Surface Transient Binding-Based Fluorescence Correlation

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B: Biophysical Chemistry and Biomolecules

Surface Transient Binding Based Fluorescence Correlation Spectroscopy (STB-FCS), a Simple and Easy-to-Implement Method to Extend the Upper Limit of Time Window to Seconds Sijia Peng, Wenjuan Wang, and Chunlai Chen J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03476 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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The Journal of Physical Chemistry

Surface Transient Binding Based Fluorescence Correlation Spectroscopy (STB-FCS), a Simple and Easy-to-Implement Method to Extend the Upper Limit of Time Window to Seconds Sijia Peng a, Wenjuan Wang b, Chunlai Chen a* a

, School of Life Sciences; Tsinghua-Peking Joint Center for Life Sciences; and Beijing

Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China. b

, School of Life Sciences and Technology Center for Protein Sciences, Tsinghua University,

Beijing, China.

AUTHOR INFORMATION Corresponding Author *[email protected]

ACS Paragon Plus Environment

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ABSTRACT. Fluorescence correlation spectroscopy is a powerful single-molecule tool, which is able to capture kinetic processes occurring at the nanosecond time scale. However, the upper limit of its time window is restricted by the dwell time of molecule of interest in the confocal detection volume, which is usually around sub-millisecond for freely-diffusing biomolecule. Here, we presented a simple and easy-to-implement method, named surface transient binding based fluorescence correlation spectroscopy (STB-FCS), which extends the upper limit of time window to seconds. We further demonstrated that STB-FCS enables to capture both intramolecular and inter-molecular kinetic processes whose time scales cross several orders of magnitude.

Introduction In the last two decades, fluorescence based single-molecule techniques have been emerging and become widely used tools to provide new insights into dynamics of biological processes and their molecular mechanisms, which are hidden in traditional ensemble measurements 1-7. To capture conformational dynamics and molecular interactions of biological processes, which occur in a broad range of time scale, techniques with suitable time resolution and detection window are needed. Imaging based single-molecule fluorescence techniques equipped with electron multiplying charge-coupled device (EMCCD) as detectors are usually used to examine dynamic processes at tens of milliseconds or slower 7-9. Using scientific complementary metal-oxide semiconductor (sCMOS) instead of EMCCD as detectors can further improve time resolution to milliseconds 10. On the other hand, fluorescence correlation spectroscopy (FCS) equipped with avalanche photon diodes (APDs) as detectors is able to


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capture kinetic processes occurring at the nanosecond time scale 11-13. However, the longest observation time of molecule of interest within the FCS detection volume, which defines the upper limit of its time window, is usually around sub-millisecond for freely-diffusing biomolecule. No doubt, new techniques to bridge the gap of detection time window between imaging based methods and FCS and to simultaneously capture two or more kinetic processes occurring at different time scales are needed. For instance, recent studies suggest that time resolution of imaging based single-molecule fluorescence measurements is insufficient to fully capture conformational dynamics of tRNAs in the PRE-translocation ribosomal complex during ongoing protein synthesis 14 and dynamics of transmembrane helix 6 in β2 adrenergic receptor in the presence of agonist 15. Techniques with broader detection window will advance research in these fields. Several FCS based techniques with broadened time window have been developed by extending dwell time of molecules in the detection volume. They include examining molecules in polyacrylamide gel matrix 16, attaching target molecules onto big objects 17-19, using dual beam fluorescence cross-correlation spectroscopy 20, and immobilizing molecules on surface 12, 21-23. In addition, molecule of interest can be trapped in solution by the Anti-Brownian trap 24 and vesicle tethering 25 to extend their observation time. However, each technique has its own advantages and disadvantages. For example, FCS with polyacrylamide gel matrix is easy to implement, but it only extends the upper limit of time window for one order of magnitude. FCS with surface immobilization (scanning FCS) provides the longest observation, but it requires a pricy and ultrastable piezo stage and its control software 12. Here, we developed a simple and easy-to-use technique, named surface transient binding based FCS (STB-FCS), to enrich the FCS toolbox. Using a conventional fluorescence confocal microscope equipped with APDs, STB-FCS extends


