pioneering work of Eschenmoser highlighted, there is no necessity for ribosidic or even furanosidic structures, and alternative polynucleotides can be constructed based on, for example, Lthreo-furanosides or even configurationally varied pyranosides.13 While their functions are typically moderated (e.g., reduced base-pairing strengths), such altered-sugar polynucleotides can still adopt relevant duplex structures via typical (e.g., Watson−Crick) patterns and can even be processed by appropriate variant enzymes, albeit at reduced rates. In DNA polymerases, an essential factor that prevents improper inclusion and extension of nucleotides appears to be governed by the preferred conformations of the furanose moiety of each incoming nucleotide during both incorporation and extension. Interestingly, as the pioneering work of Eschenmoser highlighted, there is no necessity for ribosidic or even furanosidic structures, and alternative polynucleotides can be constructed based on, for example, Lthreo-furanosides or even configurationally varied pyranosides. While their functions are typically moderated (e.g., reduced base-pairing strengths), such altered-sugar polynucleotides can still adopt relevant duplex structures via typical (e.g., Watson−Crick) patterns and can even be processed by appropriate variant enzymes, albeit at reduced rates.
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