Demystifying PIFE: The Photophysics Behind the Protein-Induced

Letter pubs.acs.org/JPCL

Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3 Elana M. S. Stennett, Monika A. Ciuba, Su Lin, and Marcia Levitus* Department of Chemistry and Biochemistry and the Biodesign Institute, Arizona State University, P.O. Box 875601, Tempe, Arizona 85287, United States S Supporting Information *

ABSTRACT: Protein-induced fluorescence enhancement (PIFE) is a term used to describe the increase in fluorescence intensity observed when a protein binds to a nucleic acid in the proximity of a fluorescent probe. PIFE using the single-molecule dye Cy3 is gaining popularity as an approach to investigate the dynamics of proteins that interact with nucleic acids. In this work, we used complexes of DNA and Klenow fragment and a combination of time-resolved fluorescence and transient spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein (PIFE). By monitoring the formation of the cis isomer directly, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.

rotein-induced fluorescence enhancement (PIFE) is a term recently popularized in the single-molecule fluorescence community to describe the increase in fluorescence intensity that occurs when a protein binds to a nucleic acid in the proximity of a fluorescent probe.1,2 So far, the phenomenon has been described and characterized using the popular singlemolecule carbocyanine dye Cy3 (Figure 1B); when linked covalently to DNA, the fluorescence intensity of this dye increases as much as 2.5-fold when a protein is bound in its proximity.1 PIFE is gaining popularity as a single-molecule approach to investigate the dynamics of proteins that diffuse or translocate along DNA because, in contrast to FRET, it does not require protein labeling. Examples of recent applications of PIFE in single-molecule biophysics include the investigation of polymerases,3−6 helicases,7−9 and other proteins that interact with nucleic acids.10−17 Despite the widespread applications of PIFE in singlemolecule biophysical research, the photophysical mechanism that leads to the protein-induced enhancement of fluorescence of Cy3-DNA has not been elucidated. We and others have hypothesized that the enhancement of fluorescence observed in the proximity of proteins is a consequence of interactions between the dye and the protein that decrease the ability of the dye to form a nonfluorescent cis isomer.2,6,18,19 To date, however, this hypothesis has not been addressed experimentally. Here, we used transient spectroscopy to detect the cis isomer directly, which allowed us to establish the needed, and so far missing, correlation between Cy3 fluorescence lifetime and cis isomer formation yield. Our results confirm the hypothesis that, for Cy3, PIFE is due to an increase in singlet-state lifetime that occurs when the photoisomerization

P

© XXXX American Chemical Society

deactivation mechanism is inhibited by steric interactions with a protein. The photophysical properties of Cy3 and other related carbocyanines have been thoroughly investigated in solution.20−25 In the ground state, formation of the cis isomer from the thermodynamically stable trans conformation is prevented by a large energy of activation (Figure 1C). However, double bond twisting can occur on the excitedstate surface, as indicated in Figure 1C. The rotation of the double bond to generate the twisted state is a nonradiative process that deactivates the excited singlet state, and therefore competes with fluorescence emission. As a consequence, conditions that result in a decrease of the rate of bond twisting (kiso, Figure 1C), such as high viscosity and low temperature, lead to longer excited state lifetimes and higher quantum yields.20,25 In the case of Cy3 covalently attached to DNA, the relatively modest fluorescence quantum yields measured in a variety of DNA constructs (ϕf ∼ 0.2−0.4) suggest that bond twisting in the excited state is only partially inhibited by interactions between the dye and the nucleic acid.27−29 This is expected to lead to a population of ground-state cis isomer (Figure 1C), and this was indeed observed in transient absorption spectroscopy studies of Cy3 bound to DNA.29 Our working hypothesis is that PIFE is the result of protein-Cy3 interactions that further reduce the rate of bond twisting in the excited state surface, resulting in a higher fluorescence quantum yield and longer excited-state lifetime. Therefore, our model predicts that Received: March 23, 2015 Accepted: April 21, 2015