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the upper limit of time window to seconds. Furthermore, for both intra-molecular and intermolecular reactions, we demonstrated that STB-FCS enables to simultaneously capture two kinetic processes whose time scales are at least three orders of magnitude apart. Methods Materials ATTO655 (NHS ester, 1 mg) was purchased from ATTO-TEC. Alexa Fluor® 488 (NHS ester, 1mg), denoted as Alexa488, was purchased from Invitrogen. Sulfo-Cyanine5 (NHS ester, 1mg), denoted as Cy5, was purchased from Lumiprobe. UltraPure™ 1M Tris-HCl pH 7.5, 5M NaCl and Nuclease-free Water were obtained from Invitrogen. Unless stated otherwise, common materials and reagents were purchased from Sigma. DNA preparation All single-stranded DNAs (ssDNAs, Table S1) were synthesized by Sangon Biotech (Shanghai, China), which were purified by HPLC. Their purity was verified by mass spectrometry. The ATTO655, Alexa488, and Cy5 labeled single-stranded DNAs were prepared via covalently conjugating N-hydroxysuccinimido (NHS) group of fluorescent dyes to an amino group on DNA following procedures from manufacturers. In brief, each fluorophore and ssDNA were mixed at 20:1 molar ratio and kept at room temperature (~23°C) overnight. Labeled DNAs were purified through three times of ethanol precipitation. All synthesized DNAs and labeled DNAs were dissolved by buffer of 50 mM Tris-HCl (pH=7.5).


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Preparation of PEG-passivated slides PEG-passivated slides were prepared according to previous procedure with minor modifications.26 In brief, slides and coverslips were sonicated at 40 ºC in the order of acetone (10 minutes), 0.2 M KOH (20 minutes), and ethanol (10 minutes). Cleaned slides and coverslips were treated with amino-silane reagents (1ml 3-Aminopropyltriethoxysilane, 5 ml acetic acid, and 94 ml methanol) at room temperature overnight and then incubated with polyethylene glycol (PEG, Laysan Bio, Inc., containing 20% w/w mPEG-Succinimidyl Valerate, MW 2,000 and 1% w/w Biotin-PEG-SC, MW 2,000) in 0.1 M sodium bicarbonate (pH 8.3) for 3 h. Slides and coverslips were dried by clean N2, put in 50 mL falcon tubes, vacuum sealed in food saver bags, and stored at -20 ºC. Experimental instrument and conditions for STB-FCS measurements PEG-passivated slides were incubated with 0.05 mg/ml streptavidin for 3 minutes. Then biotinylated DNAs were specifically attached to PEG-passivated slides via interactions between biotin and streptavidin. Different surface immobilization densities were achieved by incubated 100 pM - 100 nM of biotinylated DNAs with streptavidin treated PEG slides for 3 minutes. Unless stated otherwise, STB-FCS experiments were performed with 10 nM of labeled molecule of interest at 25 ºC in buffers containing 50 mM Tris-HCl pH 7.5 and 500 mM NaCl. Except measurements with ATTO655, an oxygen scavenging system was included in the buffer, which containing 3 mg/mL glucose, 100 µg/mL glucose oxidase (Sigma-Aldrich), 40 µg/mL catalase (Roche), 1 mM cyclooctatetraene (COT, Sigma-Aldrich), 1 mM 4-nitrobenzylalcohol (NBA, Sigma-Aldrich), and 1.5 mM 6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid (Trolox, Sigma-Aldrich – added from a concentrated DMSO stock solution).


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STB-FCS and conventional FCS measurements were performed on a home-built confocal microscope, based on a Zeiss AXIO Observer D1 fluorescence microscope with an oilimmersion objective (Zeiss, 100×, NA = 1.4), and solid state 488 nm, 532 nm, and 640 nm excitation lasers (Coherent Inc. OBIS Smart Lasers). Laser powers at the samples were ~5 µW. Fluorescence signals from the sample passed through a pinhole (diameter 50 µm) and were separated by a 50/50 beam-splitter (Thorlabs), which were further filtered by bandpass filters ET585/65m (for TMR, Chroma) and ET700/75m (for Cy5 and ATTO655, Chroma) before detected by two APDs (Excelitas, SPCM-AQRH-14). The correlation of fluorescence signals was calculated by a correlator (Correlator.com, Flex02-01D). For each experimental conditions, three or more identical replicates were performed. Each replicate was collected for five minutes. Using free Rhodamine 6G as the standard material, we calibrated that, in conventional FCS measurements, the lateral (ωxy) and the axial (ωz) radius of our confocal microscopy were around 350 nm and 1700 nm, respectively. We roughly estimated that = µm3 and = πxy = 0.38 µm2 during STB-FCS measurements.

