Figure 1.

DNA duplexes used as FRET standards. (A) DNA oligonucleotides used to form the FRET standards. The acceptor strand has a 5′ terminal Cy5 dye and a 3′ biotin TEG moiety for tethering the DNA. Four different DNA constructs were generated by annealing this top strand with one of four complimentary strands each containing an internal Cy3 dye at the position indicated. The numbering refers to the basepair preceding the internal Cy3. (B) Raw FRET histograms show the relative proximity ratio (E_PR) for each of the DNA constructs as labeled above their distribution: DNA19 (19 bp), DNA14 (14 bp), DNA10 (10 bp), and DNA7 (7 bp). Separate measurements are shown for each DNA duplex under optical path 3. (C) Relative proximity ratio for the four DNA duplexes measured under optical path 1 (solid), optical path 2 (shaded), and optical path 3 (open). Panel shows the mean value with the error bars indicating the width from a Gaussian fit.