1819

DOI: 10.1021/acs.jpclett.5b00613 J. Phys. Chem. Lett. 2015, 6, 1819−1823

Letter

The Journal of Physical Chemistry Letters

is exposed to the aqueous solvent when the DNA terminus is bound to the polymerase site, but experiences a nonpolar environment that restricts the dye’s rotational freedom when the 3′ end of the primer binds the exonuclease site.26,33,34 The different properties of the dansyl probe in the solvent-exposed and protein-buried environments allowed the authors to determine that the terminus of the primer strand shuttles between the pol and exo sites even in the absence of mismatched bases.26 For matched sequences, the fraction of primer ends in the exo site has been shown to be as high as 70%,35 and depends strongly on DNA sequence and buffer conditions.33,35 In this work, we take advantage of this partitioning, and we note that the interpretation of our data does not require a precise knowledge of the fraction of strands that reside in either active site. Here, we used this well-characterized model system to test the hypothesis that protein-buried Cy3 molecules are brighter than solvent-exposed ones (PIFE), and that the increase in fluorescence quantum yield and lifetime correlates with a reduction in the efficiency of photoisomerization. To prevent exonuclease activity during the experiments, we employed the D424A mutant of KF, which eliminates 3′ → 5′ exonuclease activity but does not abolish DNA binding to the proofreading site. Control experiments were performed with the D355A/ E357A mutant, which abolishes both exonuclease activity and DNA binding to the exonuclease site.36 For this work, we designed a primer/template DNA construct containing a Cy3 molecule attached to the 5′ terminus of a 6 nt long primer bound to a 15 nt template (Figure 1A). Based on the work of D. Millar, the Cy3 molecule is expected to experience a protein-buried or solvent-exposed environment when the DNA terminus binds to the exonuclease site or the polymerase site, respectively. Indeed, experiments using the D424A-KF mutant are consistent with the prediction that the terminus of the primer strand spends a fraction of the time bound to the exonuclease site, pushing the dye into a protein-buried environment. The addition of 1.25 equiv of D424A-KF to the DNA primer/template construct resulted in an increase of the average fluorescence lifetime of Cy3-DNA from 0.65 to 0.74 ns (Figure 2A and Table 1). This increase in lifetime indicates that the environment experienced by the Cy3 molecule when the DNA terminus of the primer is bound to the exonuclease site suppresses isomerization to a considerable extent. Consistent with this interpretation, the time-resolved intensity decays measured with the D355A/E357A mutant that abolishes exo-site binding are almost identical to the decays measured in the absence of protein (Figure 2B). In this case, the dye remains in a solvent-exposed environment at all times, and therefore the addition of protein does not result in significant changes in the photophysics of Cy3. The results of the time-resolved fluorescence anisotropy (r(t)) experiments using the D424A-KF mutant (both pol- and exo-site binding) provide further evidence of the existence of two environments that result in two populations of Cy3 molecules with distinct fluorescence lifetimes. The r(t) decay measured for the Cy3-DNA/D424A-KF complex (Figure 2C) displays the dip-and-rise behavior that characterizes two populations of molecules: one with a short fluorescence lifetime and fast rotational motion, and another with a long fluorescence lifetime and slow rotational motion in the time scale of fluorescence.33,37 Because the fluorescence lifetime of Cy3 is significantly longer for the protein-buried population than for the fraction exposed to the solvent, the contribution of

Figure 1. (A) The transition of the primer terminus from the polymerase site of KF (P) to the exonuclease site (E) shifts the dye (indicated as a star) from a solvent-exposed to a protein-buried environment, as indicated by the arrows.26 (B) Structure of Cy3. The angle θ indicates the coordinate for isomerization. (C) Potential energy surface describing the formation of the cis isomer from the excited state. Excitation of the thermodynamically stable trans isomer produces a singlet excited state, from which fluorescence occurs (green arrow). Double bond twisting deactivates the excited state (red arrow, kiso), and generates a partially twisted intermediate (90°), a fraction of which decays to the ground state cis isomer (180°). The blue arrows at 550 and 570 nm indicate the wavelengths used in the transient absorbance experiments to monitor the trans and cis isomers, respectively.

conditions that lead to PIFE (increased fluorescence quantum yield and lifetime) should also result in a decrease of the population of ground-state Cy3 cis isomers. To test this hypothesis, we chose the complex of the Klenow fragment (KF) with DNA as a model DNA−protein system. This system has been well-characterized using a variety of biophysical and structural techniques, making the interpretation of our spectroscopic data straightforward. Klenow is a fragment of DNA polymerase from Escherichia coli that retains the 5′ → 3′ polymerase (pol) and the proofreading 3′ → 5′ exonuclease (exo) activities.30,31 These activities occur in two separate active sites that are separated by approximately 30 Å, and it has been well-established that proofreading involves shuttling the DNA to the exonuclease site, a transition that displaces the DNA toward the surface of the protein by about 8-bp (Figure 1A).32,33 Our experimental design takes advantage of previous work by D. Millar and collaborators, who used a DNA primer/ template construct containing a dansyl probe located eight nucleotides from the 3′ end of the primer strand to investigate the partitioning of the DNA between the two active sites of the enzyme. In this work, the authors demonstrated that the probe 1820