xy

= 0.59

Measurements of surface density of biotinylated DNA Surface densities of DNAs were measured by mixing biotinylated DNAs with 0.1-1% Cy5 labeled biotinylated DNAs and counting numbers of Cy5 fluorescence spots per field with a total internal reflection fluorescence (TIRF) microscope. Measurements of association and dissociation rate constants All experiments were performed at 25℃ on a home-built objective-type TIRF microscope, whose configuration was described elsewhere in details 9. Biotinylated ssDNA (Bio-


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IMA) was attached to PEG-passivated slides, decorated with biotin-PEG and streptavidin. Then 20 nM of TMR labeled ssDNA (TMR-TBA1) was injected into flow chambers with 50 mM TrisHCl pH 7.5, 0 - 500 mM NaCl, an oxygen scavenging system, whose composition was described above. To extract association and dissociation rate constants between two ssDNA strands from TIRF-based single-molecule fluorescence recordings, we measured the waiting time before TMR-TBA1 binding on surface immobilized Bio-IMA (τoff) and the dwell time of TMR-TBA1 remaining bound on immobilized Bio-IMA (τon) (Figure S1). Then, we calculated association rate kass=1/([TMR-TBA1]·τoff), dissociation rate kdis=1/τon, and dissociation constant Kd = kdis/kass, respectively. Fluorescence lifetime Time-correlated single photon counting (TCSPC) data sets were acquired on an inverted OLYMPUS FV 1200 microscope equipped with a Picoquant picoHarp 300 controller. Samples were excited with picosecond 561 nm laser pulsed at 40 MHz. Fluorescence emission passed through a 615/60 filter and was detected by a MPD SPAD detector (PicoQuant, Germany). Fluorescence lifetime was analyzed using SymPhoTime64 software. Results Proof of principle The principle of STB-FCS is shown in Figure 1A. The laser beam used in STB-FCS measurements is focused on the glass surface, which is passivated by polyethylene glycol and coated with probe molecules through biotin-streptavidin interaction according to previous


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procedure 9, 26. Fluorophore labeled molecules of interest are present in the solution and can transiently bind with surface-immobilized probe molecules. Under such experimental design, assuming that (1) characteristic relaxation time of surface transient binding between molecules of interest and probe molecules ( ) is significantly longer than the characteristic diffusion time of molecules of interest near surface ( ), (2) fluorescence brightness remains the same upon binding, and (3) no other kinetic process contributes, the fluorescence autocorrelation function is given by =

D

D STB

+

STB

D STB

∙

!"#

$D = ∙ %&'( $STB = $)*)+ ∙

Eq.1

Eq.2 ,-./ 01

= $*)+ ∙ ,

,-./

-./ 01

= 2*)+ ∙ ,

,-./

-./ 01

Eq.3

where $D , $STB , $)*)+ , and $*)+ denote the average numbers of freely-diffusing molecules, surface transiently-bound molecules, unbound probe molecules, and total probe molecules, respectively, in the focal volume, and are effective illuminated volume in solution and

effective illuminated area on surface, respectively, 34 is dissociation constant between molecules

of interest and probe molecules, and 2*)+ is surface density of probe molecules. A twodimensional diffusion term 1 +

6

6

was used here, which is a good approximation to a three-

dimensional diffusion system with the common confocal illumination conditions 27. Clearly, both freely-diffusing and transiently-bound molecules contribute to , which correspond to the first and second terms in Eq. 1, respectively. When the concentration of molecules of interest (%&'( ) is close to or higher than 34 , increasing %&'( would decrease the relative ratio of two


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amplitudes in Eq. 1, defined by $STB /$D . On the other hand, when %&'( ≪ 34 , Eq. 3 can be simplified as $STB = $*)+ ∙

,-./ 01

= 2*)+ ∙

,-./ 01

Eq.4

Then, value of $STB /$D highly depends on surface density (2*)+ ) and dissociation constant (34 ) and is not affected by the value of %&'( .

