Graphene Oxide Complexes for Photothermal

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Conjugated Polymer/Graphene Oxide Complexes for Photothermal Activation of DNA Unzipping and Binding to Protein Dawei Li, Dong Gao, Junjie Qi, Ran Chai, Yong Zhan, and Chengfen Xing ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00047 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Conjugated Polymer/Graphene Oxide Complexes for Photothermal Activation of DNA Unzipping and Binding to Protein Dawei Li,a Dong Gao,b Junjie Qi,a Ran Chai,b Yong Zhanb and Chengfen Xing*a,b a

Institute of Polymer Science and Engineering College of Chemical Engineering Hebei

University of Technology, Tianjin 300130, (P. R. China) b

Key Laboratory of Hebei Province for Molecular Biophysics Institute of Biophysics Hebei

University of Technology, Tianjin 300401, (P. R. China) KEYWORDS Conjugated Polymer, graphene oxide, photothermal activation, FRET, single-stranded binding protein.

ABSTRACT

DNA-protein interactions control DNA transcription, recombination, restriction and replication, which plays an important role in regulating life activities. We developed a new strategy to photothermally regulate DNA unzipping and binding to single-stranded binding protein (SSBP) based on the hybrid systems of graphene oxide (GO) and conjugated polymer. GO is applied as

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the photothermal modulator to activate the unzipping of dsDNA into ssDNA. Upon near-infrared (NIR) laser irradiation, the thermal melting transition of DNA is promoted, resulting in much farther distance between poly[(9,9-bis(6 ′ -N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP) and fluorescein labeled at the terminus of the DNA in comparison to that of dsDNA before NIR irradiation in the presence of GO. Therefore, the FRET efficiency for PFP/fluorescein becomes weaker upon irradiation. Moreover, FRET efficiency could readily recover when the unzipped DNA hybridizes with SSBP. In this strategy, PFP/GO serves as detector and activator for the DNA unzipping and interacting with proteins, providing a facile and effective method for the regulation and detection of DNA-protein interactions.

Introduction

The interactions and structural changes of biomolecules are an important part of the life process and the intracellular processes are closely related to the DNA-protein interactions.1-5 As a protein that specifically binds to DNA, single-stranded binding protein (SSBP) extracted from sulfolobus solfataricus binds 20 bases in the form of homologous through hydrophobic and π-π interaction and protect ssDNA from damage.6-8 As an essential basic protein in life process, SSBP plays an important role in DNA replication, repair, and recombination.9-11 So detection of SSBP is of great importance in studying the function of protein,12, 13 treatment of related diseases,14 and biological detection.15, 16 Recent studies revealed mitochondrial diseases, liver cancer and some neurological diseases are also associated with the interactions between SSBP and DNA,17, 18 as well as the structural change in DNA.12, 14 Therefore, the regulation of the interactions between


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Scheme 1. Schematic Detection and Activation of DNA Unzipping and Hybridization with SSBP based on PFP/GO Composite System.

SSBP and DNA has always been the focus of attention for researchers.15 However, Most of current techniques are limited to detect the presence of SSBP and cannot achieve the regulation of interaction between SSBP and DNA.13, 16, 19, 20 So developing new therapy strategy to regulate DNA and proteins interaction is becoming an important issue.21, 22 Photothermal effect is an emerging alternative to thermally stimulate cellular processes by applying near-infrared (NIR) light-adsorbing agents to convert energy to heat,23-26 leading to the deep penetration and increased local temperature, which is promising for control the thermosensitive processes. Due to the high penetration depth, noninvasiveness and minimized adverse side effect, photothermal effect is emerging as a promising method for cancer therapy and bimolecular regulation.27-29 Herein, we designed a strategy to photothermally stimulate the DNA unzipping and regulate DNA-protein interactions by combining the advantage of GO and PFP (poly[(9,9-bis(6’-N,N,Ntrimethylammonium)hexyl)-fluorenylene phenylene dibromide]). GO, as a single-atom-thicklayered material with excellent biocompatibility and feasible surface functionalization ability,3034

has been widely concerned in biological applications,35-39 and the excellent photothermal


