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A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

Kumar N, Maiti S - Nucleic Acids Res. (2008)

Bottom Line: We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol.Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition.Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.

ABSTRACT
Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5-18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

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CD spectra of quadruplexes (10 μM) obtained in 10 mM sodium cacodylate buffer, 100 mM KCl, pH 7.4.
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Figure 1: CD spectra of quadruplexes (10 μM) obtained in 10 mM sodium cacodylate buffer, 100 mM KCl, pH 7.4.

Mentions: This study aims to elucidate the thermodynamic profiles of G-quadruplexes formed by naturally occurring sequences present in the promoter regions of various proto-oncogenes. These sequences were sorted using Quadfinder (42), which employs a consensus uni-molecular G-quadruplex sequence motif of the form GxLy1GxLy2GxLy3Gx, where x denotes the G-stretch and Ly1, Ly2 and Ly3 denote the length of loops 1, 2 and 3, respectively. The selected sequences belong to different regions of proto-oncogenes, which have been correlated to genomic instability in human malignancies by Eddy and co-workers (13). The sequences investigated in our study have three G-quartets, as they are the shortest G-tracts that form quadruplexes with reasonable stability, for which the influence of total loop length (5–18 bases) on the quadruplex stability can be evaluated. Our dataset includes 62 quadruplex-forming sequences present in the promoter regions of various proto-oncogenes located at different positions with respect to transcription start site (TSS) (SI Table 1). Based on the loop length, we classified the sequences with total loop length ranging from 5 to 18 bases (tabulated in Table 1). We initially examined the folding topologies of these sequences through CD spectroscopy. Though CD does not provide direct evidence for the structural features of quadruplexes, the spectra obtained provide valuable information about the structural characteristics of quadruplexes and agrees with the topology obtained by direct methods such as NMR (50,51). The characteristic CD signals arise from G-G stacking between G-quartets, the strength of which mainly depends on the conformation of guanine bases around the glycosidic bond (syn or anti). The difference in the orientation of glycosidic torsion conformation gives rise to parallel and antiparallel structures (8–10). The CD signature comprising of a positive peak at 262 nm and a negative peak at 240 nm typically indicates a parallel conformation and signature with a positive peak at 295 nm and a negative peak at 238 nm indicates an antiparallel conformation. However, the presence of both these signatures indicates mixed conformation. The CD signature obtained for all the sequences has been tabulated in SI Table 1 and the spectra are provided in Supplementary Data. The information provided in SI Table 1 shows that out of the 62 sequences investigated in this study, two sequences adopted predominantly antiparallel conformation, 38 sequences adopted predominantly parallel conformation and the remaining 22 sequences adopted both parallel and antiparallel signatures. The characteristic CD spectra of a few sequences are shown in Figure 1. The CD spectra provided in Figure 1 show that c-MYC (Q21), c-KIT (Q30), WNT 3 (Q2), AKT 2 (Q34), MYB (Q35) have a predominantly parallel population and a small antiparallel population, whereas VEGF (Q1), PDFB (Q4) and PIM 1 (Q9), adopt only the parallel topology. Our results for the CD spectra of c-MYC (Q21), c-KIT (Q30), VEGF (Q1) and PDFB (Q4) are in agreement with the topology reported in literature (6,7,50–52). An interesting observation was made from the CD spectra of the quadruplex-forming sequences from the HCK protooncogene, Q24 and Q25 (Supplementary Data, SI Table 1), which possess the same total loop length of 10 bases and loop composition, but display variation in base distribution among the three loops. In Q24, the number of bases in loops L1, L2 and L3 is 2, 2 and 6, respectively, while in Q25, these are 2, 6 and 2, respectively. This minor difference in loop distribution results in a remarkable difference in the CD spectra. Q24 adopts a predominant parallel conformation, whereas Q25 adopts a mixed conformation (Supplementary Data). Detailed inspection of the CD spectra of all these sequences shows that sequences with shorter loop lengths such as, VEGF (Q1), WNT 3 (Q2), WNT5A (Q3), PDGFB (Q4) adopt mainly parallel structures, while longer loops such as, RALB (Q59), EGFR (Q60), VAV 1 (Q61), FGF 6 (Q62) adopt mixed structures comprising both parallel and antiparallel characteristics. A few quadruplexes with longer loops such as, FLI (Q48), THRA (Q52), KRAS (Q57) and THPO (Q58) also adopted a parallel conformation. For the given data set, we observed that only two sequences, FGR (Q49) and FES (Q53), adopted an antiparallel fold. It is worthy to mention that for few sequences the assignment of bases is in the loops is arbitrary, and it is also likely that sequences that have been assigned with longer loops could also fold to adopt topologies with short loop lengths. Examination of the CD spectra of all the naturally occurring sequences studied here shows a predominance of the parallel topology, hinting toward a bias for the parallel fold over the antiparallel form as a recognition motif in biology.Figure 1.


