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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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Length-dependent quadruplex formation in ILPR sequences. (A) Phosphorimager scan of native PAGE (10%) of ILPR sequences of different lengths (n = 1–4). Lane M contains nonstructure forming oligonucleotides of 84, 56 and 28 nt in length. The position of bands in lanes ILPRn = 2–4 are consistent with the formation of intramolecular G-quadruplexes. Intermolecular and intramolecular structures are marked with (**) and (*) respectively. (B) Circular dichroism (CD) spectra of ILPR sequences (ILPRn = 2,4) in 10 mM Tris–HCl pH 7.8, 100 mM KCl. Peaks at ∼265 nm and ∼295 nm are indicative of a parallel and an antiparallel folded G-quadruplex topology respectively or a hybrid of the two (39). (C) Phosphorimager scan of native PAGE (10%) of the ILPR 56-nt single G-quadruplex forming mutant 1Q56 (see ‘Materials and Methods’ section for sequence) and wild-type ILPRn = 4. The 1Q56 contains a 5′ end that is the single G-quadruplex forming ILPRn = 2 sequence which is followed by a nonstructure forming 28-nt sequence.
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Figure 2: Length-dependent quadruplex formation in ILPR sequences. (A) Phosphorimager scan of native PAGE (10%) of ILPR sequences of different lengths (n = 1–4). Lane M contains nonstructure forming oligonucleotides of 84, 56 and 28 nt in length. The position of bands in lanes ILPRn = 2–4 are consistent with the formation of intramolecular G-quadruplexes. Intermolecular and intramolecular structures are marked with (**) and (*) respectively. (B) Circular dichroism (CD) spectra of ILPR sequences (ILPRn = 2,4) in 10 mM Tris–HCl pH 7.8, 100 mM KCl. Peaks at ∼265 nm and ∼295 nm are indicative of a parallel and an antiparallel folded G-quadruplex topology respectively or a hybrid of the two (39). (C) Phosphorimager scan of native PAGE (10%) of the ILPR 56-nt single G-quadruplex forming mutant 1Q56 (see ‘Materials and Methods’ section for sequence) and wild-type ILPRn = 4. The 1Q56 contains a 5′ end that is the single G-quadruplex forming ILPRn = 2 sequence which is followed by a nonstructure forming 28-nt sequence.

Mentions: First, we used native gel electrophoresis to show that the ILPRn=2,4 form compact structures. Figure 2A shows the ILPR oligonucleotides of various lengths separated on a native gel with 100 mM KCl both in the gel and the running buffer. The ILPRn=2-4 (see ‘Materials and Methods’ section for sequences) showed an increased mobility compared to nonstructure-forming oligonucleotides of identical sizes (Figure 2A, lane M), indicating the formation of intramolecular structures. Upon quantitation of the bands in the gel, ILPRn=2–4 formed ∼99% intramolecular structures, with the exception of n=3 which formed ∼5% intermolecular structures. However, ILPRn=1 containing only two guanine stretches, incapable of forming an intramolecular G-quadruplex, showed mobilities consistent with either an intermolecular structure (see ** in Figure 2A) or the single stranded DNA form (see * in Figure 2A). Then, we used CD spectroscopy to confirm that the intramolecular structures observed in ILPRn=4 are G-quadruplexes. G-quadruplexes with a parallel folded topology have been shown in most cases to have a positive peak at ∼265 nm and a negative peak at ∼240 nm; while antiparallel folded topologies are generally associated with a positive ∼295-nm peak and a negative peak at ∼260 nm (39). In case of ILPRn=2 that is capable of forming one G-quadruplex, the CD spectrum in buffer containing 100 mM KCl is consistent with an antiparallel quadruplex structure (Figure 2B). However, with the addition of a second G-quadruplex forming unit to the ILPRn=2, which forms ILPRn=4, under identical conditions the CD spectrum implies the presence of parallel and antiparallel quadruplex forms (Figure 2B, see discussions below). This result demonstrates that the quadruplex conformation of ILPRn=4 may not be a simple addition of two ILPRn=2 quadruplex units. In addition, CD spectra of ILPRn=2,4 in 100 mM LiCl were absent of any quadruplex type features, demonstrating the dependence of quadruplex formation in the ILPR sequences on the monovalent cation (Supplementary Figure S1).Figure 2.


