31 July 1998 Ž . Chemical Physics Letters 292 1998 15–21 Heterogeneity in single DNA conformational fluctuations Lars Edman ) , Stefan Wennmalm, Fredrik Tamsen, Rudolf Rigler Department of Medical Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden Received 19 January 1998; in final form 13 April 1998 Abstract Non-exponential behaviour in bulk measurements of two-state molecular reactions can have different origins. The probability density for the time which one of two states is occupied during different observation interval times can be used to reveal system characteristics if single molecules are studied. An evaluation of new experimental data from single DNA molecules labeled with a fluorescent conformation probe proves the heterogeneity of the ensemble system. Also, the anomalicity is concluded to be low for single events. The evaluation method can be applied in other experiments where two-state reversible processes are studied for isolated systems. q 1998 Elsevier Science B.V. All rights reserved. 1. Introduction Recently, we have reported experimental data showing two-state conformational fluctuations in sin- wx gle DNA molecules chained on a surface 1 and wx wx freely diffusing in solution 2 . In Ref. 2 , the diffu- Ž sion time of the sample molecule oligonucleotide . primers was reduced by attaching a 7.3-kB-long M13 phage DNA strand with a sequence comple- mentary to the sample molecule. Hence, the observa- tion time interval when a molecule diffused through the detection volume was prolonged. The transitions can be observed by spectroscopic means since the Ž . base guanine G quenches the fluorescence of the Ž . sensor molecule tetramethylrhodamine by an elec- tron transfer process. Hence, the fluorophore senses Ž conformational changes in a base-sequence DNA or . oligonucleotide that originates from a G being situ- wx ated close to a rhodamine molecule. In Ref. 2 freely diffusing fluorescently labeled oligonucleotides were ) Corresponding author. studied on the single molecule level. All single molecules were observed during a fixed time inter- val. This allowed for equivalent comparisons be- Ž . tween all single molecule events SME with regard to the total time a molecule resided in each of two detectable states during the fixed time-measuring intervals. The fraction of time a molecule resided in one of the states spanned from 0 to unity for the w x collected data 2,3 . The question was raised whether all molecules shared the same transition time charac- teristics or if different molecules could be ascribed different relaxation rates; however, no final decision wx could be made. In Ref. 1 dye-tagged DNA molecules attached to a surface were studied with regard to the same general conformational process. The observed fluctuations indicated a heterogeneic situation where each of the three measured molecules had its own exponential transition rate. wx The data in Ref. 2 also indicated that the transi- tion process, as seen for many molecules, could be well fitted with a stretched exponential relaxation Ž Ž . b . law ; exp y tk , with b s 0.44 rather than b s 0009-2614r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII: S0009-2614 98 00633-2