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effect of GO attracted enormous attention in tumor therapy.40-43 Moreover, conjugated polymers exhibit the characteristics of strong light absorption and signal amplification,44-50 which could significantly improve the detection sensitivity.51-56 As illustrated in Scheme 1, the weak interaction between fluorescein (dsDNA-Fl) labeled double-stranded DNA and GO lead to the desorption of dsDNA-FI from GO surface, affording weak fluorescence quenching of dsDNA-Fl and close distance between dsDNA-Fl and free PFP, which gives rise to the strong FRET efficiency for PFP/dsDNA-Fl. Upon NIR laser irradiation (process a), the photothermal effect of GO produces the local heating and leads to the unzipping of dsDNA-Fl to ssDNA-Fl. Due to the strong π-π stacking interaction between GO surface and ssDNA-Fl, ssDNA-Fl was strongly adsorbed on the GO surface, resulting in further distance between PFP and ssDNA-Fl and weaker FRET efficiency in comparison to that of dsDNA-Fl before NIR irradiation. However, in the presence of SSBP (process b), ssDNA-Fl was specifically hybridized with SSBP, resulting in the desorption of dsDNA-Fl from GO and recovered FRET. The PFP/GO composite system is featured by the following characteristics: First, the photothermal effect of GO could produce a local temperature higher than 50 °C under NIR laser irradiation to “cook” dsDNA into ssDNA, thus facilitating the DNA-protein interactions. Second, the PFP/GO composite system is served as both detector and activator for the DNA unzipping and hybridization with target protein. Third, the utilization of light-harvesting conjugated polymer enhances the detection sensitivity and signal-to-noise ratio by applying FRET strategy. Experimental Materials and Instruments


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PFP was prepared by referring the previous literature.49 GO was purchased from Suzhou TANFENG graphene Tech Co., Ltd and sonicated for 3 hours at 300 W power.57 Oligonucleotides and SSBP were purchased from Sangon Biotechnology (Shanghai) Co., Ltd without further purification. Other proteins, Na2HPO4·12H2O, NaH2PO4·2H2O and NaOH were purchased from Aladdin (Shanghai) Co., Ltd without further purification. Utralpure water was obtained from Milli-Q gradient system (18.2 MΩ·cm). 808 nm NIR laser was generated from high power laser generator (Hi-Tech optoelectronics Co., Ltd.). Temperature and NIR thermal images were captured by infrared thermal camera (C.A-73, Chauvin Arnoux Group, France). UV absorbance curve were recorded by UV spectrophotometer with peltier temperature control unit (Specord250 Plus, analytikjena, Germany). DNA annealing was performed by PCR instrument (EasyCycler, analytikjena, Germany). Fluorescene spectra were measured by using Multifunction Microplate Reader SpectraMax i3x from Molecular Devices, LLC. (USA). Methods Preparation of the Phosphate Buffer Solution (PB): Na2HPO4•12H2O and NaH2PO4•2H2O were added to ultrapure water to prepare 200 mL of 0.2 M mother liquor which contained 162 mL of 0.2 M Na2HPO4 and 38 mL of 0.2 M NaH2PO4. The pH was adjusted to 7.4 by adding NaOH. Various concentrations of PB were prepared by diluting mother liquor. Annealing

Process

of

DNA:

10

µL

of

two

complementary

ssDNA(FITC-5´-

GCTCACTTAGGTTCTCATC-3´ and 5´-AGATGAGAACCTAAGTGAGC-3´) with the same concentration (100 µM) were added to 80 µL of 12.5 mM PB solution in PCR tube. The temperature of the solution was raised to 60 °C at a rate of 4 °C/s and held for 30 min. Then the solution was cooled to 25 °C at a rate of 0.1 °C/s.58


