A general approach to break the concentration barrier in single-molecule imaging

Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion, and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA-replication protein Flap endonuclease 1 (Fen1) with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.


Supplementary Figure 2: mKikGR is efficiently photoactivated.
Supplementary Figure 2: (a) We first used the known photophysical properties of mKikGR 1 to calculate the predicted photoconversion efficiency of mKikG to mKikR. The efficiency of photoconversion, which we refer to as photoactivation, is dependent on the 405-nm irradiance (power, in units of W cm -2 ) and its duration (in units of s). The decay constant for the loss of the green state (τ p ) can be derived for a particular irradiance using the following relation 2 : where Φ p is the quantum yield of photoconversion of mKikGR, σ λ is the absorption cross-section of the green state at wavelength λ, I λ is the irradiance with λ, h is Planck's constant, and c is the speed of light. The values of the photoconversion quantum yield and the absorbance cross-  At the indicated times, samples were withdrawn, and DNA was subjected to electrophoresis on two alkaline-agarose gels (2%) run side-by-side. A long phosphor imager exposure of the gels (top panels) was used to visualize short nascent strands, which includes Okazaki fragments (~120 bp) (bracket). The high MW nascent strands in the well saturated the phosphor screen (red), and a short exposure (bottom panels) was used to quantify these. Note that Fen1 depletion from LSS resulted in increase in Okazaki fragments during replication, and this increase was reversed by the addition of Fen1 KikGR . (i) Quantification of Okazaki fragments. The ratio of radioactivity in short (< 300 bp in size) and long (> 1,000 bp) DNA fragments (as in Supplementary Fig. 3h) was measured for each sample and normalized to the value of the mock-depleted control (mean ± s.e.m., n = 4 except ΔFen1 time points 60 and 90 minutes, n = 3).  Supplementary Fig. 4cii). The comparison of Supplementary Figures 4cii and 4dii shows a much higher signal to noise ratio in the latter.

