Figure 1
The DNA used is a partial duplex: a
single stranded DNA tail (
) is attached to an end of 40mer duplex. The donor (
:Cy3) and acceptor (
:Cy5) are attached as shown. A biotin (
) that tightly binds to Streptavidin protein is attached to the DNA for immobilization to a quartz slide coated with Streptavidin. The sequences are
-Cy5-GCCCTGCTGCCGACCAACGATGGTTACATTCCCGCTGCTG-
biotin and
-CAGCAGCGGGAATGTAACCATCTTGGTCGGCAGCAGGGC-Cy3-
-
. Experiments were performed at room temperature in a buffer (20 mM Tris, pH 7.5, 6 mM NaCl, 1.7 mM
, 10% (
) glycerol). (a) Fluorescence time records of donor (dotted line) and acceptor (solid line) of a single DNA in an aerated sample (0.1 s bin): For measurements at much lower laser intensities, larger time bins were used. (b) Fluorescence time records in a deaerated sample (0.4 s bin).
was enzymatically removed [
12,
13,
22]. Note the time scale differences between (a) and (b). A cartoon illustrates stepwise photodestruction of the acceptor that results in the delay between the acceptor signal cessation and donor signal appearance.
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