Inspired by DNA-PAINT 28-29, a super-resolution imaging technique based on transient interactions between DNA strands, we picked DNA oligonucleotides to serve as probe molecules for STB-FCS measurements (Table S1). Labeled single-stranded DNAs (ssDNAs) can transiently bind with immobilized probe DNAs for several seconds by forming 9 or 10 base pairs between them 28. Dissociation constants between target and probe DNAs can be fine-tuned by adjusting number and GC content of base pairs. If needed, DNAs with different sequences can serve as orthogonal probes with minimal cross-talk between each other. To validate its concept, we performed STB-FCS measurements while modulating surface density (2*)+ ) and dissociation constant (34 ) (Figures 1B-E). When the laser beam was focused in solution or when there was no immobilized probe molecule during STD-FCS measurements, only tetramethyl-rhodamine (TMR) labeled freely-diffusing DNA (TMR-TBA1, Table S1) contributed. Normalized FCS curves of freely-diffusing DNA in solution and close to surface were the same within experimental error, from which we extracted that diffusion times ( ) of TMR-TBA1 in solution and near surface were both 190 ± 10 µs. In the presence of 500 mM NaCl, when density of immobilized DNA (Bio-IMA, Table S1) increased, a population contributed by transiently-bound molecules on surface started to emerge and became the


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majority at 2*)+ ≥270 µm-2 (Figure 1B). Increasing concentration of TMR-TBA1 from 1 nM to 50 nM caused a minor decrease in the population contributed by transient binding (Figure S2A), which agrees with our prediction based on Eqs. 1-3 as 34 between TMR-TBA1 and Bio-IMA is 66 ± 2 nM (Table S2). Under our illumination condition, the photon emission rate was 9.3 ± 0.9 kHz per molecule during STB-FCS measurements with the highest density of immobilized DNA, which was similar as the emission rate of 10.0 ± 0.2 kHz per molecule while performing FCS measurements in solution. In addition, the fluorescence lifetime of transiently-bound TMRTBA1 on surface (3.84 ± 0.02 ns) was almost the same as the fluorescence lifetime of freelydiffusing TMR-TBA1 in solution (3.73 ± 0.02 ns). These results prove that the brightness of TMR labeled DNA remains almost the same upon binding. The characteristic relaxation time of surface transient binding between molecules of interest and probe molecules ( ) is given by = :

1;< :=4?@ and >A@@ measured by a single-molecule

total internal reflection fluorescence (TIRF) microscope (Table S2). At optimal concentrations (~ 10 nM) to perform STB-FCS measurements, >4?@ is larger than >A@@ ∙ %&'( . Therefore, varying


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concentration of TMR-TBA1 from 1 nM to 50 nM only causes minor changes on , whereas D remains the same (Figure S2B).

Next, we decreased the binding affinity between TMR-TBA1 and Bio-IMA by decreasing concentration of NaCl from 500 mM to 0 mM, which increased their dissociation constants for ~30 folds (Table S2). Consistent with our prediction, weaker binding affinity led to less contribution from surface transiently-bound molecules and a smaller (Figures 1D-E). We did notice that diffusion time ( ) extracted from STB-FCS curves decreased from ~200 µs to ~60µs, when 2*)+ increased from 7.1 µm-2 to 680 µm-2 or when concentration of NaCl

increased from 0 mM to 500 mM (Figures 1C and 1E). However, of TMR-TBA1 near surface

was not affected by immobilized non-complementary ssDNA at high density (Bio-IMB1, Table S1, Figure S3). Therefore, we speculated that decreasing of might be caused by contributions of a fast dynamic process of surface transiently-bound molecules or due to fitting errors when contributions of freely-diffusing molecules decrease. Using values of 34 and 2*)+ measured by a single-molecule total internal reflection fluorescence (TIRF) microscope (details in SI), we estimated the contributions of surface transiently-bound molecules, defined as $STB /$STB +

$D , through Eqs. 2 and 3. In general, our estimation agreed with measured values (Figure 1F).

Unevenness of surface-immobilized probe and inaccuracy of estimating and will cause deviation between measured and predicted values. Therefore, it is better to perform STB-FCS measurements under low 34 and high 2*)+ , so that $STB is significantly larger than $D and $STB /$STB + $D is less sensitive to variations of other factors.

