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Utilising the left-helical conformation of L-DNA for analysing different marker types on a single universal microarray platform.

Hauser NC, Martinez R, Jacob A, Rupp S, Hoheisel JD, Matysiak S - Nucleic Acids Res. (2006)

Bottom Line: Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however.Typical results for the measurement of transcript level variations, genotypic differences and DNA-protein interactions are presented.However, on the basis of the characteristic features of L-DNA, also other applications of this molecule type are discussed.

View Article: PubMed Central - PubMed

Affiliation: Genomics-Proteomics-Systemsbiology, Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik Nobelstrasse 12, 70569 Stuttgart, Germany. nicole.hauser@igb.fraunhofer.de

ABSTRACT
L-DNA is the perfect mirror-image form of the naturally occurring d-conformation of DNA. Therefore, L-DNA duplexes have the same physical characteristics in terms of solubility, duplex stability and selectivity as D-DNA but form a left-helical double-helix. Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however. We analysed some of the properties that are typical for L-DNA. For all the differences, L-DNA is chemically compatible with the D-form of DNA, so that chimeric molecules can be synthesized. We take advantage of the characteristics of L-DNA toward the establishment of a universal microarray that permits the analysis of different kinds of molecular diagnostic information in a single experiment on a single platform, in various combinations. Typical results for the measurement of transcript level variations, genotypic differences and DNA-protein interactions are presented. However, on the basis of the characteristic features of L-DNA, also other applications of this molecule type are discussed.

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Nuclease sensitivity of L-DNA. Single-stranded and double-stranded molecules were digested with commonly used exonucleases and endonucleases. The DNA-sequences are shown. Black letters stand for D-DNA, blue letters indicate L-DNA. While all D-DNA is degraded by all four enzymes used, there is no apparent cleavage of L-DNA with either enzyme. (a) lane 5, the remaining 20mer L-DNA oligomer of the originally 40 nt long molecule is only weakly visible due to smearing effects caused by the presence of the buffer/enzyme cocktail. In (b) lane 5 no digestion of the L-DNA duplex was observed. In panel (c) S1-nuclease digested the D-DNA portion of the chimeric molecule, the L-DNA part, however, remained untouched, lane 5. The three bands in lanes 1 and 4 (d) represent the two single-stranded oligomers and the duplex.
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fig5: Nuclease sensitivity of L-DNA. Single-stranded and double-stranded molecules were digested with commonly used exonucleases and endonucleases. The DNA-sequences are shown. Black letters stand for D-DNA, blue letters indicate L-DNA. While all D-DNA is degraded by all four enzymes used, there is no apparent cleavage of L-DNA with either enzyme. (a) lane 5, the remaining 20mer L-DNA oligomer of the originally 40 nt long molecule is only weakly visible due to smearing effects caused by the presence of the buffer/enzyme cocktail. In (b) lane 5 no digestion of the L-DNA duplex was observed. In panel (c) S1-nuclease digested the D-DNA portion of the chimeric molecule, the L-DNA part, however, remained untouched, lane 5. The three bands in lanes 1 and 4 (d) represent the two single-stranded oligomers and the duplex.

Mentions: We also investigated the activity of nucleases on single-stranded and double-stranded L-DNA molecules. Single-stranded DNA was subjected to a treatment with Escherichia coli exonuclease I, which degrades single-stranded DNA starting from the 3′-terminus. D-DNA was digested entirely. In chimeric molecules, however, only the D-DNA portion was removed, while the L-DNA remained intact (Figure 5a). Incubation of double-stranded DNA with T7-exonuclease, which removes nucleotides from the 5′ end of double-stranded DNA, lead to the degradation of the D-DNA duplex, as expected. However, no digestion of the L-DNA duplex was observed even with an excess of enzyme (Figure 5b).


Utilising the left-helical conformation of L-DNA for analysing different marker types on a single universal microarray platform.

Hauser NC, Martinez R, Jacob A, Rupp S, Hoheisel JD, Matysiak S - Nucleic Acids Res. (2006)

Nuclease sensitivity of L-DNA. Single-stranded and double-stranded molecules were digested with commonly used exonucleases and endonucleases. The DNA-sequences are shown. Black letters stand for D-DNA, blue letters indicate L-DNA. While all D-DNA is degraded by all four enzymes used, there is no apparent cleavage of L-DNA with either enzyme. (a) lane 5, the remaining 20mer L-DNA oligomer of the originally 40 nt long molecule is only weakly visible due to smearing effects caused by the presence of the buffer/enzyme cocktail. In (b) lane 5 no digestion of the L-DNA duplex was observed. In panel (c) S1-nuclease digested the D-DNA portion of the chimeric molecule, the L-DNA part, however, remained untouched, lane 5. The three bands in lanes 1 and 4 (d) represent the two single-stranded oligomers and the duplex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636439&req=5

fig5: Nuclease sensitivity of L-DNA. Single-stranded and double-stranded molecules were digested with commonly used exonucleases and endonucleases. The DNA-sequences are shown. Black letters stand for D-DNA, blue letters indicate L-DNA. While all D-DNA is degraded by all four enzymes used, there is no apparent cleavage of L-DNA with either enzyme. (a) lane 5, the remaining 20mer L-DNA oligomer of the originally 40 nt long molecule is only weakly visible due to smearing effects caused by the presence of the buffer/enzyme cocktail. In (b) lane 5 no digestion of the L-DNA duplex was observed. In panel (c) S1-nuclease digested the D-DNA portion of the chimeric molecule, the L-DNA part, however, remained untouched, lane 5. The three bands in lanes 1 and 4 (d) represent the two single-stranded oligomers and the duplex.
Mentions: We also investigated the activity of nucleases on single-stranded and double-stranded L-DNA molecules. Single-stranded DNA was subjected to a treatment with Escherichia coli exonuclease I, which degrades single-stranded DNA starting from the 3′-terminus. D-DNA was digested entirely. In chimeric molecules, however, only the D-DNA portion was removed, while the L-DNA remained intact (Figure 5a). Incubation of double-stranded DNA with T7-exonuclease, which removes nucleotides from the 5′ end of double-stranded DNA, lead to the degradation of the D-DNA duplex, as expected. However, no digestion of the L-DNA duplex was observed even with an excess of enzyme (Figure 5b).

Bottom Line: Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however.Typical results for the measurement of transcript level variations, genotypic differences and DNA-protein interactions are presented.However, on the basis of the characteristic features of L-DNA, also other applications of this molecule type are discussed.

View Article: PubMed Central - PubMed

Affiliation: Genomics-Proteomics-Systemsbiology, Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik Nobelstrasse 12, 70569 Stuttgart, Germany. nicole.hauser@igb.fraunhofer.de

ABSTRACT
L-DNA is the perfect mirror-image form of the naturally occurring d-conformation of DNA. Therefore, L-DNA duplexes have the same physical characteristics in terms of solubility, duplex stability and selectivity as D-DNA but form a left-helical double-helix. Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however. We analysed some of the properties that are typical for L-DNA. For all the differences, L-DNA is chemically compatible with the D-form of DNA, so that chimeric molecules can be synthesized. We take advantage of the characteristics of L-DNA toward the establishment of a universal microarray that permits the analysis of different kinds of molecular diagnostic information in a single experiment on a single platform, in various combinations. Typical results for the measurement of transcript level variations, genotypic differences and DNA-protein interactions are presented. However, on the basis of the characteristic features of L-DNA, also other applications of this molecule type are discussed.

Show MeSH