Limits...
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.

Show MeSH
Calculation of  via UV and HPLC analyses. (A) UV absorption spectra of GG dimer (blue line), SeGG dimer (black line), and SeGSeG dimer (red line); (B) RP-HPLC analysis of SeGG dimer at 260 nm (blue line) and 360 nm (red line); (C) RP-HPLC analysis of SeGSeG dimer at 260 nm (blue line) and 360 nm (red line).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602767&req=5

Figure 3: Calculation of via UV and HPLC analyses. (A) UV absorption spectra of GG dimer (blue line), SeGG dimer (black line), and SeGSeG dimer (red line); (B) RP-HPLC analysis of SeGG dimer at 260 nm (blue line) and 360 nm (red line); (C) RP-HPLC analysis of SeGSeG dimer at 260 nm (blue line) and 360 nm (red line).

Mentions: To determine the extinction coefficient of DNA SeG by comparing with the native nucleotide, we synthesized and purified GG, SeGG and SeGSeG dimers, and their UV spectra are presented in Figure 3. By taking the advantage of HPLC separation and UV analysis, we have developed this useful HPLC–UV approach to accurately measure and calculate the extinction coefficients of the base-modified nucleotides. Our experimental results indicate that SeG in DNA absorbs at both 254 nm and 360 nm (λmax = 267 nm and 360 nm) while native G (λmax = 254 nm) does not absorb at 360 nm (Figure 3A). The SeG λmax values of SeGG (λmax = 359 nm) and SeGSeG (λmax = 361 nm) are virtually identical; the average λmax of SeG is 360 nm. Since the extinction coefficient of G at 260 nm ( = 1.22 × 104 M−1cm−1) is known (30), we performed HPLC analysis of SeGG and SeGSeG under both 260 nm and 360 nm (Figure 3B and C), and their peak areas were quantified, respectively. First, the absorption ratio at 260 nm and 360 nm of SeGSeG (/) was calculated and determined as α value. In Figure 3B, the α value is used to calculate the 260-nm absorption contribution from SeG of the SeGG. The net 260-nm absorption from G of the SeGG () is obtained by subtraction of the SeG 260-nm contribution from the total SeGG absorption at 260 nm (Figure 3B). Thus, we deduced Equation (3) from Equation (1) and (2) presenting the SeGG. Since can be directly measured and can be accurately calculated from this SeGG in Figure 3B, we determined as 2.3 × 104 M−1cm−1. Similarly, from Equation (2) and (4) presenting the SeGSeG, we deduced Equation (5) and calculated the ratio of /in Figure 3C, thereby accurately determining as 5.3 × 103 M−1 cm−1.12345Excitingly, we observed that invisible DNA turns into colored DNA via the single atom replacement with selenium, while natural DNAs are colorless. Comparing with the native deoxyguanosine nucleotide (UV λmax = 254 nm, ε = 1.22 × 104 M−1 cm−1; 30), the UV spectrum of the 6-Se-deoxyguanosine nucleotide (λmax = 360 nm, ε = 2.3 × 104 M−1 cm−1) reveals a higher absorption and a large red-shift over 100 nm, thereby leading to the appearance of yellow color. This Se-nucleotide visualization is probably due to the ease of the delocalization of the selenium electrons on the nucleobase, requiring less energy for the electron excitation, thereby resulting in the large UV red-shift. In contrast, the 6-S-deoxyguanosine nucleotide (6-S-dG, λmax = 339 nm; 24) shows a smaller red-shift from the deoxyguanosine nucleotide and remains colorless.Figure 3.


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)

Calculation of  via UV and HPLC analyses. (A) UV absorption spectra of GG dimer (blue line), SeGG dimer (black line), and SeGSeG dimer (red line); (B) RP-HPLC analysis of SeGG dimer at 260 nm (blue line) and 360 nm (red line); (C) RP-HPLC analysis of SeGSeG dimer at 260 nm (blue line) and 360 nm (red line).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602767&req=5

Figure 3: Calculation of via UV and HPLC analyses. (A) UV absorption spectra of GG dimer (blue line), SeGG dimer (black line), and SeGSeG dimer (red line); (B) RP-HPLC analysis of SeGG dimer at 260 nm (blue line) and 360 nm (red line); (C) RP-HPLC analysis of SeGSeG dimer at 260 nm (blue line) and 360 nm (red line).
Mentions: To determine the extinction coefficient of DNA SeG by comparing with the native nucleotide, we synthesized and purified GG, SeGG and SeGSeG dimers, and their UV spectra are presented in Figure 3. By taking the advantage of HPLC separation and UV analysis, we have developed this useful HPLC–UV approach to accurately measure and calculate the extinction coefficients of the base-modified nucleotides. Our experimental results indicate that SeG in DNA absorbs at both 254 nm and 360 nm (λmax = 267 nm and 360 nm) while native G (λmax = 254 nm) does not absorb at 360 nm (Figure 3A). The SeG λmax values of SeGG (λmax = 359 nm) and SeGSeG (λmax = 361 nm) are virtually identical; the average λmax of SeG is 360 nm. Since the extinction coefficient of G at 260 nm ( = 1.22 × 104 M−1cm−1) is known (30), we performed HPLC analysis of SeGG and SeGSeG under both 260 nm and 360 nm (Figure 3B and C), and their peak areas were quantified, respectively. First, the absorption ratio at 260 nm and 360 nm of SeGSeG (/) was calculated and determined as α value. In Figure 3B, the α value is used to calculate the 260-nm absorption contribution from SeG of the SeGG. The net 260-nm absorption from G of the SeGG () is obtained by subtraction of the SeG 260-nm contribution from the total SeGG absorption at 260 nm (Figure 3B). Thus, we deduced Equation (3) from Equation (1) and (2) presenting the SeGG. Since can be directly measured and can be accurately calculated from this SeGG in Figure 3B, we determined as 2.3 × 104 M−1cm−1. Similarly, from Equation (2) and (4) presenting the SeGSeG, we deduced Equation (5) and calculated the ratio of /in Figure 3C, thereby accurately determining as 5.3 × 103 M−1 cm−1.12345Excitingly, we observed that invisible DNA turns into colored DNA via the single atom replacement with selenium, while natural DNAs are colorless. Comparing with the native deoxyguanosine nucleotide (UV λmax = 254 nm, ε = 1.22 × 104 M−1 cm−1; 30), the UV spectrum of the 6-Se-deoxyguanosine nucleotide (λmax = 360 nm, ε = 2.3 × 104 M−1 cm−1) reveals a higher absorption and a large red-shift over 100 nm, thereby leading to the appearance of yellow color. This Se-nucleotide visualization is probably due to the ease of the delocalization of the selenium electrons on the nucleobase, requiring less energy for the electron excitation, thereby resulting in the large UV red-shift. In contrast, the 6-S-deoxyguanosine nucleotide (6-S-dG, λmax = 339 nm; 24) shows a smaller red-shift from the deoxyguanosine nucleotide and remains colorless.Figure 3.

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