Lastly, we tested two other transient binding DNA pairs, TMR-TBB/Bio-IMB1 (9 base pairs, Table S1) and TMR-TBB/Bio-IMB2 (10 base pairs), which gave of 2.0 ± 0.2 s and 7


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± 1 s, respectively (Figure 1G). In summary, STB-FCS is a simple and flexible design, which extends the upper limit of time window to seconds. Intra-molecular dynamics captured by STB-FCS Next, we demonstrated the ability of STB-FCS to capture intra-molecular processes. In our model system (Figure 2A), ssDNA Bio-IMA served as immobilized probe to transiently interact with ATTO655 labeled ssDNA TBA2 (ATTO655-TBA2, Table S1). ATTO655 has been known to blink between fluorescent and non-fluorescent states, which can be further accelerated by the presence of reductant and oxidant, such as ascorbic acid (AA) and methylviologen (MV) 30

. STB-FCS measurements were performed by measuring fluorescence signals of ATTO655 at

different concentrations of AA and MV (Figure 2B). High probe density (680 µm-2) and high NaCl concentration (500 mM) were used to ensure that contributions of surface transientlybound molecules dominated STB-FCS curves. We extracted kinetic information from STB-FCS curves via the following function = D

B

C

D1 + blk ∙

H C EFG

B

I + STB ∙

!"#

D1 + blk ∙

H C EFG

B

I

Eq.6

where D , blk , and STB are amplitudes of diffusion, blinking, and surface binding reactions,

respectively, and J is a stretch exponent 31-32, because a stretched-exponential form was needed

to well fit the FCS curves. Within experimental errors, D = 0, which indicated that contribution of diffusion is negligible here. The mean relaxation time of ATTO655 blinking, 〈blk 〉 (Figure 2C), is calculated via 〈blk 〉 = blk /JΓJ

Eq.7


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where ΓJ

is the gamma function 32. As expected, when concentrations of AA and MV

increased from 10 µM to 3.3 mM, remained almost constant, whereas 〈blk 〉 decreased ~200

folds. Our observations agreed well with previous report that high concentrations of AA and MV lead to fast blinking rates of ATTO655 30. In addition, 〈blk 〉 of the fastest blinking process we captured was 1.1 ± 0.1 ms, which was still several folds slower than diffusion process of ssDNA. Together, our results demonstrated the feasibilities to apply STB-FCS to examine intramolecular processes, which were beyond the detection time window of conventional FCS measurements. Interestingly, when ATTO655 was labeled on DNA strand TBB (Table S1), the stretch exponent J was not needed to describe its blinking process (Figures 2D and 2E). Such phenomena suggested that blinking of ATTO655 is likely to be influenced by its nearby nucleotides. Thymine might have different effects on blinking of ATTO655 from adenine and guanine, which leads ATTO655-TBB to display a smaller blk and a more homogeneous blinking kinetics than ATTO655-TBA2. At high concentration of AA and MV, blinking of ATTO655 is mainly dominated by its interactions with AA and MV, which leads ATTO655-TBB and ATTO655-TBA2 to present similar values of blk . Although values of ATTO655-TBB/Bio-

IMB1 and ATTO655-TBB/Bio-IMB2 were different by 3-4 folds, their blk values agreed with each other very well (Figure 2E), which supported that the blinking and binding kinetics were being measured independently and accurately. Inter-molecular dynamics captured by STB-FCS Lastly, we combined STB-FCS with fluorescence resonance energy transfer (FRET) to

capture inter-molecular interactions. Three ssDNAs were used in our system (Figure 3A). DNA


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strand Bio-IMA still served as immobilized probe to transiently capture Cy5 labeled strand TBA2 (Cy5-TBA2, Table S1). In addition, an Alexa488 labeled strand (CM7A2, CM8A2, or CM9A2, Table S1) was present, which can form 7, 8 or 9 base pairs with Cy5-TBA2. The autocorrelation curves of Cy5 signals were collected under 488 nm laser illumination (Figure 3B). Under such experimental conditions, Cy5 had to be in FRET pair with Alexa488 to produce fluorescence signals (Figure S4), so that only Cy5-TBA2 strand in Cy5/Alexa488 duplex form with Alexa488 labeled strand contributed to FCS curves. Clearly, there were two components in FCS curves (Figure 3B). The slow component should correspond to the reaction of Cy5/Alexa488 duplex formation, whereas the fast component was likely to be caused by DNA breathing at the end of duplex 33. The function to describe STB-FCS shown in Figure 3B is = D D

