Hui Ting Lee

DNA complexes containing joined triplex and duplex motifs: Melting behavior of intramolecular and bimolecular complexes with similar sequences

Our laboratory is interested in predicting the thermal stability and melting behavior of nucleic acids from knowledge of their sequence. One focus is to understand how sequence, duplex and triplex stabilities, and solution conditions affect the melting behavior of complex DNA structures, such as intramolecular DNA complexes containing triplex and duplex motifs. Nucleic acid oligonucleotides (ODNs), as drugs, present an exquisite selectivity and affinity that can be used in antigene and antisense strategies for the control of gene expression. In this work, we try to answer the following question: How does the molecularity of a DNA complex affect its overall stability and melting behavior? We used a combination of temperature-dependent UV spectroscopy and calorimetric (DSC) techniques to investigate the melting behavior of DNA complexes with a similar helical stem sequence, TC(+)TC(+)TC(+)T/AGAGAGACGCG/CGCGTCTCTCT, but formed with different strand molecularity. We determined standard thermodynamic profiles, and the differential binding of protons and counterions accompanying their unfolding. The formation of a DNA complex is accompanied by a favorable free energy term resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions. As expected, acidic pH stabilized each complex by allowing protonation of the cytosines in the third strand; however, the percentage of protonation increases as the molecularity decreases. The results help in the design of oligonucleotide sequences as targeting reagents that could effectively react with DNA or RNA sequences involved in human diseases, thereby increasing the feasibility of using the antigene and antisense strategies, respectively, for therapeutic purposes.

Thermodynamic contributions of the reactions of DNA intramolecular structures with their complementary strands.

One focus of our research is to further our understanding of the physico-chemical properties of unusual DNA structures and their interaction with complementary oligonucleotides. We have investigated three types of reactions involving the interaction of intramolecular DNA complexes with their complementary single strands of varied length. Specifically, we have used a combination of isothermal titration (ITC) and differential scanning (DSC) calorimetry and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of an i-motif, G-quadruplex, and triplex with their complementary strands. The enthalpies for each reaction are measured directly in ITC titrations and compared with those obtained indirectly from Hess cycles using DSC unfolding data. All reactions investigated yielded favorable free energy contributions, indicating that each single strand is able to invade and disrupt the corresponding intramolecular DNA complex. These favorable free energy terms are enthalpy driven, which result from a compensation of exothermic contributions, due to the formation of additional base-pair stacks (or base-triplet stacks) in the duplex product (or triplex product), immobilization of electrostricted water by the base-pair and base-triplet stacks, and the removal of structural water from the reactant single strands; and endothermic contributions from the disruption of base-base stacking interactions of the reactant single strands. This investigation of nucleic acid reactions has provided new methodology, based on physico-chemical principles, to determine the molecular forces involved in the interactions between DNA nucleic acid structures. This methodology may be used in targeting reactions for the control of gene expression.

A Thermodynamic Approach for the Targeting of Nucleic Acid Structures Using Their Complementary Single Strands

The main focus of our investigations is to further our understanding of the physicochemical properties of nucleic acid structures. We report on a thermodynamic approach to study the reaction of a variety of intramolecular nucleic acid structures with their respective complementary strands. Specifically, we have used a combination of isothermal titration (ITC) and differential scanning calorimetry (DSC) and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of a triplex, G-quadruplex, hairpin loops, pseudoknot, and three-arm junctions with their complementary strands. Reaction enthalpies are measured directly in ITC titrations, and compared with those obtained indirectly from Hess cycles using DSC unfolding data. All reactions investigated yielded favorable free energy contributions, indicating that each single strand is able to invade and disrupt the corresponding intramolecular DNA structure. These favorable free energy terms are enthalpy-driven, resulting from a favorable compensation of exothermic contributions due to the formation of additional base-pair stacks in the duplex product, and endothermic contributions, from the disruption of base stacking contributions of the reactant single strands. The overall results provide a thermodynamic approach that can be used in the targeting of nucleic acids, especially the secondary structures formed by mRNA, with oligonucleotides for the control of gene expression.

