Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR.
 

Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR.

Related Articles Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR.

Nucleic Acids Res. 2017 Sep 06;45(15):9178-9192

Authors: Nußbaumer F, Juen MA, Gasser C, Kremser J, Müller T, Tollinger M, Kreutz C

Abstract
We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2'deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min-1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s-1 and with a backward rate constant of 10.6 s-1.


PMID: 28911104 [PubMed - in process]



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