B

C

+ STB ∙

!"#

I B1 − Pbre + Pbre ∙

EQR

C S1 − Pdup + Pdup ∙

1TU

V

Eq.8 where bre and dup are relaxation times of DNA breathing and duplex formation reactions,

respectively, Pbre is average fraction of DNA duplex in its open form during DNA breathing, and Pdup is average fraction of Cy5 labeled DNA in its single-stranded form in the equilibrium of

duplex formation. As shown in the example of ATTO655 blinking, contribution of diffusion was negligible under our experimental condition. In addition, Pdup is close to 1 and bre < dup < STB . Therefore, a good approximation of Eq. 8 is = bre ∙

EQR

+ dup ∙

1TU

Eq.9


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where bre and dup are amplitudes of DNA breathing and duplex formation reactions, respectively. Eq. 9 was used to extract kinetic information from STB-FCS curves shown in Figure 3B. We found that dup increased from 0.10 ± 0.04 s to 1.7 ± 0.3 s, when number of base pairs increased from 7 to 9 and the Cy5/Alexa488 duplex became more stable (Figure 3C). On the other hand, within our experimental error, bre remained almost constant around 0.5 ± 0.2 ms (Figure 3C), which was consistent with reported DNA breathing lifetime at end of duplex measured by Cy3/Cy5 FRET pair (~0.3 ms) 33. Using a single-molecule TIRF microscope, we measured dup values were 0.083 ± 0.001 s, 0.34 ± 0.01 s, and 1.4 ± 0.1 s (Figure 3C) when the numbers of base pairs formed between the Alexa488 labeled strand and surface-immobilized Cy5 labeled TBA2-Bio were 7, 8, and 9, respectively. Our results derived from STB-FCS curves agreed well with results from TIRF measurements. In all, combining with FRET, STB-FCS enabled to simultaneously capture two kinetic processes, whose time scales were three orders of magnitude apart. Discussion Combining photon-counting detectors and permanent immobilization of molecule of interest on the surface, several techniques, including FCS on individual immobilized molecule 30, 33

and single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy 21,

have been developed to capture dynamics of individual molecules with high time resolution and long observation time. However, these techniques usually require pricy and sophisticated instruments to locate individual molecules and to capture their single-molecule florescence signals. With these low-throughput detection methods, it is labor intensive to get statistical meaningful results and it is difficult to resolve minor populations within heterogeneous samples.


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Moreover, in practice, these techniques are incapable to capture transient inter-molecular events which happen at low frequency, such as transient duplex formation shown in Figure 3. Because each immobilized molecule can only be detected for several seconds before photobleaching, it is almost impossible to obtain enough transient single-molecule events within reasonable data collecting time. Instead of examining immobilized molecules one at a time, scanning FCS developed by the Zhao group 12 scans the detection volume on surface containing immobilized molecule of interest. Scanning FCS provides a high-throughput detection approach. However, it requires to scan at rate ~100 nm/s or slower to extend detection window to seconds. A specialized equipment with a piezo stage is required to achieve such slow scanning rate, because most commercial scanning confocal fluorescence microscopes can only reach ~10 µm/s. Through transient binding between target molecules and surface immobilized probe molecules to increase dwell times of target molecules in the focal volume, STB-FCS extends the upper limit of time window to seconds, which is similar as the upper limit of scanning FCS. The major advantage of STB-FCS is that it requires no specialized instrument and is fully compatible with all conventional FCS apparatuses. Therefore, STB-FCS is extremely helpful when a confocal microscope equipped with a piezo stage is not available. In theory, based on the principle of STB-FCS as shown in Figure 1A and described by Eqs. 1-3, contributions of freelydiffusing molecules cannot be fully eliminated and can only be minimized. Therefore, caution should be exercised when designing STB-FCS measurements and interpreting their results. However, in practice, contributions of diffusion are negligible when experiments are designed to perform at low 34 (between target and probe molecules) and high 2*)+ . Such experimental conditions can be easily achieved as demonstrated in examples shown in Figures 2 and 3.


