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Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe C.

Edwards TE, Cekan P, Reginsson GW, Shelke SA, Ferré-D'Amaré AR, Schiemann O, Sigurdsson ST - Nucleic Acids Res. (2011)

Bottom Line: To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis.These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system.This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.

View Article: PubMed Central - PubMed

Affiliation: Emerald BioStructures, Bainbridge Island, WA 98110, USA.

ABSTRACT
Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼ 20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.

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Related in: MedlinePlus

Overlay of the 1.7 Å resolution crystal structure of a DNA containing the nitroxide spin-labeled deoxycytosine analog Ç (gray carbon backbone) with a crystal structure containing deoxycytosine at the same position (PDB ID 1DPL, green carbon backbone) (38).
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Figure 5: Overlay of the 1.7 Å resolution crystal structure of a DNA containing the nitroxide spin-labeled deoxycytosine analog Ç (gray carbon backbone) with a crystal structure containing deoxycytosine at the same position (PDB ID 1DPL, green carbon backbone) (38).

Mentions: The 1.7 Å resolution crystal structure of the decamer duplex DNA containing Ç at position 2 was initially refined with an abasic site at this position. The resulting omit /Fo/−/Fc/ electron density map showed unambiguous electron density for the Ç nucleobase (Figure 4a). The simulated-annealing omit 2/Fo/−/Fc/ electron density map calculated with phases from the refined model was of excellent quality and revealed unambiguously that Ç forms a standard base pair with dG9 of the opposite strand with three ∼2.8 Å long hydrogen bonds (Figure 4b and c). The structure of the nucleic acid helix containing Ç superimposes closely on a previously reported structure containing a deoxycytidine residue at this position (38) (Figure 5), indicating that Ç does not perturb the DNA structure relative to a standard deoxycytidine residue. EPR spectroscopic analysis showed that Ç had decreased mobility in duplex DNA relative to single-stranded (12,16) or bulged (39) sites and could be used to measure single strand to duplex transitions during folding (15), implying Ç forms a base pair with dG. Furthermore, thermal denaturation experiments showed that the Ç•dG pairing had a similar melting temperature to a dC•dG pair, but showed a decrease in the melting temperature of 10–15°C when Ç was paired with dA, dT or dC (12). Combined with the EPR spectroscopy and thermal denaturation results, the high-resolution crystal structure described here shows that Ç is a non-perturbing cytosine analog that forms a Watson–Crick base pair with dG as designed.Figure 4.


Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe C.

Edwards TE, Cekan P, Reginsson GW, Shelke SA, Ferré-D'Amaré AR, Schiemann O, Sigurdsson ST - Nucleic Acids Res. (2011)

Overlay of the 1.7 Å resolution crystal structure of a DNA containing the nitroxide spin-labeled deoxycytosine analog Ç (gray carbon backbone) with a crystal structure containing deoxycytosine at the same position (PDB ID 1DPL, green carbon backbone) (38).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105401&req=5

Figure 5: Overlay of the 1.7 Å resolution crystal structure of a DNA containing the nitroxide spin-labeled deoxycytosine analog Ç (gray carbon backbone) with a crystal structure containing deoxycytosine at the same position (PDB ID 1DPL, green carbon backbone) (38).
Mentions: The 1.7 Å resolution crystal structure of the decamer duplex DNA containing Ç at position 2 was initially refined with an abasic site at this position. The resulting omit /Fo/−/Fc/ electron density map showed unambiguous electron density for the Ç nucleobase (Figure 4a). The simulated-annealing omit 2/Fo/−/Fc/ electron density map calculated with phases from the refined model was of excellent quality and revealed unambiguously that Ç forms a standard base pair with dG9 of the opposite strand with three ∼2.8 Å long hydrogen bonds (Figure 4b and c). The structure of the nucleic acid helix containing Ç superimposes closely on a previously reported structure containing a deoxycytidine residue at this position (38) (Figure 5), indicating that Ç does not perturb the DNA structure relative to a standard deoxycytidine residue. EPR spectroscopic analysis showed that Ç had decreased mobility in duplex DNA relative to single-stranded (12,16) or bulged (39) sites and could be used to measure single strand to duplex transitions during folding (15), implying Ç forms a base pair with dG. Furthermore, thermal denaturation experiments showed that the Ç•dG pairing had a similar melting temperature to a dC•dG pair, but showed a decrease in the melting temperature of 10–15°C when Ç was paired with dA, dT or dC (12). Combined with the EPR spectroscopy and thermal denaturation results, the high-resolution crystal structure described here shows that Ç is a non-perturbing cytosine analog that forms a Watson–Crick base pair with dG as designed.Figure 4.

Bottom Line: To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis.These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system.This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.

View Article: PubMed Central - PubMed

Affiliation: Emerald BioStructures, Bainbridge Island, WA 98110, USA.

ABSTRACT
Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼ 20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.

Show MeSH
Related in: MedlinePlus