Effects of the conformation of single-stranded DNA on renaturation and aggregation

Sedimentation behavior is used to follow strand combination in T7 DNA and to analyze the nature of the product formed. Complementary single strands of DNA apparently combine under any condition where the DNA double helix is stable. The rate of strand combination is most strongly influenced by the ionic strength and the degree of folding of the single strands; the nature of the product formed is determined by the degree of folding of the reactants. Temperature affects the reaction primarily through its influence on folding, except at temperatures near Tm, where its effect on the stability of base pairing becomes dominant. Folding of the single strands (Studier, 1969) can decrease the rate of strand combination by orders of magnitude, by reducing the possibilities for pairing between complementary strands. Folding also inhibits the zippering up of joined strands, thus leading to multiple nucleations and consequent aggregation, as well as to the formation of complexes containing part native and part folded regions. If such nucleated complexes are placed under conditions which disrupt folding but do not separate strands, extensive zippering up occurs, but at least some aggregates do not dissociate. Perfect renaturation, that is, strand combination which produces perfectly native DNA, requires only that the single strands be unfolded and the native structure stable. Under such conditions, only those associations which put the two strands in register are stable, and once joined, the strands zipper up completely. No stable intermediates are detected. For unfolded molecules, the rate of renaturation increases with approximately the cube of the ionic strength, at least until 0.2 m-NaCl. The implications of these findings for the preparation and storage of singlestranded DNA and for studies of hybridization and homology are discussed. Perfect renaturation is found under quasi-physiological conditions; it seems only logical that the same factors which affect this reaction in vitro will also apply in vivo.