Supplementary
(e) To learn whether repeated photoactivation and imaging of Fen1 KikR damages replication forks, we compared the rate of bubble growth in a field of view subjected to PhADE imaging (as in Fig. 2c) to an estimate of the growth rate in non-imaged fields. The replication bubble growth rate in non-imaged fields was estimated by dividing the mean size of bubbles in these fields (12,200 ± 900 bp (± error estimate)) by the time allowed for elongation (25 minutes). However, there is an uncertainty about the time of replication initiation in these non-imaged fields. Initiation may have occurred by the time of p27 Kip addition or been delayed by ~ 5 minutes, as observed for most bubbles imaged with PhADE (data not shown). Assuming that initiation occurred between 0 and 5 minutes after NPE addition and that elongation continued at a constant rate until 25 minutes, the non-imaged replication bubbles grew at a rate of 490-610 bp min -1 (blue horizontal zone). These values are consistent with the mean growth rate of 520 ± 220 bp min -1 rate measured from bubbles imaged in real time ( Fig. 2d) suggesting that the imaging conditions used do not alter fork progression. Because damage from activation and imaging may accumulate over time, average rates derived from the entire imaging period may not reveal fork slowing towards the end of the experiment. Therefore, we analyzed the mean growth rate of PhADE-imaged replication bubbles as a function of time during 4-minute windows between 6 and 20 minutes (black squares). Only marginal slowing occurred after 12 minutes of imaging (after 72 frames or the 17 minute time point), indicating that photo-induced fork stalling was not significant when data collection was limited to fewer than 72 frames.  Figure 3a, to those determined from staining of Dig-dUTP incorporation. At equivalent times after NPE addition, we quantified the distances between the centers of neighboring replication bubbles (center-to-center, C-to-C distances) detected via D179A Fen1 KikR PhADE (orange circles) and α-Dig staining (black squares, buffer; blue triangles, 2 µM D179A Fen1 KikGR ). The profiles from the PhADE movies essentially reproduce the α-Dig profiles, except for a ~2-minute delay. The delay is consistent with a bubble-growth rate of 520 ± 220 bp min -1 (Fig. 2d) and our observation that D179A Fen1 KikR only detects bubbles longer than 1 kbp (Supplementary Fig.  5b and data not shown). The similarity of the α-Dig and D179A Fen1 KikR profiles suggests that the latter eventually detects all bubbles and is a reliable readout for the dynamics of replication initiation and elongation. We also note that C-to-C distances as measured by α-Dig imaging are the same in the presence and absence of D179A Fen1 KikGR (compare black squares and blue triangles), further confirming that this protein has no adverse effects on replication dynamics.   Fig. 2a). Re-exposure to 405-nm light was found to keep previously activated molecules in the fluorescent state (Supplementary Fig. 7b) and allowed the same molecule to be imaged for more consecutive frames. Prolonging the fluorescent state in this manner is important because we define the "on-time" as the number of consecutive frames that a specific location on DNA remains fluorescent, and we compare this value to the photodarkening of a Biotin-KikR standard imaged in the same way. Of note, re-exposure of the sample to 405nm light also activated new molecules that bound to DNA during the waiting time. If these new molecules were to bind in the same location as a molecule that had dissociated, the on-time measurement might reflect multiple Fen1 KikR binding events. However, given the low fractional occupancy of the replication bubbles by Fen1 KikR in each frame of these experiments (<25% in Fig. 4d), the likelihood of a new binding event at the same location is low (25% x 25% = 6.3%). , maximally 20% of the DNA can be chromatinized. Therefore, a possible interpretation of the small inter-origin distance detected during PhADE imaging is that it results from incomplete chromatinization of the DNA template. To test this idea, we compared the static replication profiles of singly-tethered (unconstrained, presumably fully chromatinized) and doubly-tethered (constrained, partially chromatinized) λ DNA molecules. (a) Licensed, doublytethered (black, same as in Supplementary Fig. 5a) or singly-tethered (orange) λ DNA was exposed to NPE containing dig-dUTP for 2 to 13 minutes. At the indicated times, reactions were stopped and replication intermediates were detected with α-Dig and the center-to-center distance between neighboring bubbles was measured (mean ± error estimate) and graphed. There was little difference in minimum C-to-C distances between singly-tethered and doubly-tethered molecules. Interestingly, the minimum C-to-C distance was delayed by 5 minutes on the unconstrained molecules, implying that chromatin structure affects replication timing, possibly by delaying access of one or more initiation factors to the DNA. (b) The replication bubble lengths were measured as in Supplementary Fig. 8a and graphed (black, same data as in Fig.  2e). The data show that there was little difference in the length of α-Dig tracts. These observations suggest that chromatin structure does not affect fork rate or spacing of initiations in this system. As seen for the C-to-C distances, there was a 5-minute delay in bubble growth on singly-tethered versus doubly-tethered DNA. Our results therefore suggest that chromatin structure affects temporal but not spatial parameters of origin firing.  Figure 2e and Supplementary Figure 5a, > 50 λ DNA molecules (> 2.43 Mbp of DNA) were analyzed for the average % replication, number of bubbles per DNA, size of the bubbles (µm), and the center-tocenter distances between adjacent bubbles (µm). Table 2: Replication profiles of singly-tethered λ DNA.

Supplementary Table 2:
For each time point of the experiment presented in Supplementary  Figure 8, > 50 λ DNA molecules (> 2.43 Mbp of DNA) were analyzed for the average % replication, number of bubbles per DNA, size of the bubbles (µm), and the center-to-center distances between adjacent bubbles (µm).

Cloning and site-direct mutagenesis
Fen1-6his for antibody production was cloned as follows. Xenopus laevis Fen1a (xFen1) was amplified from pET28 2xFLAG-Fen1-GST-His 5 using primers C (5′-CTA TCC ATG GGA ATT CAC GGT TTG GCC AAA C-3′) and E (5′-TTT TCT CGA GTT AAT GAT GGT GAT GTT GAT GTT TAC CCC TCT TGA ACT TTC CTG C-3′) (IDT DNA) with flanking restriction sites and a C-terminal 6xhis tag, and the double digest was cloned into pET28b between the NcoI and XhoI sites. The ORF of the resulting plasmid pET28b-xFen1-6xhis was verified by sequencing (GeneWiz).

