Reshaping the Energy Landscape Transforms the Mechanism and

Dec 15, 2017 - Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal ...
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Reshaping the energy landscape transforms the mechanism and binding kinetics of DNA threading intercalation Andrew G Clark, M. Nabuan Naufer, Fredrik Westerlund, Per Lincoln, Ioulia Rouzina, Thayaparan Paramanathan, and Mark C Williams Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01036 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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Biochemistry

Reshaping the energy landscape transforms the mechanism and binding kinetics of DNA threading intercalation Andrew G. Clark†, M. Nabuan Naufer†, Fredrik Westerlund‡, Per Lincoln§, Ioulia RouzinaÅ, Thayaparan Paramanathanc*, Mark C. Williams†* †

Department of Physics, Northeastern University, Boston, USA Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden § Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden Å Department of Chemistry and Biochemistry, Ohio State University, Columbus, USA cDepartment of Physics, Bridgewater State University, Bridgewater, USA *Corresponding Authors: [email protected], [email protected]

‡

ABSTRACT: Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single molecule techniques, we quantify the binding of a small metalorganic ruthenium threading intercalator (4,4-B), and compare its binding characteristics to a similar molecule with significantly larger threading moieties (4,4-P). The binding affinity is the same for both molecules, while comparison of the binding kinetics reveals significantly faster kinetics for 4,4-B. However, these kinetics are still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that binding affinity is modulated independently by the intercalating section and binding kinetics is modulated by the threading moiety. In order to thread DNA, 4,4-P requires a “lock mechanism”, in which a large length increase of the DNA duplex is required both for association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that 4,4-B requires a large DNA length increase for association, but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association, but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared to conventional intercalation and will pave the way for rational design of therapeutic drugs based on DNA threading intercalation. INTRODUCTION Developing new anticancer therapies that target DNA requires molecules that display high binding affinity to DNA as well as slow dissociation kinetics from DNA.1-3 Anticancer drugs that target DNA have been shown to covalently cross link DNA,4 non-covalently bind to specific sequences or grooves,5-7 or bind non-covalently via intercalation and base stacking interactions.8 In light of this observation, several ruthenium based complexes have been synthesized that demonstrated effective mutagenic9 and anticancer properties,10, 11 and a few have even entered clinical trials.12-14 Polypyridyl ruthenium complexes with high binding affinities, synthesized in the 1980s, were shown to bind to DNA via intercalation.15 These metalorganic ruthenium complexes were later modified by covalently linking two ligands in order to further increase DNA binding affinity.16 This resulted in a group of new ruthenium based compounds (Examples shown in Figure 1A and 1B) that bind to DNA via a modified form of intercalation known as threading, which involves forcing a bulky ruthenium center through double-stranded DNA (dsDNA).17-19 Threading intercalators display even higher binding affinity, with dissociation constants in the nanomolar range, as well as particularly slow dissociation kinetics, with unbinding occurring on the order of hours to days.20, 21 It is the large positive charge, the strong stacking of aromatic group with the DNA bases, as well as the favorable interaction of the ligand ancillary groups with the intercalated duplex that lead to such high affinity. The strong DNA deformation required for both binding and unbinding of such threading intercalators leads to its slow dissociation.21 There is great potential for molecules that bind to DNA via threading to serve as anticancer drugs, with some current anticancer drugs such as nogalamycin having been identified as threading intercalators.22 Identifying the specific molecular characteristics that affect DNA threading intercalation is of critical importance as it allows for improved rational drug design.23 Polypyridyl ruthenium complexes are ideally suited for studying the impact of molecular characteristics on DNA threading intercalation due to the ability to create molecules that differ from each other by only one characteristic, such as chirality, steric

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D FRPSOH[ WLPHV PRUH IOH[LEOH than dsDNA, but an order of magnitude stiffer than the saturated 4,4-P/DNA complex.30 The stretch modulus of the 4,4-B '1$ FRPSOH[ ZDV PHDVXUHG WR EH ±65 pN, which is again significantly smaller than the stretch modulus for dsDNA and also smaller than the stretch modulus of the 4,4-P/DNA complex.21 Interestingly, the effective elastic properties of the 4,4-B/DNA complex are strikingly similar to the properties measured for the monomeric 4-P/DNA complex (Supporting Information Table S1).28 These properties are an indication of the level of deformation of the DNA duplex when bound by the ligand. Thus they provide insight into the effect that steric bulk has on threading-induced DNA deformation. The sterically smaller 4,4-B deforms the DNA far less than its parent molecule 4,4-P, and the deformation is closer to the monomeric 4-P, which intercalates DNA without threading. Crystal structures for 4,4-P, 4-B, and ,-P all indicate that one of the ruthenium centers sits in the DNA minor groove, which results in the distortion of the duplex.33-35 These effective elastic properties indicate that the smaller 4,4-B sits more compactly in the minor groove, resulting in less duplex deformation when bound compared to 4,4-P. Characterization of the binding kinetics was performed using the total relaxation rate, ktot (Supporting Information Equation 1). Figure 2A represents sample data obtained at 29.8 pN and increasing concentrations of 4,4-B. At each force the relaxation rate was plotted as a function of concentration, and fit to a linear function (Figure 2C). Each linear fit returned the force-dependent association rate ka, and the off rate koff (Supporting Information (TXDWLRQ ). The association rates and off rates were then plotted as a function of force (open circles, Figure 2D, and 2E, respectively) and also used to determine the force-dependent dissociation constant Kd (Supporting Information Equation 5). The dissociation constants were also plotted as a function of force (open circles, Figure 2F). All three binding parameters (ka, koff, and Kd) were fit to an exponential force dependence (broken lines, Figure 2D, 2E, and 2F) in order to extrapolate to the zero force binding limit (Supporting Information Equations 4 and 6). Data points at 10 pN and 40 pN in Fig. 2D-F were also obtained by measuring koff directly (Supporting Information Figure S1).

