Abstract
We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristic identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.
Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.
Footnotes
↵† tritemio{at}gmail.com
↵‡ michalet{at}chem.ucla.edu
1 In this document, we will indifferently refer to detector counts as “photons” or “counts”, since there is no way to distinguish between them at the individual count level.
2 Note that Eq. (SI.19) uses the fact that the “gap” regions discussed in Appendix 5 have a negligible influence.
3 Other possibilities are, for instance, to use only donor channel photons (“donor emission burst search”, or DemBS: all photons recorded by the donor channel, irrespective of the laser alternation period they were emitted in), or acceptor channel photons (“acceptor emission burst search”, or AemBS: same as above, but for acceptor photons), donor excitation period photons (“donor excitation burst search”, or DexBS: all photons recorded by either channels but limited to the donor laser on period), or acceptor excitation period photons (“acceptor excitation burst search”, or AexBS: same as above, but for the acceptor laser). Any of these burst searches can be combined using logic AND or OR operations. The AND combination of two searches simply keeps the burst parts overlapping in both searches (in other words, their intersection, ∩). The OR combination of two searches returns all bursts found in either search, fusing any two overlapping bursts into a single, larger burst (their union, ⋃). For instance, the “dual channel burst search” (DCBS) defined in ref. 21 corresponds to the intersection (OR operation) of a donor excitation and an acceptor excitation burst searches: DCBS = DexBS AND AexBS.
4 Starting with this appendix, notations in this Supporting Material depart from the simpler notations introduced the main text in order to better distinguish several related quantities.
5 In the main text, this quantity is called nDem (or nD). Quantity nAem (or nA) in the main text corresponds to in this document. corresponds to nAA.
6 Eq. (SI.50) only applies to zero-FRET samples. For a doubly-labeled sample characterized by a non-zero FRET efficiency E, it needs to be modified into: This expression reduces to Eq. (SI.50) for E = 0, but is indeterminate for a 100% FRET efficiency sample (E = 1), for which . In this case, Eq. (SI.55) is a preferable expression. Obviously, a donor-only molecule will not contribute any direct acceptor excitation signal (i.e. Dir = 0 for these molecules).
7 By construction, N D + N A = FD.
8 At this stage, it is possible to apply the donor leakage and acceptor direct excitation corrections to obtain an EPR-histogram rather than a PRH
9 A similar term accounting for a potential vertical shift can be included, but, as for the diffusion component of the ACF which was not necessary in this study, the ACF is much less sensitive to this effect than to a lateral shift.