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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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Representative graphs showing effect of loop length on thermodynamic stability (ΔG37) of quadruplex.
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Figure 4: Representative graphs showing effect of loop length on thermodynamic stability (ΔG37) of quadruplex.

Mentions: Both enthalpy and entropy in turn influence G, which is an indicator of thermodynamic stability. It is well documented in literature that increases in loop length results in decrease in quadruplex stability; however this observation is reported mainly for model sequences (29–34). A recent study using model sequences has shown that total loop length ≤5 has a major effect on quadruplex stability, and an addition of single nucleotide significantly affects the thermodynamic stability (34). Another study using model sequences with long central loops (6–9 bases) and two side loops composed of single base has shown that pyrimidines provide more stability over adenines and the composition of the central loop can minimize the destabilizing effect of a long central loop on quadruplex stability (29). This is in agreement with another study which demonstrated that, for intramolecular quadruplexes with G3 tracts and single base loops, a T to A loop substitution in reduced stability by 8°C (33). Intriguingly, the thermodynamic data obtained for quadruplexes formed by naturally occurring sequences having different loop lengths show that there is no clear correlation of loop length with the thermodynamic stability. Figure 4 depicts the variability in the thermodynamic stability with respect to the loop length. Quadruplexes with a total loop length of 5 formed by WNT 5A (Q3), VEGF (Q1) and WNT 3 (Q2) have thermodynamic stability ranging from −3.7 to −15.6 kcal/mol, respectively. Similarly, wide variations in the thermodynamic stability (−1.8 to −9.3 kcal/mol) were observed among quadruplex formed by sequences with loop size of 7. Likewise, no significant correlation of loop length and thermodynamic stability was observed for other data sets with longer loops. The lack of correlation between the thermodynamic analysis of quadruplexes formed by model sequences and genomic sequences with varied loop length can be attributed to the loop composition. For the model sequences, the loop length and composition are usually homogeneous. However, the naturally occurring sequences show random occurrences of purines and/or pyrimidines in the loops between the G stretches, which substantially influence the quadruplex thermodynamic stabilities. For instance, PIM1 (Q9) and FGF 3 (Q10) have total loop lengths of 7 with similar loop composition display remarkably different thermodynamic stabilities of −9.3 and −1.8 kcal/mol, respectively. Apart from total loop length and loop composition, the number of bases in the central loop is critical for quadruplex stability (32–35). We observed that for smaller loops, quadruplexes with a single base central loop are much more stable and the stability decreased upon increasing the bases in the central loop, as observed in case of WNT 3 (Q2) and WNT 5A (Q3) with thermodynamic stability of −15.6 and −3.7 kcal/mol, respectively. Our results shows that the majority (32) of quadruplex-forming sequences of protoncogenes have ΔG37 values approximately within the range of −1.8 to −8.6 kcal/mol. Only five genes from our data set have high ΔG37 values (≥−9.0 kcal/mol). These genes include WNT 3 (Q2), MYB (Q35), AKT 2 (Q34), PDGFB (Q4) and PIM 1 (Q9) which form quadruplex structures having 1–3 bases in the central loop and have ΔG37 as −15.6, −10.4, −9.6, −9.6 and −9.3 kcal/mol, respectively. Potential quadruplex-forming sequences of promoter regions of c-MYC, c-KIT, VEGF and PDGFB have served as paradigms for quadruplex-mediated gene expression regulation (6,7,51). The structure adopted by promoter regions c-MYC (Q21), c-KIT (Q30), VEGF (Q1) and PDGFB (Q4) have thermodynamic stability of −3.2, −3.6, −6.5 and −9.6 kcal/mol, respectively.Figure 4.


A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

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

Representative graphs showing effect of loop length on thermodynamic stability (ΔG37) of quadruplex.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Representative graphs showing effect of loop length on thermodynamic stability (ΔG37) of quadruplex.
Mentions: Both enthalpy and entropy in turn influence G, which is an indicator of thermodynamic stability. It is well documented in literature that increases in loop length results in decrease in quadruplex stability; however this observation is reported mainly for model sequences (29–34). A recent study using model sequences has shown that total loop length ≤5 has a major effect on quadruplex stability, and an addition of single nucleotide significantly affects the thermodynamic stability (34). Another study using model sequences with long central loops (6–9 bases) and two side loops composed of single base has shown that pyrimidines provide more stability over adenines and the composition of the central loop can minimize the destabilizing effect of a long central loop on quadruplex stability (29). This is in agreement with another study which demonstrated that, for intramolecular quadruplexes with G3 tracts and single base loops, a T to A loop substitution in reduced stability by 8°C (33). Intriguingly, the thermodynamic data obtained for quadruplexes formed by naturally occurring sequences having different loop lengths show that there is no clear correlation of loop length with the thermodynamic stability. Figure 4 depicts the variability in the thermodynamic stability with respect to the loop length. Quadruplexes with a total loop length of 5 formed by WNT 5A (Q3), VEGF (Q1) and WNT 3 (Q2) have thermodynamic stability ranging from −3.7 to −15.6 kcal/mol, respectively. Similarly, wide variations in the thermodynamic stability (−1.8 to −9.3 kcal/mol) were observed among quadruplex formed by sequences with loop size of 7. Likewise, no significant correlation of loop length and thermodynamic stability was observed for other data sets with longer loops. The lack of correlation between the thermodynamic analysis of quadruplexes formed by model sequences and genomic sequences with varied loop length can be attributed to the loop composition. For the model sequences, the loop length and composition are usually homogeneous. However, the naturally occurring sequences show random occurrences of purines and/or pyrimidines in the loops between the G stretches, which substantially influence the quadruplex thermodynamic stabilities. For instance, PIM1 (Q9) and FGF 3 (Q10) have total loop lengths of 7 with similar loop composition display remarkably different thermodynamic stabilities of −9.3 and −1.8 kcal/mol, respectively. Apart from total loop length and loop composition, the number of bases in the central loop is critical for quadruplex stability (32–35). We observed that for smaller loops, quadruplexes with a single base central loop are much more stable and the stability decreased upon increasing the bases in the central loop, as observed in case of WNT 3 (Q2) and WNT 5A (Q3) with thermodynamic stability of −15.6 and −3.7 kcal/mol, respectively. Our results shows that the majority (32) of quadruplex-forming sequences of protoncogenes have ΔG37 values approximately within the range of −1.8 to −8.6 kcal/mol. Only five genes from our data set have high ΔG37 values (≥−9.0 kcal/mol). These genes include WNT 3 (Q2), MYB (Q35), AKT 2 (Q34), PDGFB (Q4) and PIM 1 (Q9) which form quadruplex structures having 1–3 bases in the central loop and have ΔG37 as −15.6, −10.4, −9.6, −9.6 and −9.3 kcal/mol, respectively. Potential quadruplex-forming sequences of promoter regions of c-MYC, c-KIT, VEGF and PDGFB have served as paradigms for quadruplex-mediated gene expression regulation (6,7,51). The structure adopted by promoter regions c-MYC (Q21), c-KIT (Q30), VEGF (Q1) and PDGFB (Q4) have thermodynamic stability of −3.2, −3.6, −6.5 and −9.6 kcal/mol, respectively.Figure 4.

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