Unfolding thermodynamics of DNA pyrimidine triplexes with different molecularities

Nucleic acid oligonucleotides (ODNs), as drugs, present an exquisite selectivity and affinity that can be used in antigene and antisense strategies for the control of gene expression. In this work we try to answer the following question: How does the molecularity of a DNA triplex affect its overall stability and melting behavior? To this end, we used a combination of temperature-dependent UV spectroscopy and calorimetric (differential scanning calorimetry) techniques to investigate the melting behavior of DNA triplexes with a similar helical stem, TC+TC+TC+T/AGAGAGA/TCTCTCT, but formed with different strand molecularity. We determined standard thermodynamic profiles and the differential binding of protons and counterions accompanying their unfolding. The formation of a triplex is accompanied by a favorable free energy term, resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions, i.e., the folding of a particular triplex is enthalpy driven. The magnitude of the favorable enthalpy contributions corresponds to the number and strength of the base-triplet stacks formed, which are helped by stacking contributions due to the incorporation of dangling ends or loops. Triplex stability is in the following order: monomolecular > bimolecular > trimolecular; this is explained in terms of additional stacking contributions due to the inclusion of loops. As expected, acidic pH stabilized all triplexes by allowing protonation of the cytosines in the third strand; however, the percentage of protonation increases as the molecularity decreases. The results help to choose adequate solution conditions for the study of triplexes containing different ratios of CGC+ and TAT base triplets and to aid in the design of oligonucleotide sequences as targeting reagents that could effectively react with mRNA sequences involved in human diseases, thereby increasing the feasibility of using the antisense strategy for therapeutic purposes.

The size of the internal loop in DNA hairpins influences their targeting with partially complementary strands.

Targeting of noncanonical DNA structures, such as hairpin loops, may have significant diagnostic and therapeutic potential. Oligonucleotides can be used for binding to mRNA, forming a DNA/RNA hybrid duplex that inhibits translation. This kind of modulation of gene expression is called the antisense approach. In order to determine the best strategy to target a common structural motif in mRNA, we have designed a set of stem-loop DNA molecules with sequence: d(GCGCTnGTAAT5GTTACTnGCGC), where n = 1, 3, or 5, “T5” is an end loop of five thymines. We used a combination of calorimetric and spectroscopy techniques to determine the thermodynamics for the reaction of a set of hairpins containing internal loops with their respective partially complementary strands. Our aim was to determine if internal- and end-loops are promising regions for targeting with their corresponding complementary strands. Indeed, all targeting reactions were accompanied by negative changes in free energy, indicating that reactions proceed spontaneously. Further investigation showed that these negative free energy terms result from a net balance of unfavorable entropy and favorable enthalpy contributions. In particular, unfolding of hairpins and duplexes is accompanied by positive changes in heat capacity, which may be a result of exposure of hydrophobic groups to the solvent. This study provides a new method for the targeting of mRNA in order to control gene expression.

Interaction of DNA Intramolecular Structures with Their Complementary Strands: A Thermodynamic Approach for the Control of Gene Expression

The folding of mRNA sequences into secondary/tertiary structures plays an important role in RNA and DNA function and expression. Disruption of these structures can potentially be used in the control of gene expression. However, a detailed understanding of the physicochemical properties of nucleic acid structures is needed before this targeting approach can be used. In this chapter, we have examined six intramolecular DNA structures and have investigated their reaction thermodynamics with single strands partially complementary to their stems and loops. We measured the heat of each reaction directly using isothermal titration calorimetry. These are compared with the heat measured indirectly using Hess cycles obtained from differential scanning calorimetric unfolding thermodynamic profiles. Each reaction yielded favorable free energy terms that were enthalpy driven, indicating each complementary strand was able to disrupt the intramolecular complex. In short, we have developed a thermodynamic approach that can be used in the control of gene expression that targets the loops of secondary structures formed by mRNA.