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Derivatization of DNAs with selenium at 6-position of guanine for function and crystal structure studies.

Salon J, Jiang J, Sheng J, Gerlits OO, Huang Z - Nucleic Acids Res. (2008)

Bottom Line: We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm (epsilon = 2.3 x 10(4) M(-1) cm(-1)), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature.Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H.Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.

ABSTRACT
To investigate nucleic acid base pairing and stacking via atom-specific mutagenesis and crystallography, we have synthesized for the first time the 6-Se-deoxyguanosine phosphoramidite and incorporated it into DNAs via solid-phase synthesis with a coupling yield over 97%. We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm (epsilon = 2.3 x 10(4) M(-1) cm(-1)), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature. Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H. The crystal structure determination and analysis reveal that the overall structures of the native and Se-modified nucleic acid duplexes are very similar, the selenium atom participates in a Se-mediated hydrogen bond (Se ... H-N), and the (Se)G and C form a base pair similar to the natural G-C pair though the Se-modification causes the base-pair to shift (approximately 0.3 A). Our biophysical and structural studies provide new insights into the nucleic acid flexibility, duplex recognition and stability. Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes.

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HPLC, MS and UV analyses of the SeG-DNAs. (A) RP-HPLC analysis of 5′-d(GAATCA-SeG-GTGTC)-3′ [monitored at 260 nm (blue) and 360 nm (red)]. The sample was analyzed on a Welchrom XB-C18 column (4.6 × 250 mm, 5 μ) at a flow of 1.0 ml/min and with a linear gradient of 5 to 50% B in 10 min, with a retention time of 7.6 min. Buffer A: 10mM TEAAc (pH 7.1); B: 60% acetonitrile in 10 mM TEAAc (pH 7.1). (B) MS analysis of 5′-d(GT-SeG-TACAC)-3′.Molecular formula: C78H99N30O45P7Se; [M+H]+: 2473.8 (calcd: 2473.6). (C)UV spectra of the SeG-DNAs containing one SeG (ATG-SeG-TGCAC, black), two SeGs (ATG-SeG-T-SeG-CAC, red), and three SeGs (AT-SeG-SeG-T-SeG-CAC, pink).
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Figure 2: HPLC, MS and UV analyses of the SeG-DNAs. (A) RP-HPLC analysis of 5′-d(GAATCA-SeG-GTGTC)-3′ [monitored at 260 nm (blue) and 360 nm (red)]. The sample was analyzed on a Welchrom XB-C18 column (4.6 × 250 mm, 5 μ) at a flow of 1.0 ml/min and with a linear gradient of 5 to 50% B in 10 min, with a retention time of 7.6 min. Buffer A: 10mM TEAAc (pH 7.1); B: 60% acetonitrile in 10 mM TEAAc (pH 7.1). (B) MS analysis of 5′-d(GT-SeG-TACAC)-3′.Molecular formula: C78H99N30O45P7Se; [M+H]+: 2473.8 (calcd: 2473.6). (C)UV spectra of the SeG-DNAs containing one SeG (ATG-SeG-TGCAC, black), two SeGs (ATG-SeG-T-SeG-CAC, red), and three SeGs (AT-SeG-SeG-T-SeG-CAC, pink).

Mentions: To measure the coupling efficiency of the 6-Se-G phosphoramidite (3), we synthesized 5′-DMTr-SeGG dinucleotide, analyzed it by RP-HPLC (Figure 1), and compared it with the native 5′-DMTr-GG synthesis and analysis (Supplementary Data), which indicated a high coupling yield (over 97%). Typical HPLC, UV and MS analyses of the SeG-DNAs are shown in Figure 2. More MS data of the synthesized SeG-DNAs are shown in Table 1. The purified Se-DNAs are yellow colored, which is the first observation of the colored DNAs containing the 6-Se-deoxyguanosine. In addition, our synthesis and analysis indicated that the 6-Se-G functionality of the Se-DNAs is relatively stable under aqueous conditions and air.Figure 1.