DOI: 10.1021/acs.jpclett.5b00613 J. Phys. Chem. Lett. 2015, 6, 1819−1823

Letter

The Journal of Physical Chemistry Letters

Figure 2. Time-resolved fluorescence (A,B) and anisotropy (C,D) decays of Cy3-DNA (black) and Cy3-DNA bound to KF (red). (E,F) Transient spectra of Cy3-DNA bound to KF before (red) or after (blue) the addition of a protease. The three plots on the right column correspond to experiments performed with the D355A/E357 mutant of KF, where the DNA terminus binds exclusively to the pol site. The three plots on the left correspond to experiments using the D424A-KF mutant, which results in partitioning between the pol and exo sites. Binding of the DNA to the exo site pulls the dye toward the protein.

spectroscopy technique. The absorption spectrum of the ground-state cis isomer of Cy3 is known to be shifted about 20 nm to the red with respect to the ground-state trans isomer,38 and therefore, it overlaps with the fluorescence spectrum of the trans dye (fluorescence from the cis conformation is negligible). Transient absorption spectroscopy measures the increase or decrease in absorbance (ΔA(λ)) after the sample is excited with a pulsed laser. Figure 2E−F shows averaged transient spectra measured in the 10−30 ns range after excitation. The data at earlier times is dominated by stimulated emission from the trans excited state, and is therefore not included in the average (Figure S1, Supporting Information). The negative ΔA signal observed around 550 nm coincides with the absorption spectrum of the trans ground state of Cy3, and is consistent with the depletion of the ground state due to absorption. It is important to note that the radiative lifetime of the singlet excited state of trans Cy3 is 2.8 ns,29 and therefore one would expect a complete recovery of the ground state (ΔA = 0) 30 ns after excitation. The fact that ΔA(550 nm) is still negative after 30 ns indicates the formation of a transient species that absorbs less efficiently than the trans ground state at 550 nm, and persists at times much greater than the lifetime of the excited state. The positive signal around 570 nm is consistent with the reported absorption spectrum of the ground state cis isomer,38−40 which reverts back to the thermodynamically stable trans isomer on time scales from microseconds to milliseconds.29 Therefore, the amplitude of the ΔA signal at 570 nm is proportional to the population of cis Cy3 in solution, and can be used to monitor the efficiency of photoisomerization of Cy3 in different environments.

Table 1. Results of Fitting the Fluorescence Intensity Decays with Three Exponential Terms: I(t) = I0∑3i=1Aie−t/τi,∑3i=1Ai = 1a

Cy3-DNA Cy3-DNA/ D424A-KF Cy3-DNA/ D355A/ E357A-KF a

A1

τ1 (ns)

A2

τ2 (ns)

A3

τ3 (ns)

⟨τ⟩ (ns)

0.43 0.41

0.28 0.25

0.44 0.46

0.77 0.82

0.13 0.13

1.46 2.03

0.65 0.74

0.44

0.30

0.46

0.83

0.10

1.59

0.67

The average lifetimes were calculated as ⟨τ⟩ = ∑3i=1Aiτi.

the first to the total anisotropy decay dominates at times greater than ca. 2 ns. The fact that r(t) increases to about 0.25 at long times (t > 6 ns) indicates that the rotational correlation time of the dye is significantly longer when the dye experiences the protein-buried environment. In contrast, the dip-and-rise is not observed in control experiments with the D355A/E357A mutant (only pol-site binding), consistent with the fact that, in this case, protein binding results in a decrease in rotational correlation time (Figure 2D) without a change in the lifetime of the dye. So far, we have demonstrated that PIFE occurs when the terminus of the DNA binds to the exonuclease site, pushing the Cy3 probe to a protein-buried environment that suppresses the nonradiative mechanisms that deactivate the excited state when the dye is exposed to the solvent. To prove that the nonradiative mechanism responsible for PIFE is indeed cis− trans isomerization on the excited-state surface, we monitored the formation of the ground-state cis isomer using a transient 1821