A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

Kumar N, Maiti S - Nucleic Acids Res. (2008)

CD spectra of quadruplexes (10 μM) obtained in 10 mM sodium cacodylate buffer, 100 mM KCl, pH 7.4.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553590&req=5

Figure 1: CD spectra of quadruplexes (10 μM) obtained in 10 mM sodium cacodylate buffer, 100 mM KCl, pH 7.4.
Mentions: This study aims to elucidate the thermodynamic profiles of G-quadruplexes formed by naturally occurring sequences present in the promoter regions of various proto-oncogenes. These sequences were sorted using Quadfinder (42), which employs a consensus uni-molecular G-quadruplex sequence motif of the form GxLy1GxLy2GxLy3Gx, where x denotes the G-stretch and Ly1, Ly2 and Ly3 denote the length of loops 1, 2 and 3, respectively. The selected sequences belong to different regions of proto-oncogenes, which have been correlated to genomic instability in human malignancies by Eddy and co-workers (13). The sequences investigated in our study have three G-quartets, as they are the shortest G-tracts that form quadruplexes with reasonable stability, for which the influence of total loop length (5–18 bases) on the quadruplex stability can be evaluated. Our dataset includes 62 quadruplex-forming sequences present in the promoter regions of various proto-oncogenes located at different positions with respect to transcription start site (TSS) (SI Table 1). Based on the loop length, we classified the sequences with total loop length ranging from 5 to 18 bases (tabulated in Table 1). We initially examined the folding topologies of these sequences through CD spectroscopy. Though CD does not provide direct evidence for the structural features of quadruplexes, the spectra obtained provide valuable information about the structural characteristics of quadruplexes and agrees with the topology obtained by direct methods such as NMR (50,51). The characteristic CD signals arise from G-G stacking between G-quartets, the strength of which mainly depends on the conformation of guanine bases around the glycosidic bond (syn or anti). The difference in the orientation of glycosidic torsion conformation gives rise to parallel and antiparallel structures (8–10). The CD signature comprising of a positive peak at 262 nm and a negative peak at 240 nm typically indicates a parallel conformation and signature with a positive peak at 295 nm and a negative peak at 238 nm indicates an antiparallel conformation. However, the presence of both these signatures indicates mixed conformation. The CD signature obtained for all the sequences has been tabulated in SI Table 1 and the spectra are provided in Supplementary Data. The information provided in SI Table 1 shows that out of the 62 sequences investigated in this study, two sequences adopted predominantly antiparallel conformation, 38 sequences adopted predominantly parallel conformation and the remaining 22 sequences adopted both parallel and antiparallel signatures. The characteristic CD spectra of a few sequences are shown in Figure 1. The CD spectra provided in Figure 1 show that c-MYC (Q21), c-KIT (Q30), WNT 3 (Q2), AKT 2 (Q34), MYB (Q35) have a predominantly parallel population and a small antiparallel population, whereas VEGF (Q1), PDFB (Q4) and PIM 1 (Q9), adopt only the parallel topology. Our results for the CD spectra of c-MYC (Q21), c-KIT (Q30), VEGF (Q1) and PDFB (Q4) are in agreement with the topology reported in literature (6,7,50–52). An interesting observation was made from the CD spectra of the quadruplex-forming sequences from the HCK protooncogene, Q24 and Q25 (Supplementary Data, SI Table 1), which possess the same total loop length of 10 bases and loop composition, but display variation in base distribution among the three loops. In Q24, the number of bases in loops L1, L2 and L3 is 2, 2 and 6, respectively, while in Q25, these are 2, 6 and 2, respectively. This minor difference in loop distribution results in a remarkable difference in the CD spectra. Q24 adopts a predominant parallel conformation, whereas Q25 adopts a mixed conformation (Supplementary Data). Detailed inspection of the CD spectra of all these sequences shows that sequences with shorter loop lengths such as, VEGF (Q1), WNT 3 (Q2), WNT5A (Q3), PDGFB (Q4) adopt mainly parallel structures, while longer loops such as, RALB (Q59), EGFR (Q60), VAV 1 (Q61), FGF 6 (Q62) adopt mixed structures comprising both parallel and antiparallel characteristics. A few quadruplexes with longer loops such as, FLI (Q48), THRA (Q52), KRAS (Q57) and THPO (Q58) also adopted a parallel conformation. For the given data set, we observed that only two sequences, FGR (Q49) and FES (Q53), adopted an antiparallel fold. It is worthy to mention that for few sequences the assignment of bases is in the loops is arbitrary, and it is also likely that sequences that have been assigned with longer loops could also fold to adopt topologies with short loop lengths. Examination of the CD spectra of all the naturally occurring sequences studied here shows a predominance of the parallel topology, hinting toward a bias for the parallel fold over the antiparallel form as a recognition motif in biology.Figure 1.

Bottom Line: We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol.Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition.Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.

ABSTRACT
Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5-18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (DeltaG(37)) ranging from -1.7 to -15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex-duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.

Show MeSH