Limits...
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.

Show MeSH
UV cooling profile of quadruplexes monitored at 295 nm along with nonlinear least square curve fitting (in red). The experiment was performed in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl. The cooling rate was 0.15°C/min. The thermodynamic parameters obtained by van't Hoff analysis are provided in the inset.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553590&req=5

Figure 2: UV cooling profile of quadruplexes monitored at 295 nm along with nonlinear least square curve fitting (in red). The experiment was performed in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl. The cooling rate was 0.15°C/min. The thermodynamic parameters obtained by van't Hoff analysis are provided in the inset.

Mentions: Further investigations were carried out only with the 40 sequences which displayed predominantly one type of conformation to determine their thermodynamic profile by van't Hoff analysis. We also performed the UV melting of the remaining 22 sequences which showed a mixed population. The data for these sequences are presented in Supplementary Data, and can be used as a resource for future investigations; however, these 22 sequences have been excluded from the present analysis. UV melting of these quadruplexes showed characteristic hypochromic absorbance profile at 295 nm, which showed reversible heating and cooling curves, thus indicating that the system is in thermodynamic equilibrium. We performed concentration-dependent UV melting on all 40 sequences and observed that the melting temperatures were concentration independent (data not shown), which indicates that they form intramolecular structures. Representative UV melting profiles of a few of these quadruplexes is presented in Figure 2; the melting profile of rest of the sequences has been provided in Supplementary Data. In Figure 2, the upper panel displays melting profiles of quadruplexes from the promoters of c-MYC (Q21), c-KIT (Q30), VEGF (Q1) and PDGFB (Q4) which have melting temperature, Tm of 75.0, 71.6, 80.8 and 83.0°C, respectively. We identified new quadruplex-forming sequences which demonstrated remarkable stability and are presented in the lower panel of Figure 2. These sequences belong to promoter regions of AKT 2 (Q34), MYB (Q35), PIM 1 (Q9), WNT 3 (Q2) which exhibit Tm of 87.0, 88.0, 82.8 and 86.0°C, respectively. We found several examples where sequences with the same loop length display different Tm values. For example, VEGF (Q1), WNT 3 (Q2) and WNT 5A (Q3) have total loop lengths of 5 bases and Tms of 80.8, 86.0 and 65.0°C, respectively. It can also be seen that structures with different loop lengths display similar melting temperatures. For example, JUN B (Q30), MAF (Q42), FES (Q53) and FGF 6 (Q62) have total loop length of 11, 13, 14 and 18 bases, respectively, but all exhibit a Tm of 70.0°C. The melting profiles obtained for sequences Q6, Q7, Q16 and Q17 showed very broad transitions suggestive of multiple quadruplex conformations. However, the UV melting data for these sequences were concentration independent, thereby indicating intramolecular quadruplex formation. Moreover, the CD and gel studies also showed that these sequences each adopt a single conformation. The multiple transitions in the UV melting profiles of these structures, suggest the presence of conformations which have similar CD spectra and similar mobility but different melting temperatures. These sequences were not considered for further analysis.Figure 2.


A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

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

UV cooling profile of quadruplexes monitored at 295 nm along with nonlinear least square curve fitting (in red). The experiment was performed in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl. The cooling rate was 0.15°C/min. The thermodynamic parameters obtained by van't Hoff analysis are provided in the inset.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: UV cooling profile of quadruplexes monitored at 295 nm along with nonlinear least square curve fitting (in red). The experiment was performed in 10 mM sodium cacodylate buffer, pH 7.4, 100 mM KCl. The cooling rate was 0.15°C/min. The thermodynamic parameters obtained by van't Hoff analysis are provided in the inset.
Mentions: Further investigations were carried out only with the 40 sequences which displayed predominantly one type of conformation to determine their thermodynamic profile by van't Hoff analysis. We also performed the UV melting of the remaining 22 sequences which showed a mixed population. The data for these sequences are presented in Supplementary Data, and can be used as a resource for future investigations; however, these 22 sequences have been excluded from the present analysis. UV melting of these quadruplexes showed characteristic hypochromic absorbance profile at 295 nm, which showed reversible heating and cooling curves, thus indicating that the system is in thermodynamic equilibrium. We performed concentration-dependent UV melting on all 40 sequences and observed that the melting temperatures were concentration independent (data not shown), which indicates that they form intramolecular structures. Representative UV melting profiles of a few of these quadruplexes is presented in Figure 2; the melting profile of rest of the sequences has been provided in Supplementary Data. In Figure 2, the upper panel displays melting profiles of quadruplexes from the promoters of c-MYC (Q21), c-KIT (Q30), VEGF (Q1) and PDGFB (Q4) which have melting temperature, Tm of 75.0, 71.6, 80.8 and 83.0°C, respectively. We identified new quadruplex-forming sequences which demonstrated remarkable stability and are presented in the lower panel of Figure 2. These sequences belong to promoter regions of AKT 2 (Q34), MYB (Q35), PIM 1 (Q9), WNT 3 (Q2) which exhibit Tm of 87.0, 88.0, 82.8 and 86.0°C, respectively. We found several examples where sequences with the same loop length display different Tm values. For example, VEGF (Q1), WNT 3 (Q2) and WNT 5A (Q3) have total loop lengths of 5 bases and Tms of 80.8, 86.0 and 65.0°C, respectively. It can also be seen that structures with different loop lengths display similar melting temperatures. For example, JUN B (Q30), MAF (Q42), FES (Q53) and FGF 6 (Q62) have total loop length of 11, 13, 14 and 18 bases, respectively, but all exhibit a Tm of 70.0°C. The melting profiles obtained for sequences Q6, Q7, Q16 and Q17 showed very broad transitions suggestive of multiple quadruplex conformations. However, the UV melting data for these sequences were concentration independent, thereby indicating intramolecular quadruplex formation. Moreover, the CD and gel studies also showed that these sequences each adopt a single conformation. The multiple transitions in the UV melting profiles of these structures, suggest the presence of conformations which have similar CD spectra and similar mobility but different melting temperatures. These sequences were not considered for further analysis.Figure 2.

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