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Direct experimental evidence for quadruplex-quadruplex interaction within the human ILPR.

Schonhoft JD, Bajracharya R, Dhakal S, Yu Z, Mao H, Basu S - Nucleic Acids Res. (2009)

Bottom Line: These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem.Additional evidence for the QQI was provided by DMS footprinting on the ILPR(n)(=4) that identified specific guanines only protected in the presence of a neighboring G-quadruplex.There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, School of Biomedical Sciences, Kent State University, Kent, OH 44242, USA.

ABSTRACT
Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPR(n)(=4)). Mobilities of ILPR(n)(=4) in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPR(n)(=4) most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPR(n)(=2)). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPR(n)(=4) at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex-quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPR(n)(=4) that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.

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Relative differences between 265 nm (parallel) and 295 nm (antiparallel) peaks for different lengths of ILPR, Oxytricha telomere, and Human telomere of one or two possible adjacent G-quadruplexes. Values were calculated by taking the difference between the 295 nm and 265 nm peaks. The resulting values were then normalized to the 295 nm peak for comparison. Values that are positive indicate a majority of antiparallel and the negative values a majority of parallel, while values near zero indicate equal intensities of both. Values from CD spectra corresponding to quadruplex structures for telomere DNA show a monotonous increase with length while the values of the ILPR drastically differ with change in repeat length (*). Values for long telomeric sequences were calculated from published results (25). Recent human telomeric CD from Petraccone et al. also showed a similar trend (34).
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Figure 3: Relative differences between 265 nm (parallel) and 295 nm (antiparallel) peaks for different lengths of ILPR, Oxytricha telomere, and Human telomere of one or two possible adjacent G-quadruplexes. Values were calculated by taking the difference between the 295 nm and 265 nm peaks. The resulting values were then normalized to the 295 nm peak for comparison. Values that are positive indicate a majority of antiparallel and the negative values a majority of parallel, while values near zero indicate equal intensities of both. Values from CD spectra corresponding to quadruplex structures for telomere DNA show a monotonous increase with length while the values of the ILPR drastically differ with change in repeat length (*). Values for long telomeric sequences were calculated from published results (25). Recent human telomeric CD from Petraccone et al. also showed a similar trend (34).

Mentions: As mentioned before, the CD spectra showed a dramatic change between ILPRn=2 and ILPRn=4. This change is entirely different from that observed in the spectra between one and two G-quadruplex containing units in human and Oxytricha telomeres under nearly identical conditions (25,34) (Figure 3). In contrast to the ILPR sequences, as the length of Oxytricha (25) and Human telomere (25,34) sequences increased, the relative distribution of the CD peaks at 265 nm and 295 nm that suggested parallel and antiparallel strands respectively, remained almost identical (Figure 3). The length-dependent CD features in ILPR provides strong evidence to our hypothesis that conformation of two quadruplexes in ILPRn=4 cannot be extracted directly from individual quadruplex structures as suggested by CD signals in telomeres (25,34).Figure 3.


Direct experimental evidence for quadruplex-quadruplex interaction within the human ILPR.

Schonhoft JD, Bajracharya R, Dhakal S, Yu Z, Mao H, Basu S - Nucleic Acids Res. (2009)

Relative differences between 265 nm (parallel) and 295 nm (antiparallel) peaks for different lengths of ILPR, Oxytricha telomere, and Human telomere of one or two possible adjacent G-quadruplexes. Values were calculated by taking the difference between the 295 nm and 265 nm peaks. The resulting values were then normalized to the 295 nm peak for comparison. Values that are positive indicate a majority of antiparallel and the negative values a majority of parallel, while values near zero indicate equal intensities of both. Values from CD spectra corresponding to quadruplex structures for telomere DNA show a monotonous increase with length while the values of the ILPR drastically differ with change in repeat length (*). Values for long telomeric sequences were calculated from published results (25). Recent human telomeric CD from Petraccone et al. also showed a similar trend (34).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691825&req=5

Figure 3: Relative differences between 265 nm (parallel) and 295 nm (antiparallel) peaks for different lengths of ILPR, Oxytricha telomere, and Human telomere of one or two possible adjacent G-quadruplexes. Values were calculated by taking the difference between the 295 nm and 265 nm peaks. The resulting values were then normalized to the 295 nm peak for comparison. Values that are positive indicate a majority of antiparallel and the negative values a majority of parallel, while values near zero indicate equal intensities of both. Values from CD spectra corresponding to quadruplex structures for telomere DNA show a monotonous increase with length while the values of the ILPR drastically differ with change in repeat length (*). Values for long telomeric sequences were calculated from published results (25). Recent human telomeric CD from Petraccone et al. also showed a similar trend (34).
Mentions: As mentioned before, the CD spectra showed a dramatic change between ILPRn=2 and ILPRn=4. This change is entirely different from that observed in the spectra between one and two G-quadruplex containing units in human and Oxytricha telomeres under nearly identical conditions (25,34) (Figure 3). In contrast to the ILPR sequences, as the length of Oxytricha (25) and Human telomere (25,34) sequences increased, the relative distribution of the CD peaks at 265 nm and 295 nm that suggested parallel and antiparallel strands respectively, remained almost identical (Figure 3). The length-dependent CD features in ILPR provides strong evidence to our hypothesis that conformation of two quadruplexes in ILPRn=4 cannot be extracted directly from individual quadruplex structures as suggested by CD signals in telomeres (25,34).Figure 3.

Bottom Line: These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem.Additional evidence for the QQI was provided by DMS footprinting on the ILPR(n)(=4) that identified specific guanines only protected in the presence of a neighboring G-quadruplex.There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, School of Biomedical Sciences, Kent State University, Kent, OH 44242, USA.

ABSTRACT
Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPR(n)(=4)). Mobilities of ILPR(n)(=4) in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPR(n)(=4) most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPR(n)(=2)). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPR(n)(=4) at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex-quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPR(n)(=4) that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.

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