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Measurement of Photothermal Effect of GO: GO (80 µg/mL, 100 µL) was added to 96-well plates at room temperature. Then the plates were irradiated under NIR laser with different power density (2.5-4 W/cm2) for 10 min. GO with different concentrations (0-100 µg/mL) were also measured with a power density of 3.5 W/cm2. The temperature was recorded once every 30 s by using Infrared thermal camera. Measurement of Tm Value of dsDNA: 20 µL of 1 µM dsDNA and 180 µL of 12.5 mM PB (pH = 7.4) were added to micro-quartz cuvette and shook slightly at room temperature. Subsequently, the temperature range of the peltier temperature control unit was adjusted to 37-46 °C and then the absorbance of 260 nm was measured every 0.1 °C by using UV spectrophotometer. Activation and Detection of the Unzipping of dsDNA: 93 µL of 12.5 mM PB and 10 µM dsDNA-Fl with different volume (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 µL) were added to 96-well plates at room temperature, and the fluorescence spectra of the solutions were measured with the excitation wavelength of 480 nm. Then, 4 µL of 2 mg/mL GO was evenly added to each solution and the fluorescence spectra of the solutions were measured after 5 min. Subsequently, the solutions were irradiated by NIR laser with the power of 3.5 W/cm2 for 10 min after the solutions were cooled to room temperature and the fluorescence spectra of the solutions were measured again. Finally, 1.5 µL of 100 µM PFP was evenly added to each solution and incubated for 5 min. The excitation wavelength was changed to 375 nm, and the fluorescence spectrum of the system was measured. Same experimental procedure was also performed for the control experiment that without irradiation. Detection Sensitivity for dsDNA: 1 µL of 1 µM dsDNA-Fl and 1.75 µM of GO was added to 95.25 µL of 12.5 mM PB and incubated for 5 min. Then the solution was irradiated for 10 min


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under NIR laser with the power density of 5 W/cm2. When the solution was cooled to room temperature, 2 µL of 10µM PFP was evenly added and the solution was incubated for 5 min. Finally, the fluorescence of the system was measured with the excitation wavelength of 375 nm. The same experimental procedure was also performed for the control experiment without irradiation. Regulation of the Interaction between SSBP and DNA: To two wells was added 89.5 µL of PB and 1.5 µL of 10 µM dsDNA-Fl or ssDNA-Fl before 4 µL of 2 mg/mL GO was added. Then the fluorescence spectra of two wells was measured with excitation wavelength of 480 nm after incubated for 5 min. 4 µL of 100 µg/mL SSBP was added to one of the wells and then the two wells were immediately irradiated under NIR laser with the power density of 3.5 W/cm2 for 8 min. Then, fluorescence spectra were measured again after the solutions were cooled to room temperature. Finally, 1µL of 100 µM PFP was added to each well and incubated for 5 min. The fluorescence spectra of the solutions were measured with the excitation wavelength of 375 nm. The control experiment without irradiation was performed by following the same procedure. Specific Detection of Proteins: To four wells was added 89.5 µL of PB, 1.5 µL of 10 µM dsDNA-Fl and 4 µL of 2 mg/mL GO. After incubating for 5 min, 4 µL of 100 µg/mL SSBP, ligase, thrombin, lysozyme, BSA and trypsin were added to different wells. Subsequently, four wells were irradiated under NIR laser with power density of 3.5 W/cm2 for 8 min. 1 µL of 100 µM PFP was added to each well when the solution was cooled to room temperature and then incubated for 5 min. Finally, the FRET ratio of I520

nm/I420 nm

and fluorescence spectra were

measured with the excitation wavelength of 375 nm.


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Results and Discussion

Figure 1. a) Infrared thermal images of GO aqueous solution with various concentrations exposed to NIR laser irradiation and b) temperature profiles of GO aqueous solution with various concentrations versus irradiation time. The power density is 3.5 W/cm2. Measurements were performed in PB (12.5 mM, pH = 7.4).

In order to characterize the efficiency of photothermal effect of GO, NIR thermal images of different concentrations of GO aqueous solution, which were irradiated under NIR laser irradiation with the power density of 3.5 W/cm2, were collected. As shown in Figure 1a, the temperature of the GO aqueous solution increases with the increase of the concentration upon exposure to the NIR laser at 3.5W/cm2 for 10 min. GO aqueous solution with concentration of 100 µg/mL exhibits the temperature rising by 20 °C while the temperature of the control group without GO slightly rises by 5 °C. Moreover, the temperature increases with the irradiation time. Sharp temperature increase can be obtained within 5 min irradiation (Figure 1b). The temperature of GO aqueous solution (80 µg/mL) irradiated under different power densities (Figure S1a) was measured as well. The temperature of GO aqueous solution increased with the increasing power density and a density of 3.5 W/cm2 could cause temperature enhancement to 44.8 °C upon 10 min irradiation. Moreover, Figure S1b shows that PFP and DNA produces little effect on the photothermal effect of GO. These results confirm the excellent photothermal


