It is common practise in antisense technology to view third strand binding to be controlled by the same principles which are found to determine the stability of the double helix. In contrast to this view based on a general consideration of the various forces contributing to the binding energy of the third strand it was proposed that the dominant contributions will originate from electrostatic interactions. These electrostatic contributions can be subdivided into sequence independent repulsive forces between the negatively charged backbones and into sequence dependent attractive forces between the positively charged protonated Hoogsteen cytosines and the backbone phosphates. The observable changes in the stability of triple helices should be a reflection of the number (global composition) and distribution (local composition) of cytosines in the third strand. To this aim two families of 38-mer oligonucleotides were synthesized, which have as a common design feature a linear array of 10 homopurine bases followed by 10 homopyrimidine bases as Watson & Crick complementary strand to the homopurine region and ending in a 10 homopyrimidine residue stretch which binds to the W&C helix via Hoogsteen base-pairing. This arrangement of homopurine and homopyrimidine sections with connecting pyrimidine linkers allows the formation of intramolecular triple helices of predetermined stoichiometry and strand orientation. Physical (UV-spectroscopy, CD-spectroscopy and fluorimetry) and biochemical techniques (P1-nuclease digestion) have been used to show that the oligonucleotides undergo a stepwise folding process from a random coil into a hairpin with 3'dangling tail and then into a intramolecular triple helix. This folding occurs as a function of pH and/or ionic strength. The effect of local and global composition on the stability of the three conformational transitions has been evaluated from a comparison of the melting temperatures and the behavior of the phase boundaries of the different oligonucleotides. As the result of this thesis the following general rules emerge: The stability of the third strand depends on the particular combination of sequence, pH and ionic strength. At physiological conditions (pH 7.1, 150 mM Na⁺) thymines and cytosines contribute equally to the stability (global effect) provided that the cytosines are spaced by more than one thymine. (local effect). Below pH 7.1 (150 mM Na⁺) the stability increases linearly with the number of cytosines and at pH above pH 7.1 ( 150 mM Na⁺) it decreases. At ionic strength below 400 mM Na⁺ (pH 6. 75) the stability increases with the number of cytosine while above 400 mM Na⁺ (pH 6. 75) it decreases. Based on these results a rational approach for the design of oligonucleotide third strands and the choice of appropriate environmental conditions for the formation of a particular triple helix becomes feasible