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)

Length-dependent quadruplex formation in ILPR sequences. (A) Phosphorimager scan of native PAGE (10%) of ILPR sequences of different lengths (n = 1–4). Lane M contains nonstructure forming oligonucleotides of 84, 56 and 28 nt in length. The position of bands in lanes ILPRn = 2–4 are consistent with the formation of intramolecular G-quadruplexes. Intermolecular and intramolecular structures are marked with (**) and (*) respectively. (B) Circular dichroism (CD) spectra of ILPR sequences (ILPRn = 2,4) in 10 mM Tris–HCl pH 7.8, 100 mM KCl. Peaks at ∼265 nm and ∼295 nm are indicative of a parallel and an antiparallel folded G-quadruplex topology respectively or a hybrid of the two (39). (C) Phosphorimager scan of native PAGE (10%) of the ILPR 56-nt single G-quadruplex forming mutant 1Q56 (see ‘Materials and Methods’ section for sequence) and wild-type ILPRn = 4. The 1Q56 contains a 5′ end that is the single G-quadruplex forming ILPRn = 2 sequence which is followed by a nonstructure forming 28-nt sequence.
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Related In: Results  -  Collection

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Figure 2: Length-dependent quadruplex formation in ILPR sequences. (A) Phosphorimager scan of native PAGE (10%) of ILPR sequences of different lengths (n = 1–4). Lane M contains nonstructure forming oligonucleotides of 84, 56 and 28 nt in length. The position of bands in lanes ILPRn = 2–4 are consistent with the formation of intramolecular G-quadruplexes. Intermolecular and intramolecular structures are marked with (**) and (*) respectively. (B) Circular dichroism (CD) spectra of ILPR sequences (ILPRn = 2,4) in 10 mM Tris–HCl pH 7.8, 100 mM KCl. Peaks at ∼265 nm and ∼295 nm are indicative of a parallel and an antiparallel folded G-quadruplex topology respectively or a hybrid of the two (39). (C) Phosphorimager scan of native PAGE (10%) of the ILPR 56-nt single G-quadruplex forming mutant 1Q56 (see ‘Materials and Methods’ section for sequence) and wild-type ILPRn = 4. The 1Q56 contains a 5′ end that is the single G-quadruplex forming ILPRn = 2 sequence which is followed by a nonstructure forming 28-nt sequence.
Mentions: First, we used native gel electrophoresis to show that the ILPRn=2,4 form compact structures. Figure 2A shows the ILPR oligonucleotides of various lengths separated on a native gel with 100 mM KCl both in the gel and the running buffer. The ILPRn=2-4 (see ‘Materials and Methods’ section for sequences) showed an increased mobility compared to nonstructure-forming oligonucleotides of identical sizes (Figure 2A, lane M), indicating the formation of intramolecular structures. Upon quantitation of the bands in the gel, ILPRn=2–4 formed ∼99% intramolecular structures, with the exception of n=3 which formed ∼5% intermolecular structures. However, ILPRn=1 containing only two guanine stretches, incapable of forming an intramolecular G-quadruplex, showed mobilities consistent with either an intermolecular structure (see ** in Figure 2A) or the single stranded DNA form (see * in Figure 2A). Then, we used CD spectroscopy to confirm that the intramolecular structures observed in ILPRn=4 are G-quadruplexes. G-quadruplexes with a parallel folded topology have been shown in most cases to have a positive peak at ∼265 nm and a negative peak at ∼240 nm; while antiparallel folded topologies are generally associated with a positive ∼295-nm peak and a negative peak at ∼260 nm (39). In case of ILPRn=2 that is capable of forming one G-quadruplex, the CD spectrum in buffer containing 100 mM KCl is consistent with an antiparallel quadruplex structure (Figure 2B). However, with the addition of a second G-quadruplex forming unit to the ILPRn=2, which forms ILPRn=4, under identical conditions the CD spectrum implies the presence of parallel and antiparallel quadruplex forms (Figure 2B, see discussions below). This result demonstrates that the quadruplex conformation of ILPRn=4 may not be a simple addition of two ILPRn=2 quadruplex units. In addition, CD spectra of ILPRn=2,4 in 100 mM LiCl were absent of any quadruplex type features, demonstrating the dependence of quadruplex formation in the ILPR sequences on the monovalent cation (Supplementary Figure S1).Figure 2.

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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