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Figure 2. a) Fluorescence emission spectra of the dsDNA-Fl and dsDNA-Fl/GO before or after irradiation under NIR laser irradiation with the power density of 3.5 W/cm2 for 10 min. [dsDNA-Fl] = 0.15 µM, [GO] = 80 µg/ml. Measurements were performed in PB (12.5 mM, pH = 7.4). The excitation wavelength is 480 nm. b) Fluorescence emission spectra of PFP/GO and PFP/dsDNA-Fl/GO before or after irradiation. [dsDNA-Fl] = 0.15 µM, [GO] = 80 µg/ml, [PFP] = 1.5 µM in repeat units (RUs). The excitation wavelength is 375 nm. c) FRET ratio (I520 nm/I420 nm) of the PFP/dsDNA-Fl/GO before or after irradiation as a function of dsDNA-Fl concentrations. Error bars were calculated as standard deviations of data from three seperate measurements. [GO] = 80 µg/ml, [PFP] = 1.5 µM in repeat units (RUs). d) Agarose gel electrophoresis (2.5%) analysis of dsDNA/GO before or after irradiation. [dsDNA] = 0.2 µM, [GO] = 80 µg/mL, [ssDNA] = 1.0 µM.

conversion capacity of the GO aqueous solution. The Tm of dsDNA was measured (Figure S2) to be 42.2 °C, which can be reached by irradiating the GO solution with the concentration of 80 µg/mL under NIR laser at 3.5 W/cm2 for 10 min. To confirm the photothermal activation of GO in unzipping of DNA, dsDNA-Fl, which was obtained by annealing of a single-stranded complementary DNA strand to ssDNA-Fl, and


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ssDNA-Fl were used to indicate the unzipping changes of dsDNA to ssDNA. As shown in Figure 2a, 41.4% of the fluorescence of dsDNA-Fl is quenched in the presence of GO, which is attributed to the fluorescence quenching effect caused by the absorption of a small amount of dsDNA-Fl. Then, after irradiating the dsDNA-Fl in the presence of GO under NIR laser with the power density of 3.5 W/cm2 for 10 min, the fluorescence intensity at 520 nm is 4.7 times lower than that before irradiation. The high temperature produced by photothermal effect of GO causes thermal melting transition of dsDNA into ssDNA, and therefore the ssDNA-Fl adsorbs onto the GO surface, leading to the efficient fluorescence quenching of ssDNA-Fl. Moreover Figure S3 shows that irradiation and heating have no effect on dsDNA-Fl fluorescence. DLS analysis (Figure S4a) exhibits slight increase in GO particle size after laser irradiation, further illustrating the photothermal activation of GO in unzipping of DNA. Addition of PFP to the aqueous solution of dsDNA-Fl in the presence of GO exhibits 6.4 times lower signal for dsDNAFl/GO/PFP after NIR laser irradiation than that before NIR laser irradiation (Figure 2b), which is attributed to the resulting reduced FRET ratio (I520 nm/I420 nm) between free PFP and fluorescein. Figure 2c shows the FRET ratio change versus various concentrations of dsDNA-Fl before and after irradiation. Before irradiation, FRET ratio is increased with the increasing concentration of dsDNA-Fl and remains substantially constant after 0.3 µM. While the FRET ratio after irradiation is much lower in the range 0-0.4 µM and the concentration of 0.15 µM gives the most obvious difference. The increased fluorescence difference between before and after irradiation caused by addition of PFP reveals the signal amplification induced by conjugated polymer. The lowest concentration for dsDNA to be photothermally activated by NIR laser and detected by using FRET strategy is 0.01 µM (Figure S5), demonstrating that the PFP/GO system provides a strategy to activate and detect DNA structure changes with high sensitivity. Figure 2d shows the


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Figure 3. Fluorescence emission spectra of a) dsDNA-Fl/GO and b) ssDNA-Fl/GO with and without SSBP before or after NIR laser irradiation. The excitation wavelength is 480 nm. Fluorescence emission spectra of c) PFP/dsDNA-Fl and d) PFP/ssDNA-Fl in the presence of GO with and without SSBP. [dsDNA-Fl] = [ssDNA-Fl] = 0.15 µM, [SSBP] = 4 µg/mL, [GO] = 80 µg/ml, [PFP] = 1.0 µM in repeat units (RUs). The excitation wavelength is 375 nm. Measurements were performed in PB (12.5 mM, pH = 7.4). e) SDS-PAGE (15%) analysis of SSBP before or after irradiation in the presence of dsDNA and GO. [dsDNA] = 0.3 µM, [SSBP] = 4 µg/mL, [GO] = 80 µg/ml.

