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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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Plots showing dependence of relative thermodynamic stability of duplex and quadruplex on total loop length. The encircled points display lower ΔΔG37 values suggesting a significant contribution from competing quadruplex structures.
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Figure 5: Plots showing dependence of relative thermodynamic stability of duplex and quadruplex on total loop length. The encircled points display lower ΔΔG37 values suggesting a significant contribution from competing quadruplex structures.

Mentions: Emerging evidence from bioinformatic data analysis indicates a wide distribution of quadruplex-forming sequences in the human genome with an average incidence of 1 quadruplex in 10 000 bases (27). Our study shows that the sequences in the promoter region of proto-oncogenes adopt quadruplex structures which exhibit variable thermodynamic stability ranging from −1.7 to −15.6 kcal/mol. These findings raise a discussion over whether biological function is associated with quadruplex structures having lower thermodynamic stability, which undergoes facile formation and disruption, or is it accomplished, by quadruplex structures with high thermodynamic stability. Our attempt to elucidate the thermodynamic profile of the quadruplexes formed by naturally occurring sequences also invites a systematic effort to integrate the thermodynamics and biological relevance of quadruplexes that would allow better understanding of quadruplex-mediated regulatory mechanism. In the human genome, a number of sites with potential quadruplex-forming structures have been estimated and some of these potential sites for quadruplex formation have been correlated with gene function (13). Quadruplex formation at specific human promoter regions has suggested the notion that quadruplexes might serve as potential regulatory motifs. However, these speculations have only been supported by analysis of either synthetic oligonucleotides or supercoiled DNA. To comprehend how quadruplex formation contributes in gene regulation, it is essential to take into account the contribution of the competing Watson–Crick duplex structure. For quadruplex formation, G-rich regions must be released from the duplex DNA, which occurs under transient denaturation during replication, transcription and recombination. The interconversion of quadruplex and duplex DNA is dependent on the relative stability of these competing secondary structures. In the current study we have examined the role of loop length on thermodynamic stability of quadruplex formed by naturally occurring sequences in human proto-oncogene promoters. However, it is necessary to compare thermodynamic stability of quadruplexes with their respective duplexes to understand duplex–quadruplex interconversion. It is difficult to obtain the thermodynamic parameters involved in duplex formation from the same sequences and their respective complementary strand by UV melting studies, as it includes the contributions from both duplex and quadruplex. We therefore obtained the thermodynamic profiles for the duplexes through a nearest neighbor (NN) method (48,49). This method assumes that the stability of a given base pair depends on the identity and orientation of the neighboring base pairs. During the past decades numerous studies have been performed to calculate thermodynamic parameters of a given duplex under specified experimental conditions by this method. It is well established that NN method allows evaluation of the thermal stability and thermodynamic parameters of duplexes with precision and data are in agreement with the experimental data. Hyther is a tool that allows calculation of nucleic acid hybridization thermodynamics using NN method. Using Hyther, we calculated the thermodynamic profile of duplexes formed by G-rich sequence with their respective complementary strand with the strand concentration and buffer conditions (Tables 2–4) used in this study. The important parameter that dictates the predominance of either the duplex or quadruplex is the relative free energy difference, the ΔΔG37 between the duplex and quadruplex forms. It is noteworthy that in all the cases ΔΔG37 values are negative indicating that duplex is predominant structure in the competition (Tables 2–4). We also observed an increase in duplex stability upon increasing the loop length (Tables 2–4). The relative free energy difference, the ΔΔG37 between duplex and quadruplex structure increases (∼−8.1 kcal/mol to −35.1 kcal/mol) upon increasing the loop length. The greater the negative magnitude of ΔΔG37, the higher is the predominance of duplex at equilibrium. As shown in Figure 5, only the quadruplexes formed by WNT 3 (Q2), PDGFB (Q4), VEGF (Q1) and WNT 5A (Q3) demonstrate less negative ΔΔG37 values of −8.1 kcal/mol, −12.1 kcal/mol, −13.3 kcal/mol and −16.0 kcal/mol, respectively. These lower ΔΔG37 values indicate that although the duplex is more stable than the quadruplex, the contribution from competing quadruplex population is high, which leads to less negative ΔΔG37 values.Figure 5.


A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes.

