Limits...
Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA.

Dierckx A, Dinér P, El-Sagheer AH, Kumar JD, Brown T, Grøtli M, Wilhelmsson LM - Nucleic Acids Res. (2011)

Bottom Line: To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A(T)) and its photophysical characterization inside DNA.A(T) shows promising properties compared to the widely used adenine analogue 2-aminopurine.In conclusion, A(T) shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, University of Gothenburg, S-41296 Gothenburg, Sweden.

ABSTRACT
To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A(T)) and its photophysical characterization inside DNA. A(T) shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, A(T) shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that A(T) only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that A(T) shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, A(T) shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

Show MeSH

Related in: MedlinePlus

Putative stabilized AT.A base pair. Only the sugar attached to AT is shown and its triazole and pentyl chain are coloured grey. R1, R2 and R3 represent the rest of the DNA structure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105426&req=5

Figure 6: Putative stabilized AT.A base pair. Only the sugar attached to AT is shown and its triazole and pentyl chain are coloured grey. R1, R2 and R3 represent the rest of the DNA structure.

Mentions: Despite this possible change in conformation, AT still shows base pairing capacity to thymine and seems to be stacked reasonably well in the DNA duplex in that case. This can be concluded from a limiting anisotropy value of 0.34 which was recorded for AT in duplexes AA and TA in viscous sucrose solutions. The difference compared to the fundamental anisotropy of AT in a vitrified matrix (0.38) was calculated to be due to an internal wobble of AT in the DNA duplex of 16°. This value is higher than the estimated wobble for the natural canonical bases (∼5°), but lower than the corresponding value for the intercalating dye ethidium bromide (21°) (51). Furthermore, melting experiments were performed to examine the base pairing specificity of AT. Three of the modified sequences (GA, CT, CA) were annealed with strands containing an adenine, guanine or cytosine opposite of AT instead of a thymine. Melting temperatures recorded for AT-cytosine/guanine mismatches reveal an average drop in melting temperatures of 16°C, almost twice as large compared to an AT-thymine match (8°C). The AT-cytosine/guanine mismatch melting temperatures are in line with the average destabilization (14°C) recorded for a single-base mismatch of adenine–adenine in these duplexes compared to their natural matching counterparts. It is therefore reasonable to assume that AT exhibits some hydrogen-bonding with thymine but shows virtually no base-pairing with guanine or cytosine. Surprisingly, the same mismatch experiment performed for an AT-adenine mismatch showed on average no further destabilization of the duplexes (8°C) compared to the AT-thymine case. This suggests that AT is able to form equally strong base pairs with thymine and adenine. To the best of our knowledge, no similar findings have been previously reported for other adenine analogues. The putative AT.A base pair in Figure 6 would have a similar overall shape to a Watson–Crick base pair and would have good stacking interactions with surrounding base pairs and, thus, constitutes a plausible structure. Protonation of N(1) of adenine would provide a second hydrogen bond. Additionally a possible third weakly stabilizing C-H—N hydrogen bond to H(2) on adenine may be formed (58). It should be mentioned that the pKa of N(1) of adenine is approximately 4.0. However, in double-stranded DNA, if it is or has the possibility of being involved in H-bonding, it can be raised much higher. Thus, the proposed base pair structure is presently speculative and future high-resolution structural studies will be necessary to accurately determine the base pairing properties of AT.Figure 6.


Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA.

Dierckx A, Dinér P, El-Sagheer AH, Kumar JD, Brown T, Grøtli M, Wilhelmsson LM - Nucleic Acids Res. (2011)

Putative stabilized AT.A base pair. Only the sugar attached to AT is shown and its triazole and pentyl chain are coloured grey. R1, R2 and R3 represent the rest of the DNA structure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105426&req=5

Figure 6: Putative stabilized AT.A base pair. Only the sugar attached to AT is shown and its triazole and pentyl chain are coloured grey. R1, R2 and R3 represent the rest of the DNA structure.
Mentions: Despite this possible change in conformation, AT still shows base pairing capacity to thymine and seems to be stacked reasonably well in the DNA duplex in that case. This can be concluded from a limiting anisotropy value of 0.34 which was recorded for AT in duplexes AA and TA in viscous sucrose solutions. The difference compared to the fundamental anisotropy of AT in a vitrified matrix (0.38) was calculated to be due to an internal wobble of AT in the DNA duplex of 16°. This value is higher than the estimated wobble for the natural canonical bases (∼5°), but lower than the corresponding value for the intercalating dye ethidium bromide (21°) (51). Furthermore, melting experiments were performed to examine the base pairing specificity of AT. Three of the modified sequences (GA, CT, CA) were annealed with strands containing an adenine, guanine or cytosine opposite of AT instead of a thymine. Melting temperatures recorded for AT-cytosine/guanine mismatches reveal an average drop in melting temperatures of 16°C, almost twice as large compared to an AT-thymine match (8°C). The AT-cytosine/guanine mismatch melting temperatures are in line with the average destabilization (14°C) recorded for a single-base mismatch of adenine–adenine in these duplexes compared to their natural matching counterparts. It is therefore reasonable to assume that AT exhibits some hydrogen-bonding with thymine but shows virtually no base-pairing with guanine or cytosine. Surprisingly, the same mismatch experiment performed for an AT-adenine mismatch showed on average no further destabilization of the duplexes (8°C) compared to the AT-thymine case. This suggests that AT is able to form equally strong base pairs with thymine and adenine. To the best of our knowledge, no similar findings have been previously reported for other adenine analogues. The putative AT.A base pair in Figure 6 would have a similar overall shape to a Watson–Crick base pair and would have good stacking interactions with surrounding base pairs and, thus, constitutes a plausible structure. Protonation of N(1) of adenine would provide a second hydrogen bond. Additionally a possible third weakly stabilizing C-H—N hydrogen bond to H(2) on adenine may be formed (58). It should be mentioned that the pKa of N(1) of adenine is approximately 4.0. However, in double-stranded DNA, if it is or has the possibility of being involved in H-bonding, it can be raised much higher. Thus, the proposed base pair structure is presently speculative and future high-resolution structural studies will be necessary to accurately determine the base pairing properties of AT.Figure 6.

Bottom Line: To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A(T)) and its photophysical characterization inside DNA.A(T) shows promising properties compared to the widely used adenine analogue 2-aminopurine.In conclusion, A(T) shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, University of Gothenburg, S-41296 Gothenburg, Sweden.

ABSTRACT
To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A(T)) and its photophysical characterization inside DNA. A(T) shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, A(T) shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that A(T) only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that A(T) shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, A(T) shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

Show MeSH
Related in: MedlinePlus