Monitoring the conformational fluctuations of DNA hairpins using single-pair fluorescence resonance energy transfer

We present single-pair fluorescence resonance energy transfer (spFRET) obse rvations of individual opening and closing events of surface-immobilized DN A hairpins. Two glass-surface immobilization strategies employing the bioti n-streptavidin interaction and a third covalent immobilization strategy inv olving formation of a disulfide bond to a thiol-derivatized glass surface a re described and evaluated. Results from image and time-trace data from sur face-immobilized molecules are compared with those from freely diffusing mo lecules which are unperturbed by surface interactions. Using a simple two-s tate model to analyze the open and closed time distributions for immobilize d hail-pins, we calculate the lifetimes of the two states. For hairpins wit h a loop size of 40 adenosines and a stem size of either seven or nine base s, the respective closed-state Lifetimes are 45 +/- 2.4 and 103 +/- 6.0 ms, while the respective open-state lifetimes are 133 +/- 5.5 and 142 +/- 22 m s. These results show that the open state of the hairpin is favored over th e closed state of the hairpin under these conditions, consistent with previ ous diffusion fluorescence correlation spectroscopy (FCS) experiments on po ly(A)-loop hairpins. The measured open-state lifetime is about 30 times lon ger than the calculated 3 ms open-state lifetime for both hairpins based on a closing rate scaling factor derived from a previous FCS study for hairpi ns in diffusion with 12-30 thymidines in their loops. As predicted, the clo sed-state lifetime is dependent on the stem length and is independent of th e loop characteristics. Our findings indicate that current models should co nsider sequence dependence in calculating ssDNA thermostability. The surfac e immobilization chemistries and ether experimental techniques described he re should prove useful for studies of single-molecule populations and dynam ics.