Egg Extract and Immunological Methods
High-speed supernatant (HSS) extract, nucleoplasmic extract (NPE), low-speed supernatant (LSS) and sperm chromatin were prepared from unfertilized Xenopus laevis eggs as described previously 6,7 . Chromatin spin-downs and bulk replication assays were performed as previously described 7,8 . For Western blotting, we used previously described antibodies against XlRCC1 9 , Xl PCNA 10 , Hs PCNA (Santa Cruz sc-056), HsFen1 (Cell Signaling Technology) 5 and 6His Tag (AbDSerotec MCA1396). For xFen1 antibody production, the xFen1-6his fusion protein, was expressed from plasmid pET28b-xFen1-6xhis and purified under the native conditions described in the Online Methods for Fen1-KikGR. xFen1-6his was used to generate polyclonal antibodies in rabbits (Pocono Rabbit Farm and Laboratory; Canadensis, PA). α-xFen1 serum but not the pre-immune serum recognized a band 45 kDa in size in LSS (data not shown).

Flap endonuclease assay
The flap substrate was prepared using oligonucleotides (IDT DNA) as described in 11 . 2.5 pmol of the "Flap strand" was end-labeled by T4 PNK (NEB) with 32 P-γ-dATP (Perkin-Elmer). Free, labeled nucleotides were removed by gel filtration (illustra ProbeQuant TM G-50 Micro Columns, GE Healthcare). 5 pmol of "F br strand" and "F adj strand" were added and annealed in 50 µL of STE (10mM Tris 7.5, 50mM NaCl and 0.1mM EDTA) by heating to 95ºC and slow cooling to room temperature. For the cleavage assay 12 , 2 fmol of flap substrate was incubated with 0.2 pmol of Fen1 WT or mutant protein in a total volume of 20 µl of Flap Binding Buffer (50 mM Tris 8, 30 mM NaCl, 5% Glycerol, 2mM DTT, 0.2 mg ml -1 BSA and 1mM EDTA) for 10 minutes at room temperature. Next, the reaction was supplemented with 1/10 volume of 50 mM MgCl 2 and incubated a further 10 minutes at room temperature. The reactions were stopped with Gel Loading Buffer II (Ambion), heated to 80ºC for 5 minutes, and separated on 12% Urea-PAGE mini-gels at 20W for ~20 minutes. The fate of the 32 P-labeled flap strand was determined by phosphor imager analysis.

xFen1 depletion and nascent strand assay
To deplete Fen1 from LSS, 1 volume of rProtein-A Sepharose Fast Flow resin (GE Healthcare) was incubated with 3 volumes α-xFen1 or matched pre-immune serum (mock control) for 30 minutes at 4ºC. The beads were washed 4 times with 500 µl PBS and 2 times with ELB (10 mM Hepes-KOH 7.7, 50 mM KCl, 2.5 mM MgCl 2 , 250 mM Sucrose). Next, one volume of LSS supplemented with 3.3 ng µl -1 nocodazole was incubated with 0.2 volumes of antibody-bound beads for 30 minutes at 4ºC. Beads were spun down, LSS in the supernatant was collected, and the depletion procedure was repeated once. After the final round, LSS was isolated from beads by spinning through Nitex membrane and was immediately used for DNA replication.
Mock-or Fen1-depleted LSS was supplemented with an ATP regeneration system (2 mM ATP, 20 mM phosphocreatine, 5 µg ml -1 creatine kinase), Fen1 KikGR or Fen1 storage buffer, 32 Pα-dATP, and 10,000 sperm µl -1 , and reactions were moved to 22ºC to start replication. At the appropriate time, samples were withdrawn and processed differentially either for native agarose gels to determine replication efficiency 7 or denaturing agarose gels to visualized the size distribution of nascent strands 13 . For the latter, 2 µl aliquots of the reaction were transferred into 200 µl of ice-cold Buffer A (20 mM Hepes-KOH, pH 7.6, 50 mM NaCl, 5mM EDTA) supplemented with 1 mM spermidine and 1 mM spermine. After all time points were collected, the reactions were centrifuged 5 minutes at 4ºC. Pellets were resuspended in Buffer A supplemented with 0.5% SDS and 0.1 mg ml -1 RNase A to digest single-stranded RNA and incubated for 30 minutes at 37ºC. 0.5 mg ml -1 proteinase K was added to digest proteins for one hour at 37ºC. Finally, DNA was extracted with phenol:chloroform and ethanol precipitated in the presence of 10 µg of glycogen. DNA Pellets were resuspended in Alkaline Agarose Loading Buffer (100 mM NaOH, 1mM EDTA, 5% Ficoll 400, and 0.025% Bromocresol Green) and run immediately on a 2% alkaline agarose gel alongside a 32 P-labeled 100 bp ladder (NEB), 2 V cm-1 for 18 hours. Gels were dried between DE81 paper (Whatman) and exposed to a phosphor screen. A one hour exposure was used to quantify the abundant large nascent strands running greater than 1,000 bp. A 36 hour or longer exposure was needed to quantify the short nascent strands running < 300 bp in size.