direct evidence of the unzipping of dsDNA triggered by NIR laser irradiation in the presence of GO. As shown in agarose gel electrophoresis, only a 40 bp band corresponding to dsDNA is


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observed before irradiation, whereas an obvious ssDNA band of 20 bp occurs after irradiation, further suggesting the NIR laser triggers dsDNA unzipping. To imitate the intracellular processes of DNA-protein interactions, the photothermal activation of DNA-SSBP interaction was evaluated as well. As shown in Figure 3a, before irradiation, no obvious change of the fluorescence of dsDNA-Fl in the presence of GO can be observed upon the addition of SSBP. However, upon NIR laser irradiation, the SSBP-containing solution exhibits 2 times stronger fluorescence intensity than that without SSBP This is attributed to the specific binding of SSBP with the ssDNA-Fl produced by photothermally triggered unzipping of dsDNA-Fl, leading to the desorption of ssDNA-Fl on GO surface and recovery of fluorescence. The fluorescence emission spectra of ssDNA-Fl/GO with or without SSBP were also measured (Figure 3b). The hybridization with SSBP enhances the fluorescence of ssDNA-Fl in the absence or presence of laser irradiation, suggesting that dsDNA unzipping to ssDNA is photothermally triggered by NIR laser in the presence of GO and thus bound with SSBP. To improve sensitivity and signal-to-noise ratio of the detection of DNA-SSBP interaction, the fluorescence spectra of PFP/dsDNA-Fl (Figure 3c) and PFP/ssDNA-Fl (Figure 3d) in the presence of GO with or without adding SSBP before or after NIR laser irradiation were measured. In the absence of SSBP, the FRET ratio upon NIR laser irradiation for PFP/dsDNA-Fl in the presence of GO is 6.6 times lower than that before irradiation (Figure 3c). However, the FRET ratio only recovered nearly half upon adding SSBP into this irradiated solution, which results from the hybridization of ssDNA-Fl with SSBP and subsequent desorption from GO. While for the fluorescence spectra of PFP/ssDNA-Fl (Figure 3d) in the presence of GO, the FRET ratio is dependent on the exist ance of SSBP and independent on irradiation. Moreover, the effect of the addition order of SSBP on the fluorescence of the system has been checked as well. If SSBP was added after irradiation,


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Figure 4. (a) FRET ratio (I520 nm/I420 nm) and (b) fluorescence emission spectra of of the PFP/dsDNA-Fl in the presence of GO with various proteins under NIR laser irradiation with the power density of 3.5 W/cm2 for 8min. [dsDNA-Fl] = 0.15 µM, [proteins] = 4 µg/mL, [GO] = 80 µg/mL, [PFP] = 1.0 µM in repeat units (RUs). The excitation wavelength is 375 nm. Measurements were performed in PB (12.5 mM, pH = 7.4)

slight fluorescence recovery was observed (Figure S6), indicating that SSBP was difficult to bind to ssDNA-Fl adsorbed on GO surface. In addition, SDS-PAGE analysis (Figure 3e) shows that the ssDNA yielded by NIR irradiation in the presence of GO gives rise to the increased proportion of cross-linked species. The species result from tetramers or dimers produced by the combination of SSBP and ssDNA, which is consistent with the previous results.7 In order to investigate the specificity of the GO-conjugated polymer composite system to regulate the interaction between SSBP and DNA, we also measured the response of ligase, thrombin, lysozyme, BSA and trypsin under the same condition. Figure 4 shows the FRET ratio (I520 nm/I420 nm) and fluorescence emission spectra of the PFP/dsDNA-Fl in the presence of GO with various proteins under NIR laser irradiation with the power density of 3.5 W/cm2 for 8 min. In particular, the FRET ratio for the system containing SSBP is 3 times higher than those of other proteins, resulting from specific binding of SSBP to ssDNA. While, for ligase, thrombin, lysozyme, BSA and trypsin, the FRET ratio only shows a slight change compared to the blank group without protein, which is due to the little binding with DNA and the absorption on the GO


