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

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.

Selective Targeting of the Zinc Finger Domain of HIV Nucleocapsid Protein NCp7 with Ruthenium Complexes

“…This result indicates that the C‐terminal zinc‐finger domain is more reactive than the N‐terminal domain. This observation is consistent with recent reports that two zinc‐fingers of NCp7 possess different dynamic properties in which the N‐Zn knuckle is conformationally more stable, whereas the coordination exchange of Cys36 makes the C‐terminal zinc‐finger more reactive …”

“…2D 1 H‐ 15 N HSQC NMR spectra of NCp7 were recorded to further analyze the reaction of Ru‐3 (Figure B). The well‐dispersed signal of NCp7 is consistent with literature report and indicates the well‐folded protein . After incubation with Ru‐3 for 1 h, a large number of signals disappeared and a series of peaks appeared at 7.9–8.4 ppm on the 1 H dimension, which is consistent with the protein unfolding .…”

“…The well-dispersed signal of NCp7 is consistent with literature report and indicates the well-folded protein. [17] After incubation with Ru-3 for 1h,al arge number of signals disappeareda nd as eries of peaks appeared at 7.9-8.4 ppm on the 1 Hd imension, which is consistentw ith the protein unfolding. [7] This result further supports the conclusion that Ru binding disrupts the secondary structure of NCp7.…”

“…This result indicates that the C-terminal zinc-finger domain is more reactive than the N-terminal domain.T hiso bservation is consistent with recent reports that two zinc-fingers of NCp7 possessd ifferent dynamic properties in which the N-Zn knuckle is conformationallym ore stable, whereas the coordination exchange of Cys36 makest he C-terminal zinc-finger more reactive. [17,18] The compositionofr uthenium binding adducts of NCp7 was characterized by electrospray ionization mass spectra (ESI-MS). The reaction of Ru-3 showed that the bi-ruthenium binding adduct was formed quickly during the reaction (3 min) ( Figure 6).…”

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

“…However, the isotopic 15 N/ 13 C enrichment of nucleosides incorporated into oligonucleotides by solid‐phase synthesis is complex, labor‐intensive and expensive, particularly in longer sequences. Indeed, the introduction of these isotopes into nucleosides requires either modified phosphoramidite building blocks for solid phase synthesis or nucleoside triphosphate analogs for enzymatic DNA or RNA production . Since the preparation of these isotope‐enriched building blocks requires multistep synthetic protocols, the possibility of characterizing nitrogen environments by exploiting the naturally abundant 99.9 % 14 N isotope without recourse to isotopic enrichment is particularly appealing.…”