DOI: 10.1021/acs.jpclett.5b00613 J. Phys. Chem. Lett. 2015, 6, 1819−1823

The Journal of Physical Chemistry Letters

■

While the results of the time-resolved fluorescence intensity and anisotropy experiments are independent of sample concentration, the transient absorbance is proportional to the concentration of the absorbing species, and therefore a careful comparison of the efficiency of photoisomerization requires that all concentrations are matched carefully. To ensure that the concentration of Cy3-labeled DNA remained constant in experiments with and without KF, we performed measurements with DNA-KF complexes before and after adding a small volume of a protease to degrade the enzyme and expose the Cy3-DNA to the aqueous solvent. As expected, the transient amplitude due to the cis isomer was not affected by the action of the protease when the D355A/E357A mutant was used in the experiment (Figure 2F). For this mutant, the primer terminus binds exclusively to the polymerase site, and therefore the dye is water exposed and able to isomerize regardless of whether the protein is bound to the DNA or not. We note that the action of the protease was evident by a marked decrease in scattering at wavelengths close to the laser excitation wavelength (Figure S2 Supporting Material), and by the fact that the r(t) decay after addition of the protease was almost identical to that of the Cy3-DNA sample (not shown). Protease activity, in contrast, had a marked effect in the transient spectra of the Cy3-DNA/D424A-KF complex. As shown in Figure 2E, the concentration of cis isomer measured 30 ns after excitation using the Cy3-DNA/D424A-KF complex was significantly smaller than the value measured with the D355A/E357A mutant under the same conditions. In addition, the action of a protease that degrades the enzyme resulted in a marked increase in the concentration of the cis isomer. These results are consistent with the fact that the 3′ terminus of the primer visits the exonuclease site a fraction of the time, pushing the dye to a protein-buried environment that prevents photoisomerization. Isomerization requires that one-half of the molecule rotates 180° with respect to the other, so its efficiency is expected to decrease in sterically constrained environments. In summary, we have used a combination of time-resolved fluorescence and transient-spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in the close proximity of a protein (PIFE). Using the Klenow fragment as a model system, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization in conditions where the dye is sterically constrained by the protein.