α-Dig staining and measuring of replication bubbles on doubly and singly-tethered DNA
To image replication intermediates on singly-verus doubly-tethered λ DNA, slightly different procedures were used. For singly-tethered DNA, extracts and proteins compacting the DNA were removed by flow of 1 x PBS with 0.1% SDS, initially at 10 µl min -1 for 5 minutes then 50 µl min -1 for 5 minutes to stretch the DNA. SDS was then removed with a 1 minute wash with 1 x PBS. For studying replication on doubly-tethered λ DNA, extracts but not all proteins bound to DNA were removed by flushing the flow cell with ELB++ for at total of 10 minutes at 10 µl min -1 . Under these conditions, singly-tethered DNA remained compacted while doubly-tethered DNA remained extended. Next, dig-dUTP incorporation on singly-tethered or doubly-tethered λ DNA was stained with anti-digoxigenin-fluorescein Fab fragments (α-Dig) (Roche) and co-stained with SYTOX Orange (Invitrogen). Doubly-tethered DNA was imaged in the absence of flow while singly-tethered DNA was imaged at a flow rate of 100 µl min -1 of the SYTOX Orange solution to stretch the DNA. α-Dig was excited with 488-nm light (2.3 W cm -2 , 100 ms) and SYTOX Orange was excited with 568-nm light (3.5 W cm -2 , 100 ms).

Immunostaining of PCNA at individual replication forks
Following λ DNA replication in the flow cell, extracts were removed by washing with Wash Buffer 2 (10 mM Hepes 7.7, 300 mM KCl, 2.5 mM MgCl 2 with 0.1mg ml -1 BSA) for 5 minutes at 20 µl min -1 . The flow-cell surface was further washed and blocked by flow of ELB++ for 25 minutes, 20 µl min -1 . Next, Dig-dUTP incorporation was stained as described above. After washing with ELB++ for 5 minutes, PCNA was stained as follows. First 1.2 ng µl -1 α-XlPCNA IgG in ELB++ (purified from serum on rProtein-A Sepharose Fast Flow resin (GE Healthcare)) was introduced into the flow cell at 10 µl min -1 for 15 minutes. After washing with ELB++ for 5 minutes, the α-xPCNA rabbit IgG was detected with 2ng µl -1 Goat-anti-Rabbit IgG-AlexaFluor647 (Invitrogen) in ELB++, introduced into the flow cell at 10 µl min -1 for 5 minutes. Free secondary antibody was removed by flushing the flow cell with ELB++ at 10 µl min -1 for 5 minutes. Lastly, DNA was stained with SYTOX Orange as described above. α-Dig and SYTOX were detected as before. The α-PCNA signal was excited with 647-nm light (0.16 W cm -2 , 100 ms exposure).