Probing the structure of macromolecules using microsecond time-resolved fluorescence of europium chelates

Lanthanide chelates, due to their unique luminescence properties, offer a potential for improvement and simplification of fluorescence resonance energy transfer distance measurements. in this report we present a procedure for incorporation of highly-luminescent europium chelate into internal sites of ds DNA. Using this labeling strategy donor-acceptor labeled 20 bp DNA fragments were prepared containing a stretch of six A residues (A-tract) in the middle of the fragment. Distance measurement between the donor (europium chelate) and acceptor (Cy5) were performed and compared to measurements with a DNA fragment lacking the A-tract. Only small changes in measured distances were observed between these two DNA molecules. The implications of these results for existing models of A-tract induced DNA bending are discussed.