■

REFERENCES

(1) Hwang, H.; Kim, H.; Myong, S. Protein Induced Fluorescence Enhancement as a Single Molecule Assay with Short Distance Sensitivity. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 7414−7418. (2) Hwang, H.; Myong, S. Protein Induced Fluorescence Enhancement (PIFE) for Probing Protein-Nucleic Acid Interactions. Chem. Soc. Rev. 2014, 43, 1221−1229. (3) Vrtis, K. B.; Markiewicz, R. P.; Romano, L. J.; Rueda, D. Carcinogenic Adducts Induce Distinct DNA Polymerase Binding Orientations. Nucleic Acids Res. 2013, 41, 7843−7853. (4) Maxwell, B. A.; Suo, Z. Single-Molecule Investigation of Substrate Binding Kinetics and Protein Conformational Dynamics of a B-Family Replicative DNA Polymerase. J. Biol. Chem. 2013, 288, 11590−11600. (5) Markiewicz, R. P.; Vrtis, K. B.; Rueda, D.; Romano, L. J. SingleMolecule Microscopy Reveals New Insights into Nucleotide Selection by DNA Polymerase I. Nucleic Acids Res. 2012, 40, 7975−7984. (6) Luo, G.; Wang, M.; Konigsberg, W. H.; Xie, X. S. Single-Molecule and Ensemble Fluorescence Assays for a Functionally Important Conformational Change in T7 DNA Polymerase. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 12610−12615. (7) Park, J.; Myong, S.; Niedziela-Majka, A.; Lee, K. S.; Yu, J.; Lohman, T. M.; Ha, T. PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps. Cell 2010, 142, 544−555. (8) Koenig, S. L. B.; Liyanage, P. S.; Sigel, R. K. O.; Rueda, D. Helicase-Mediated Changes in RNA Structure at the Single-Molecule Level. RNA Biology 2013, 10, 133−148. (9) Galletto, R.; Tomko, E. J. Translocation of Saccharomyces Cerevisiae Pif1 Helicase Monomers on Single-Stranded DNA. Nucleic Acids Res. 2013, 41, 4613−4627. (10) Qiu, Y.; Antony, E.; Doganay, S.; Koh, H. R.; Lohman, T. M.; Myong, S. Srs2 Prevents Rad51 Filament Formation by Repetitive Motion on DNA. Nat. Commun. 2013, 4, 2281. (11) Myong, S.; Cui, S.; Cornish, P. V.; Kirchhofer, A.; Gack, M. U.; Jung, J. U.; Hopfner, K. P.; Ha, T. Cytosolic Viral Sensor RIG-I Is a 5′Triphosphate-Dependent Translocase on Double-Stranded RNA. Science 2009, 323, 1070−1074. (12) Marko, R. A.; Liu, H.-W.; Ablenas, C. J.; Ehteshami, M.; Goette, M.; Cosa, G. Binding Kinetics and Affinities of Heterodimeric Versus Homodimeric HIV-1 Reverse Transcriptase on DNA−DNA Substrates at the Single-Molecule Level. J. Phys. Chem. B 2013, 117, 4560− 4567. (13) Luo, Y.; North, J. A.; Rose, S. D.; Poirier, M. G. Nucleosomes Accelerate Transcription Factor Dissociation. Nucleic Acids Res. 2014, 42, 3017−3027. (14) Koh, H. R.; Kidwell, M. A.; Ragunathan, K.; Doudna, J. A.; Myong, S. ATP-Independent Diffusion of Double-Stranded RNA Binding Proteins. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 151−156. (15) Fischer, C. J.; Maluf, N. K.; Lohman, T. M. Mechanism of ATPDependent Translocation of E. coli UvrD Monomers Along SingleStranded DNA. J. Mol. Biol. 2004, 344, 1287−1309. (16) Nguyen, B.; Sokoloski, J.; Galletto, R.; Elson, E. L.; Wold, M. S.; Lohman, T. M. Diffusion of Human Replication Protein A Along Single-Stranded DNA. J. Mol. Biol. 2014, 426, 3246−3261. (17) Schauer, G. D.; Huber, K. D.; Leuba, S. H.; Sluis-Cremer, N. Mechanism of Allosteric Inhibition of HIV-1 Reverse Transcriptase Revealed by Single-Molecule and Ensemble Fluorescence. Nucleic Acids Res. 2014, 42, 11687−11696. (18) Levitus, M.; Ranjit, S. Cyanine Dyes in Biophysical Research: The Photophysics of Polymethine Fluorescent Dyes in Biomolecular Environments. Q. Rev. Biophys. 2011, 44, 123−151. (19) Stennett, E.; Ciuba, M.; Levitus, M. Photophysical Processes in Single Molecule Organic Fluorescent Probes. Chem. Soc. Rev. 2014, 43, 1057−1075. (20) Sundstrom, V.; Gillbro, T. Viscosity-Dependent Isomerization Yields of Some Cyanine Dyes - A Picosecond Laser Spectroscopy Study. J. Phys. Chem. 1982, 86, 1788−1794. (21) Akesson, E.; Sundstrom, V.; Gillbro, T. Solvent-Dependent Barrier Heights of Excited-State Photoisomerization Reactions. Chem. Phys. Lett. 1985, 121, 513−522.

ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental procedures and two supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org.

■

Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]; phone: +1-480-7278586. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank David Millar (Scripps Institute) for his generous gift of purified D424A-KF. 1822

