title: Deciphering Kinetic Information from Single-Molecule FRET Data That Show Slow Transitions.
abstract: Single-molecule FRET is one of the most powerful and widely used biophysical techniques in biological sciences. It, however, often suffers from limitations such as weak signal and limited measurement time intrinsic to single-molecule fluorescence measurements. Despite several ameliorative measures taken to increase measurement time, it is nearly impossible to acquire meaningful kinetic information on a molecule if conformational transitions of the molecule are ultraslow such that transition times (⟨τ⟩orig) are comparable to or longer than measurement times (δt) limited by the finite lifetime of fluorescent dye. Here, to extract a reliable and accurate mean transition time from a series of short time traces with ultraslow kinetics, we suggest a scheme called sHaRPer (serialized Handshaking Repeated Permutation with end removal) that concatenates multiple time traces. Because data acquisition frequency f and measurement time (δt) affect the estimation of mean transition time (⟨τ⟩), we provide mathematical criteria that f, δt, and ⟨τ⟩ should satisfy to make ⟨τ⟩ close enough to ⟨τ⟩orig. Although application of the sHaRPer method has a potential risk of distorting the time constants of individual kinetic phases if the data are described with kinetic partitioning, we also provide criteria to avoid such distortion. Our sHaRPer method is a useful way to handle single-molecule data with slow transition kinetics. This study provides a practical guide to use sHaRPer.
authors.affiliation: †Department of Physics, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Sung Eun
authors.initials: SE
authors.lastname: Kim
authors.affiliation: †Department of Physics, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Il-Buem
authors.initials: IB
authors.lastname: Lee
authors.affiliation: ‡Korea Institute for Advanced Study, Seoul 130-722, Republic of Korea.
authors.forename: Changbong
authors.initials: C
authors.lastname: Hyeon
authors.affiliation: †Department of Physics, Korea University, Seoul 136-713, Republic of Korea.|‡Korea Institute for Advanced Study, Seoul 130-722, Republic of Korea.
authors.forename: Seok-Cheol
authors.initials: SC
authors.lastname: Hong
mesh_terms: D004247:DNA / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D009690:Nucleic Acid Conformation
chemical_list: D004247:DNA