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

Plots showing dependence of relative thermodynamic stability of duplex and quadruplex on total loop length. The encircled points display lower ΔΔG37 values suggesting a significant contribution from competing quadruplex structures.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Plots showing dependence of relative thermodynamic stability of duplex and quadruplex on total loop length. The encircled points display lower ΔΔG37 values suggesting a significant contribution from competing quadruplex structures.
Mentions: Emerging evidence from bioinformatic data analysis indicates a wide distribution of quadruplex-forming sequences in the human genome with an average incidence of 1 quadruplex in 10 000 bases (27). Our study shows that the sequences in the promoter region of proto-oncogenes adopt quadruplex structures which exhibit variable thermodynamic stability ranging from −1.7 to −15.6 kcal/mol. These findings raise a discussion over whether biological function is associated with quadruplex structures having lower thermodynamic stability, which undergoes facile formation and disruption, or is it accomplished, by quadruplex structures with high thermodynamic stability. Our attempt to elucidate the thermodynamic profile of the quadruplexes formed by naturally occurring sequences also invites a systematic effort to integrate the thermodynamics and biological relevance of quadruplexes that would allow better understanding of quadruplex-mediated regulatory mechanism. In the human genome, a number of sites with potential quadruplex-forming structures have been estimated and some of these potential sites for quadruplex formation have been correlated with gene function (13). Quadruplex formation at specific human promoter regions has suggested the notion that quadruplexes might serve as potential regulatory motifs. However, these speculations have only been supported by analysis of either synthetic oligonucleotides or supercoiled DNA. To comprehend how quadruplex formation contributes in gene regulation, it is essential to take into account the contribution of the competing Watson–Crick duplex structure. For quadruplex formation, G-rich regions must be released from the duplex DNA, which occurs under transient denaturation during replication, transcription and recombination. The interconversion of quadruplex and duplex DNA is dependent on the relative stability of these competing secondary structures. In the current study we have examined the role of loop length on thermodynamic stability of quadruplex formed by naturally occurring sequences in human proto-oncogene promoters. However, it is necessary to compare thermodynamic stability of quadruplexes with their respective duplexes to understand duplex–quadruplex interconversion. It is difficult to obtain the thermodynamic parameters involved in duplex formation from the same sequences and their respective complementary strand by UV melting studies, as it includes the contributions from both duplex and quadruplex. We therefore obtained the thermodynamic profiles for the duplexes through a nearest neighbor (NN) method (48,49). This method assumes that the stability of a given base pair depends on the identity and orientation of the neighboring base pairs. During the past decades numerous studies have been performed to calculate thermodynamic parameters of a given duplex under specified experimental conditions by this method. It is well established that NN method allows evaluation of the thermal stability and thermodynamic parameters of duplexes with precision and data are in agreement with the experimental data. Hyther is a tool that allows calculation of nucleic acid hybridization thermodynamics using NN method. Using Hyther, we calculated the thermodynamic profile of duplexes formed by G-rich sequence with their respective complementary strand with the strand concentration and buffer conditions (Tables 2–4) used in this study. The important parameter that dictates the predominance of either the duplex or quadruplex is the relative free energy difference, the ΔΔG37 between the duplex and quadruplex forms. It is noteworthy that in all the cases ΔΔG37 values are negative indicating that duplex is predominant structure in the competition (Tables 2–4). We also observed an increase in duplex stability upon increasing the loop length (Tables 2–4). The relative free energy difference, the ΔΔG37 between duplex and quadruplex structure increases (∼−8.1 kcal/mol to −35.1 kcal/mol) upon increasing the loop length. The greater the negative magnitude of ΔΔG37, the higher is the predominance of duplex at equilibrium. As shown in Figure 5, only the quadruplexes formed by WNT 3 (Q2), PDGFB (Q4), VEGF (Q1) and WNT 5A (Q3) demonstrate less negative ΔΔG37 values of −8.1 kcal/mol, −12.1 kcal/mol, −13.3 kcal/mol and −16.0 kcal/mol, respectively. These lower ΔΔG37 values indicate that although the duplex is more stable than the quadruplex, the contribution from competing quadruplex population is high, which leads to less negative ΔΔG37 values.Figure 5.

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
Related in: MedlinePlus