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Conclusion Here, we presented STB-FCS as a simple, flexible, powerful, and easy-to-implement tool to capture both intra-molecular and inter-molecular kinetic processes. Based on a conventional fluorescence confocal microscope equipped with APDs, it extends the upper limit of time window to seconds while maintaining the high time resolution of conventional FCS measurements. In all, STB-FCS is an easy and helpful complement to previous FCS techniques, which further enriches the FCS toolbox. No doubt, STB-FCS will have broad applications in revealing kinetics of biological processes including protein-nucleic acid and protein-protein interactions and elucidating their molecular mechanisms.


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Figure 1. Proof-of-concept experiments of STB-FCS. (A) A cartoon to illustrate the principle of STB-FCS. (B) Comparison of conventional FCS curve with STB-FCS curves under different probe densities (target: TMR-TBA1 and probe: Bio-IMA). (C) D and STB extracted from STBFCS curves shown in B via Eq. 1. (D) STB-FCS curves of TMR-TBA1/Bio-IMA under different NaCl concentrations. (E) D and STB extracted from STB-FCS curves shown in D. (F)

Measured and predicted values of $STB /$STB + $D . Measured values were extracted from

STB-FCS curves in B and D. Predicted values were estimated using Eqs. 2 and 3 with =


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0.59 µm3 and =0.38 µm2. 34 and 2*)+ were measured using a TIRF microscope. (G) STBFCS curves of other target/probe DNA pairs.


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Figure 2. Application of STB-FCS to capture intra-molecular processes. (A) Experimental design to examine blinking of ATTO655 fluorescence in the presence of AA and MV. ATTO655 labeled DNA strand in fluorescent and non-fluorescent states are shown in strands with red dot and grey dot, respectively. (B) Normalized STB-FCS curves capturing blinking of ATTO655 under different AA and MV concentrations using ATTO655-TBA2/Bio-IMA pair. (C) 〈blk 〉 and STB extracted from STB-FCS curves shown in B via Eqs. 6 and 7. (D) Normalized STB-FCS

curves capturing blinking of ATTO655 under different AA and MV concentrations using ATTO655-TBB/Bio-IMB1 pair. (E) blk and STB extracted from STB-FCS curves captured using ATTO655-TBB/Bio-IMB1 (solid square) and ATTO655-TBB/Bio-IMB2 (hollow diamond) pairs via Eq. 6 by fixing J = 1.


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Figure 3. Application of STB-FCS with FRET to capture inter-molecular interactions. (A) Experimental design to examine kinetic of duplex formation between Cy5 labeled ssDNA (strand with red dot) and Alexa488 labeled ssDNA (strand with green dot). Under 488 nm laser illumination, Cy5 labeled ssDNA itself will present no fluorescence, which is shown as strand with grey dot. (B) Normalized STB-FCS curves capturing interactions between Cy5-TBA2 and Alexa488 labeled CM7A2, CM8A2, or CM9A2 ssDNA. (C) bre and dup extracted from STB-

FCS curves via Eq. 9 were shown in black dots. dup measured by a TIRF microscope was shown in red dots.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Names and sequences of DNA, rate constants of TMR-TBA1/Bio-IMA pair, and supporting figures (Figures S1-S4) (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected] Notes The authors declare no competing financial interests.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This project was supported by funds from the National Natural Science Foundation of China (31570754), Tsinghua-Peking Joint Center for Life Sciences and Beijing Advanced Innovation


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Center for Structural Biology to CC and Lab Innovation Funding from Lab and Instrument Department, Tsinghua University to WW. The authors would like to acknowledge the Cell Imaging Facility, Technology Center for Protein Sciences, Tsinghua University for the assistance of using FV1200 LSCM with Picoquant FLIM/FCS system.

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TOC Graphic


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Figure 1 207x289mm (300 x 300 DPI)



Figure 2 191x218mm (300 x 300 DPI)


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Figure 3 180x126mm (300 x 300 DPI)



TOC graphic 162x92mm (300 x 300 DPI)


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Surface Transient Binding-Based Fluorescence Correlation

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