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surface. These results demonstrate the method of photothermally regulating interaction between SSBP and DNA by GO-conjugated polymer composite system is highly specific. Conclusions In summary, we have demonstrated a hybrid system of graphene oxide (GO) and conjugated polymer for photothermal regulation DNA unzipping and binding to single-stranded binding protein (SSBP). The photothermal effect of GO realizes the “cooking” dsDNA into ssDNA, facilitating DNA-protein interactions. Moreover, the conjugate polymer/GO composite system can be served as both detector and activator for the DNA unzipping and hybridization with target protein with high detection sensitivity and signal-to-noise ratio by applying FRET strategy. Therefore, our strategy is applicable for detecting and regulating DNA-protein interactions, promising for sensing and controlling structural changes of biomolecules and intracellular processes. ASSOCIATED CONTENT Supporting Information available: Infrared thermal image of GO aqueous solution with various power densities, the absorbance curve of dsDNA at different temperature, the lowest detection concentration of dsDNA, details for fluorescence emission spectra of ssDNA-Fl/GO before and after SSBP addition. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors are grateful for the financial support of the National Natural Science Foundation of China (No. 21574037 and No. 21773054), the “100 Talents” Program of Hebei Province, China (No. E2014100004), the Natural Science Foundation of Hebei Province (No. B2017202051), the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province (No. SLRC2017028). REFERENCES (1) Duan, X.; Liu, L.; Feng, X.; Wang, S., Assemblies of Conjugated Polyelectrolytes with Proteins for Controlled Protein Photoinactivation. Adv. Mater. 2010, 22, 1602-1606. (2) Xie, Z.; Hu, S.; Blackshaw, S.; Qian, J.; Zhu, H., Systematic Characterization of ProteinDNA Interactions. Cell. Mol. Life. Sci. 2011, 68, 1657-1668. (3) Loffreda, A.; Rigamonti, A.; Barabino, S. M. L.; Lenzken, S. C., RNA-Binding Proteins in the Regulation of miRNA Activity: A Focus on Neuronal Functions. Biomolecules. 2015, 5, 2363-2387. (4) Chen, Y.; Xu, Y.; Bao, Q.; Xing, Y.; Li, Z.; Lin, Z.; Stock, J. B.; Jeffrey, P. D.; Shi, Y., Structural and Biochemical Insights into the Regulation of Protein Phosphatase 2A by Small T Antigen of SV40. Nat. Struct. Mol. Biol. 2007, 14, 527-534. (5) Kim, Y.; Spitz, G. S.; Veturi, U.; Lach, F. P.; Auerbach, A. D.; Smogorzewska, A., Regulation of Multiple DNA Repair Pathways by the Fanconi Anemia Protein SLX4. Blood. 2013, 121, 54-63. (6) Haseltine, C. A.; Kowalczykowski, S. C., A Distinctive Single-Stranded DNA-Binding Protein from the Archaeon Sulfolobus Solfataricus. Mol. Microbio. 2002, 43, 1505–1515. (7) Wadsworth, R. I. M.; White, M. F., Identification and Properties of the Crenarchaeal SingleStranded DNA Binding Protein from Sulfolobus Solfataricus. Nucleic. Acids. Res. 2001, 29, 914-920. (8) Kerr, I. D.; Wadsworth, R. I. M.; Cubeddu, L.; Blankenfeldt, W.; Naismith, J. H.; White, M. F., Insights into ssDNA Recognition by the OB Fold from a Structural and Thermodynamic Study of Sulfolobus SSB Protein. EMBO. J. 2003, 22, 2561-2570. (9) Bochkareva, E.; Belegu, V.; Korolev, S.; Bochkarev, A., Structure of the Major SingleStranded DNA-Binding Domain of Replication Protein A Suggests A Dynamic Mechanism for DNA Binding. EMBO. J. 2001, 20, 612-618. (10) Korhonen, J. A.; Gaspari, M.; Falkenberg, M., Twinkle has 5' -> 3' DNA Helicase Activity and is Specifically Stimulated by Mitochondrial Single-Stranded DNA-Binding Protein. J. Biol. Chem. 2003, 278, 48627-48632.


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