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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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Overall crystal structure of a DNA containing Ç. (a) Stick figure representation of an A-form duplex DNA crystal structure containing Ç solved at 1.7 Å resolution. For clarity, Ç is shown in light blue carbon backbone and the remainder of the DNA is shown in gray carbon backbone. (b) Final crystallographic model containing waters overlaid with the 2/Fo/−/Fc/ electron density map shown in blue mesh and contoured at 1.0 σ.
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Figure 3: Overall crystal structure of a DNA containing Ç. (a) Stick figure representation of an A-form duplex DNA crystal structure containing Ç solved at 1.7 Å resolution. For clarity, Ç is shown in light blue carbon backbone and the remainder of the DNA is shown in gray carbon backbone. (b) Final crystallographic model containing waters overlaid with the 2/Fo/−/Fc/ electron density map shown in blue mesh and contoured at 1.0 σ.

Mentions: Egli et al. (38) reported crystal structures of an A-form decamer DNA duplex with either cytosine or the phenoxazine-derived cytosine analog ‘G-clamp’ (29). These crystal structures contain a 2′-O-methoxymethyl T at position 6 that promotes formation of A-form DNA in solution rather than B-form, which is standard for DNA. To our knowledge, the 2′-O-methoxymethyl T phosphoramidite is not commercially available, and thus we first produced crystals with a C at position 2 and a 2′-O-methyl U at position 6. These crystals diffracted X-rays to better than 2.3 Å resolution in house (data not shown). Next, a DNA sample was prepared with the nitroxide spin label Ç incorporated into position 2 and a 2′-O-methyl U at position 6 (Figure 1b) and resulted in a crystal from which we determined a 1.7 Å resolution crystal structure (Figure 3, Table 1 and Supplementary Data). Crystals suitable for structure determination failed to grow from a sample containing Ç at position 2 with a dT at position 6 (i.e. no 2′-O-methyl). EPR spectra of these two Ç-containing samples, one with a 2′-O-methyl U at position 6 that should be A-form in solution and one with a dT at position 6 that should be B-form in solution, were found to be nearly identical (Supplementary Data). Thus, there is likely to be little difference in the mobility of the probe in comparison of A- and B-form DNA in solution. Inspection of the electron density maps from our 1.7 Å resolution structure obtained from the DNA sample containing Ç at position 2 and a 2′-O-methyl U at position 6 clearly showed the 2′-O-methyl group of U6 (Figure 3 and Supplementary Data).Figure 3.


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)

Overall crystal structure of a DNA containing Ç. (a) Stick figure representation of an A-form duplex DNA crystal structure containing Ç solved at 1.7 Å resolution. For clarity, Ç is shown in light blue carbon backbone and the remainder of the DNA is shown in gray carbon backbone. (b) Final crystallographic model containing waters overlaid with the 2/Fo/−/Fc/ electron density map shown in blue mesh and contoured at 1.0 σ.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Overall crystal structure of a DNA containing Ç. (a) Stick figure representation of an A-form duplex DNA crystal structure containing Ç solved at 1.7 Å resolution. For clarity, Ç is shown in light blue carbon backbone and the remainder of the DNA is shown in gray carbon backbone. (b) Final crystallographic model containing waters overlaid with the 2/Fo/−/Fc/ electron density map shown in blue mesh and contoured at 1.0 σ.
Mentions: Egli et al. (38) reported crystal structures of an A-form decamer DNA duplex with either cytosine or the phenoxazine-derived cytosine analog ‘G-clamp’ (29). These crystal structures contain a 2′-O-methoxymethyl T at position 6 that promotes formation of A-form DNA in solution rather than B-form, which is standard for DNA. To our knowledge, the 2′-O-methoxymethyl T phosphoramidite is not commercially available, and thus we first produced crystals with a C at position 2 and a 2′-O-methyl U at position 6. These crystals diffracted X-rays to better than 2.3 Å resolution in house (data not shown). Next, a DNA sample was prepared with the nitroxide spin label Ç incorporated into position 2 and a 2′-O-methyl U at position 6 (Figure 1b) and resulted in a crystal from which we determined a 1.7 Å resolution crystal structure (Figure 3, Table 1 and Supplementary Data). Crystals suitable for structure determination failed to grow from a sample containing Ç at position 2 with a dT at position 6 (i.e. no 2′-O-methyl). EPR spectra of these two Ç-containing samples, one with a 2′-O-methyl U at position 6 that should be A-form in solution and one with a dT at position 6 that should be B-form in solution, were found to be nearly identical (Supplementary Data). Thus, there is likely to be little difference in the mobility of the probe in comparison of A- and B-form DNA in solution. Inspection of the electron density maps from our 1.7 Å resolution structure obtained from the DNA sample containing Ç at position 2 and a 2′-O-methyl U at position 6 clearly showed the 2′-O-methyl group of U6 (Figure 3 and Supplementary Data).Figure 3.

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