DOI: 10.1021/acs.jpclett.5b00613 J. Phys. Chem. Lett. 2015, 6, 1819−1823

Letter

The Journal of Physical Chemistry Letters (22) Korppitommola, J. E. I.; Hakkarainen, A.; Hukka, T.; Subbi, J. An Isomerization Reaction of a Cyanine Dye in Normal-Alcohols Microscopic Friction and an Excited-State Barrier Crossing. J. Phys. Chem. 1991, 95, 8482−8491. (23) Ponterini, G.; Momicchioli, F. Trans Cis Photoisomerization Mechanism of Carbocyanines - Experimental Check of TheoreticalModels. Chem. Phys. 1991, 151, 111−126. (24) Momicchioli, F.; Baraldi, I.; Berthier, G. Theoretical-Study of Trans Cis Photoisomerism in Polymethine Cyanines. Chem. Phys. 1988, 123, 103−112. (25) Aramendia, P. F.; Negri, R. M.; Sanroman, E. TemperatureDependence of Fluorescence and Photoisomerization in Symmetrical Carbocyanines - Influence of Medium Viscosity and MolecularStructure. J. Phys. Chem. 1994, 98, 3165−3173. (26) Guest, C. R.; Hochstrasser, R. A.; Dupuy, C. G.; Allen, D. J.; Benkovic, S. J.; Millar, D. P. Interaction of DNA with the Klenow Fragment of DNA-Polymerase-I Studied by Time-Resolved Fluorescence Spectroscopy. Biochemistry 1991, 30, 8759−8770. (27) Spiriti, J.; Binder, J. K.; Levitus, M.; van der Vaart, A. Cy3-DNA Stacking Interactions Strongly Depend on the Identity of the Terminal Basepair. Biophys. J. 2011, 100, 1049−1057. (28) Harvey, B. J.; Perez, C.; Levitus, M. DNA Sequence-Dependent Enhancement of Cy3 Fluorescence. Photochem. Photobiol. Sci. 2009, 8, 1105−1110. (29) Sanborn, M. E.; Connolly, B. K.; Gurunathan, K.; Levitus, M. Fluorescence Properties and Photophysics of the Sulfoindocyanine Cy3 Linked Covalently to DNA. J. Phys. Chem. B 2007, 111, 11064− 11074. (30) Freemont, P. S.; Friedman, J. M.; Beese, L. S.; Sanderson, M. R.; Steitz, T. A. Cocrystal Structure of an Editing Complex of Klenow Fragment with DNA. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 8924− 8928. (31) Beese, L. S.; Derbyshire, V.; Steitz, T. A. Structure of DNAPolymerase-I Klenow Fragment Bound to Duplex DNA. Science 1993, 260, 352−355. (32) Cowart, M.; Gibson, K. J.; Allen, D. J.; Benkovic, S. J. DNA Substrate Structural Requirements for the Exonuclease and Polymerase Activities of Procaryotic and Phage DNA-Polymerases. Biochemistry 1989, 28, 1975−1983. (33) Carver, T. E.; Hochstrasser, R. A.; Millar, D. P. Proofreading DNA - Recognition of Aberrant DNA Termini by the Klenow Fragment of DNA-Polymerase-I. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 10670−10674. (34) Lam, W. C.; Van der Schans, E. J. C.; Joyce, C. M.; Millar, D. P. Effects of Mutations on the Partitioning of DNA Substrates between the Polymerase and 3′−5′ Exonuclease Sites of DNA Polymerase I (Klenow Fragment). Biochemistry 1998, 37, 1513−1522. (35) Datta, K.; Johnson, N. P.; von Hippel, P. H. DNA Conformational Changes at the Primer-Template Junction Regulate the Fidelity of Replication by DNA Polymerase. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 17980−17985. (36) Derbyshire, V.; Freemont, P. S.; Sanderson, M. R.; Beese, L.; Friedman, J. M.; Joyce, C. M.; Steitz, T. A. Genetic and Crystallographic Studies of the 3′,5′-Exonucleolytic Site of DNA-Polymerase-I. Science 1988, 240, 199−201. (37) Ludescher, R. D.; Peting, L.; Hudson, S.; Hudson, B. TimeResolved Fluorescence Anisotropy for Systems with Lifetime and Dynamic Heterogeneity. Biophys. Chem. 1987, 28, 59−75. (38) Chibisov, A. K.; Zakharova, G. V.; Gorner, H.; Sogulyaev, Y. A.; Mushkalo, I. L.; Tolmachev, A. I. Photorelaxation Processes in Covalently-Linked Indocarbocyanine and Thiacarbocyanine Dyes. J. Phys. Chem. 1995, 99, 886−893. (39) Jia, K.; Wan, Y.; Xia, A. D.; Li, S. Y.; Gong, F. B.; Yang, G. Q. Characterization of Photoinduced Isomerization and Intersystem Crossing of the Cyanine Dye Cy3. J. Phys. Chem. A 2007, 111, 1593−1597. (40) Stennett, E. M. S.; Ma, N.; van der Vaart, A.; Levitus, M. Photophysical and Dynamical Properties of Doubly Linked Cy3− DNA Constructs. J. Phys. Chem. B 2014, 118, 152−163. 1823

DOI: 10.1021/acs.jpclett.5b00613 J. Phys. Chem. Lett. 2015, 6, 1819−1823