DNase I cleavage of branched DNA molecules

We report here a potentially useful signature of branched DNA structures. The base 5' to the branch and the five bases flanking the 3' side of the branch site are protected from cleavage by DNase I in both three- and four-arm branched DNA molecules. Our procedure is to measure the cleavage profile for each 5'-labeled strand in a control duplex and compare this with that of the same strand in a branched structure under conditions yielding less than one cut per strand. The resulting cleavage pattern in an immobile four-arm junction is roughly 2-fold symmetric, consistent with the pattern of Fe(II)·EDTA-induced cleavage that has been observed previously. In the three-arm junction, the DNase I cleavage pattern is asymmetric, indicating lack of 3-fold symmetry. A variable pattern of protection occurs to the 5' side of the branch in some strands only for both three- and four-arm junctions, extending 2-4 residues 5' to the branch.