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The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation.

Hwang GT, Hari Y, Romesberg FE - Nucleic Acids Res. (2009)

Bottom Line: In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex.Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair.The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Kyungpook National University, Daegu 702-701, Korea.

ABSTRACT
In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context. In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair. The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.

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CD spectra of DNA duplexes. (Curve A) Fully natural and correctly paired duplex (N4:N23 = dT:dA, X7:Y20 = dA:dT); (curve B) Fully natural duplex containing a single mispair (N4:N23 = dT:dC, X7:Y20 = dA:dT); (curve C) Duplex containing a d34DMPy self pair and no mispairs (N4:N23 = dT:dA, X7:Y20 = d34DMPy:d34DMPy) and (curve D) Duplex containing a d34DMPy self pair and a single mispair (N4:N23 = dT:dC, X7:Y20 = d34DMPy:d34DMPy).
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Figure 2: CD spectra of DNA duplexes. (Curve A) Fully natural and correctly paired duplex (N4:N23 = dT:dA, X7:Y20 = dA:dT); (curve B) Fully natural duplex containing a single mispair (N4:N23 = dT:dC, X7:Y20 = dA:dT); (curve C) Duplex containing a d34DMPy self pair and no mispairs (N4:N23 = dT:dA, X7:Y20 = d34DMPy:d34DMPy) and (curve D) Duplex containing a d34DMPy self pair and a single mispair (N4:N23 = dT:dC, X7:Y20 = d34DMPy:d34DMPy).

Mentions: One global property of the duplex that could be affected by the introduction of the self pair is its conformation which might be altered by the self pair such that the duplex is more sensitive to mispairing. To examine this possibility, we used circular dichroism to characterize the correctly paired duplexes (with and without the self pair), as well as the analogous duplexes containing a dT4:dC23 mispair (Figure 2). While the self pair induces slightly stronger and red-shifted absorptions, the spectra suggest that it does not significantly perturb duplex structure. Most importantly, mispairing has little effect on the spectrum of either duplex. Thus, we conclude that the self pair-mediated hypersensitivity to mispairing does not result from a global conformational change. This conclusion is consistent with previous studies which showed that different hydrophobic unnatural base pairs (18) or even a hydrophobic shape mimic of dT paired opposite dA (31) do not significantly perturb the structure of the duplex.Figure 2.


The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation.

Hwang GT, Hari Y, Romesberg FE - Nucleic Acids Res. (2009)

CD spectra of DNA duplexes. (Curve A) Fully natural and correctly paired duplex (N4:N23 = dT:dA, X7:Y20 = dA:dT); (curve B) Fully natural duplex containing a single mispair (N4:N23 = dT:dC, X7:Y20 = dA:dT); (curve C) Duplex containing a d34DMPy self pair and no mispairs (N4:N23 = dT:dA, X7:Y20 = d34DMPy:d34DMPy) and (curve D) Duplex containing a d34DMPy self pair and a single mispair (N4:N23 = dT:dC, X7:Y20 = d34DMPy:d34DMPy).
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Related In: Results  -  Collection

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Figure 2: CD spectra of DNA duplexes. (Curve A) Fully natural and correctly paired duplex (N4:N23 = dT:dA, X7:Y20 = dA:dT); (curve B) Fully natural duplex containing a single mispair (N4:N23 = dT:dC, X7:Y20 = dA:dT); (curve C) Duplex containing a d34DMPy self pair and no mispairs (N4:N23 = dT:dA, X7:Y20 = d34DMPy:d34DMPy) and (curve D) Duplex containing a d34DMPy self pair and a single mispair (N4:N23 = dT:dC, X7:Y20 = d34DMPy:d34DMPy).
Mentions: One global property of the duplex that could be affected by the introduction of the self pair is its conformation which might be altered by the self pair such that the duplex is more sensitive to mispairing. To examine this possibility, we used circular dichroism to characterize the correctly paired duplexes (with and without the self pair), as well as the analogous duplexes containing a dT4:dC23 mispair (Figure 2). While the self pair induces slightly stronger and red-shifted absorptions, the spectra suggest that it does not significantly perturb duplex structure. Most importantly, mispairing has little effect on the spectrum of either duplex. Thus, we conclude that the self pair-mediated hypersensitivity to mispairing does not result from a global conformational change. This conclusion is consistent with previous studies which showed that different hydrophobic unnatural base pairs (18) or even a hydrophobic shape mimic of dT paired opposite dA (31) do not significantly perturb the structure of the duplex.Figure 2.

Bottom Line: In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex.Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair.The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Kyungpook National University, Daegu 702-701, Korea.

ABSTRACT
In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context. In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair. The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.

Show MeSH