Derivatization of DNAs with selenium at 6-position of guanine for function and crystal structure studies.

Salon J, Jiang J, Sheng J, Gerlits OO, Huang Z - Nucleic Acids Res. (2008)

HPLC, MS and UV analyses of the SeG-DNAs. (A) RP-HPLC analysis of 5′-d(GAATCA-SeG-GTGTC)-3′ [monitored at 260 nm (blue) and 360 nm (red)]. The sample was analyzed on a Welchrom XB-C18 column (4.6 × 250 mm, 5 μ) at a flow of 1.0 ml/min and with a linear gradient of 5 to 50% B in 10 min, with a retention time of 7.6 min. Buffer A: 10mM TEAAc (pH 7.1); B: 60% acetonitrile in 10 mM TEAAc (pH 7.1). (B) MS analysis of 5′-d(GT-SeG-TACAC)-3′.Molecular formula: C78H99N30O45P7Se; [M+H]+: 2473.8 (calcd: 2473.6). (C)UV spectra of the SeG-DNAs containing one SeG (ATG-SeG-TGCAC, black), two SeGs (ATG-SeG-T-SeG-CAC, red), and three SeGs (AT-SeG-SeG-T-SeG-CAC, pink).
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Figure 2: HPLC, MS and UV analyses of the SeG-DNAs. (A) RP-HPLC analysis of 5′-d(GAATCA-SeG-GTGTC)-3′ [monitored at 260 nm (blue) and 360 nm (red)]. The sample was analyzed on a Welchrom XB-C18 column (4.6 × 250 mm, 5 μ) at a flow of 1.0 ml/min and with a linear gradient of 5 to 50% B in 10 min, with a retention time of 7.6 min. Buffer A: 10mM TEAAc (pH 7.1); B: 60% acetonitrile in 10 mM TEAAc (pH 7.1). (B) MS analysis of 5′-d(GT-SeG-TACAC)-3′.Molecular formula: C78H99N30O45P7Se; [M+H]+: 2473.8 (calcd: 2473.6). (C)UV spectra of the SeG-DNAs containing one SeG (ATG-SeG-TGCAC, black), two SeGs (ATG-SeG-T-SeG-CAC, red), and three SeGs (AT-SeG-SeG-T-SeG-CAC, pink).
Mentions: To measure the coupling efficiency of the 6-Se-G phosphoramidite (3), we synthesized 5′-DMTr-SeGG dinucleotide, analyzed it by RP-HPLC (Figure 1), and compared it with the native 5′-DMTr-GG synthesis and analysis (Supplementary Data), which indicated a high coupling yield (over 97%). Typical HPLC, UV and MS analyses of the SeG-DNAs are shown in Figure 2. More MS data of the synthesized SeG-DNAs are shown in Table 1. The purified Se-DNAs are yellow colored, which is the first observation of the colored DNAs containing the 6-Se-deoxyguanosine. In addition, our synthesis and analysis indicated that the 6-Se-G functionality of the Se-DNAs is relatively stable under aqueous conditions and air.Figure 1.

Bottom Line: We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm (epsilon = 2.3 x 10(4) M(-1) cm(-1)), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature.Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H.Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.

ABSTRACT
To investigate nucleic acid base pairing and stacking via atom-specific mutagenesis and crystallography, we have synthesized for the first time the 6-Se-deoxyguanosine phosphoramidite and incorporated it into DNAs via solid-phase synthesis with a coupling yield over 97%. We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm (epsilon = 2.3 x 10(4) M(-1) cm(-1)), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature. Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H. The crystal structure determination and analysis reveal that the overall structures of the native and Se-modified nucleic acid duplexes are very similar, the selenium atom participates in a Se-mediated hydrogen bond (Se ... H-N), and the (Se)G and C form a base pair similar to the natural G-C pair though the Se-modification causes the base-pair to shift (approximately 0.3 A). Our biophysical and structural studies provide new insights into the nucleic acid flexibility, duplex recognition and stability. Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes.

Show MeSH