Cooperative Oligonucleotide-Directed Triple Helix Formation at Adjacent DNA Sites

Citation

Colocci, Natalia (1996) Cooperative Oligonucleotide-Directed Triple Helix Formation at Adjacent DNA Sites. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/08j6-sd37. https://resolver.caltech.edu/CaltechTHESIS:12222020-002250563

Abstract

Cooperative interactions between DNA-binding ligands are essential for their sequence specificity, binding affinity, and biological activity. Oligonucleotides can bind cooperatively to adjacent sites on double-helical DNA by triple helix formation. The study of the cooperative binding of oligonucleotides to DNA by triple helix formation is important as it provides useful information for the development of new methods leading to the sequence-specific recognition of DNA. As a first step towards this goal, the thermodynamics of the cooperative binding of oligodeoxyribonucleotides to adjacent DNA sites by triple helix formation have been determined by quantitative affinity cleavage titrations (Chapter Two). A 20-fold enhancement in equilibrium association constant is realized for an 11mer pyrimidine oligonucleotide binding in the presence of a neighboring bound site at 24 °C and pH 7.0 (25 mM TrisOAc, 10 mM NaCl, 1 mM spermine). This corresponds to an increase in binding free energy of 1.8 kcal•mol⁻¹. This cooperativity is not observed when the two binding sites are separated by one base pair. The observed cooperative energy likely arises from favorable polarization and charge-charge interactions between the terminal bases at the triple-helical junction.

In addition, the energetics of cooperative binding by oligodeoxyribonucleotides to adjacent sites by triple helix formation have been determined as a function of sequence composition at the junction (Chapter Three). The binding affinity of an 11mer in the presence of a neighboring bound oligonucleotide is enhanced by a factor of 12, 17, 61, and 127 when a 5'-TT-3', 5'-ᵐCᵐC-3', 5'-TᵐC-3', and 5'-ᵐCT-3' stack, respectively, is formed at the junction (10 mM Bis-Tris•HCl at pH 7.0, 10 mM NaCl, 250 µM spermine, 24 °C) (ᵐC designates 5-methyl-2'-deoxycytidine). These binding enhancements correspond to an interaction energy between the two oligonucleotides of 1.5, 1.7, 2.5, and 2.9 kcal•mol⁻¹, respectively. The energetic penalties for a single-base mismatch differ depending on sequence and the location of the mismatch with respect to the 5'- or 3'-side of the junction. In the case of a 5'-TT-3' stack, a T•GC mismatch on the 5'- side of the junction decreases the interaction energy from 1.5 kcal•mol⁻¹ to 0.6 kcal•mol⁻¹, whereas a T•GC mismatch on the 3'- side destroys cooperativity. For a 5'-ᵐCT-3' stack, a ᵐC•AT mismatch on the 5'-side of the junction decreases the cooperative interaction energy from 2.9 kcal•mol⁻¹ to 1.7 kcal•mol⁻¹, whereas a T•GC mismatch on the 3'-side of the junction destroys cooperativity. Most importantly, two 11mer oligonucleotides interacting through a 5'-TT -3' stack binding to adjacent sites on DNA are significantly more sensitive to single-base mismatches than the corresponding 22mer binding to the same two abutting sites.

The use of modified bases, such as 5-(1-propynyl)-2'-deoxyuridine, increases cooperativity between oligonucleotides bound to adjacent sites on DNA, presumably due to an increased stacking energy between the modified bases at the triplex junction (Chapter Four). Oligodeoxyribonucleotides containing 5-(1-propynyl)-2'-deoxyuridine and 5-methyl-2'-deoxycytidine as short as 8 nucleotides in length bind at micromolar concentrations to adjacent 8-bp sites on double-helical DNA at 24 °C and pH 7.0 (10 mM Bis-Tris•HCl, 10 mM NaCl, 1 mM spermine). Quantitative affinity cleavage titrations reveal that the binding affinity of an 8mer in the presence of a neighboring bound 8mer is enhanced by a factor of at least 40. This corresponds to a remarkable cooperative interaction energy of > 4.5 kcal•mol⁻¹. In addition, these cooperative interactions allow oligonucleotides as short as 6mers to bind to three adjacent sites on double-helical DNA at near micromolar concentrations (10 mM Bis-Tris•HCl at pH 7.0, 10 mM NaCl, 1 mM spermine, 24 °C).

Cooperativity is also observed between purine-rich oligonucleotides (Chapter Five). Quantitative DNase footprinting titration experiments show that the binding affinity of an 11mer purine-rich oligonucleotide in the presence of a neighboring bound oligonucleotide is enhanced by a factor of 81 when a 5'-GG-3' stack is formed at the junction (50 mM TrisOAc at pH 7.0, 10 mM NaCl, 10 mM MgCl₂, 24 °C). This binding enhancement corresponds to an interaction energy between the two oligonucleotides of 2.7 kcal•mol⁻¹, and is abolished when the two binding sites are separated by one base pair.

The synthesis of pyrimidine oligonucleotide analogs containing 5-(1-propynyl)- and 2'-O-allyl-modified nucleosides (Chapter Six), and the progress towards the synthesis of a novel base, N7-2'-deoxyisoinosine, designed for the recognition of AT base pairs within a parallel isomorphous purine motif (Chapter Seven), are described.

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