Psoralen

As an RNA polymerase tracks along helical DNA during transcription, positive supercoils of DNA are produced in front of the polymerase and negative supercoils are generated behind it (ref 1). These supercoils are then relaxed by topoisomerases. Matsumoto and Hirose developed a method to visualize the transcription-coupled negative supercoils in an interphase genome (ref 2). The technique relies on the ability of 4, 5', 8-trimethyl psoralen (abbreviated as psoralen below) to intercalate into DNA. Upon exposure to 365 nm light, the intercalated psoralen crosslinks DNA strands at a rate that depends on the degree of negative superhelicity in the DNA. Using biotinylated psoralen and fluorescent streptavidine, the unconstrained negative supercoils can be visualized within a cell. The psoralen reagent was further improved by replacing a linker region between psoralen and biotin with a delivery peptide derived from HIV Tat protein (ref 3). This allows rapid incorporation of thereagent into cells without prior treatment with a detergent. The method is applicable to cultured cells, tissue slices and dissected out small tissues where transcription is going on.