B

0.45 0.43

0.08

70

0.07

60

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50

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0.41

Force (pN)

Extension (nm/bp)

C

80

0.39 0.37

ktot (s-1)

A

40 30

P = 19.2 ± 10.5 nm S = 354 ± 65 pN

10 0.33

0.04 0.03 0.02

20 0.35

0.01 0

0 0

100

200

300

0.3

Time (s)

0.4

0.5

0

0.6

Kd (nM)

koff (s-1)

M-1s-1

1.E+06

0.01

10

Kd(0) = 65 ± 5 nM 4xeq = 0.29 ± 0.02 nm

koff(0) = (8.0 ± 1.0) x 10-3 s-1 xoff = -0.03 ± 0.01 nm 1

0.001

1.E+05 0

20

40

Force (pN)

30

100

0.1 ka(0) = (1.28 ± 0.13) x xon = 0.25 ± 0.01 nm

20

F

E 1.E+07 105

10

Concentration (nM)

Extension (nm/bp)

D

ka (M-1s-1)

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60

0

20

40

Force (pN)

60

0

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Force (pN)

Figure 2. Constant force measurements and analysis. (A) '1$ H[WHQVLRQV RSHQ FLUFOHV LQ WKH SUHVHQFH RI YLROHW EOXH JUHHQ \HOORZ RUDQJH DQG UHG Q0 ûû-% DV D IXQFWLRQ RI WLPH DW D FRQVWDQW “ S1 IRUFH DQG VLQJOH H[SRQHQWLDO fits (solid lines). (B) Extension of DNA whLOH UDSLGO\ VWUHWFKLQJ XS WR “ S1 DQG KROGLQJ DW FRQVWDQW IRUFH LQ WKH SUHVHQFH RI VDWXUDWHG FRQFHQWUDWLRQ RI ûû-B (solid red line). Saturated extensions obtained at various forces (dark red circles) are fit to the Worm Like Chain polymer model (broken lilac line) to obtain the effective elastic properties of the saturated DNA-Ligand complex (parameters as shown). (C) Net relaxation rate ktot obtained from the exponential fits in A (open circles) as a function of concentration with linear fit (brokeQ OLQH DW “ S1 SLQN “ S1 JUHHQ DQG “ S1 EOXH IRUFHV (D) Association rates ka obtained from the linear fits in C (circles) exhibiting exponential facilitation by force (broken line), yielding the structural change and zero force association rate (parameters as shown). (E) Off rates koff obtained from the linear fits in 2C (circles) fitted to an exponential dependence on force (broken line) yielding the structural changes required to unbind the drug (parameters as shown) and shows no facilitation by force. (F) Dissociation constants Kd estimated from the data in 2D and 2E (circles) exhibits exponential dependence on force (broken line) and yields the equilibrium binding parameters (parameters as shown). Triangle data points in D, E, and F were obtained using an alternative kinetics method, directly measuring koff, described in Supporting Information.

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DISCUSSION The returned zero force association rate obtained from the combination of kinetics analysis shown above (Figure 2) and equilibrium extension analysis discussed in the Supporting Information, ka(0) (1.21±0.12) x 105 M-1s-1, is approximately one order of magnitude larger than the value reported for 4,4-P.30 The zero force off rate koff(0) for 4,4-B was measured to be ( .1 ± 0.6) x 10- s-1 which is approximately 4 times the value reported for 4,4-P.30 These values are in agreement with the full association and dissociation times of the two ligands reported in the bulk studies.20, 36 The variation in kinetics indicates the modulating effect steric bulk has on the rate of threading into dsDNA. Interestingly, although higher affinity threading ligands have been reported,37 the zero force dissociation constants for these ligands are very similar (65±5 nM for 4,4-B, and 44±2 nM for 4,4-P). The binding affinity shows little dependence on the steric bulk of the threading subunit, which suggests that the steric bulk only affects the kinetics of binding. These measurements also reveal the DNA deformation associated with the DNA binding of 4,4-B. The exponential force dependences return values for the DNA elongation required for the association of one ligand (xon), and the elongation required for the dissociation of one ligand (xoff). From these two values, the equilibrium elongation of the DNA can be defined as 4Æeq A Æon - Æoff. For 4,4-B, the values obtained combining the kinetics analysis shown in Figure 2 and the equilibrium analysis shown in Supporting Infor28±0.02 nm. This calculated value for 4Æeq agrees within mation yield xon = 0.26±0.01 nm, and xoff -0.02±0.01 nm which gives 4xeq error with the value returned from fitting Kd as a function of force from the kinetics analysis (Fig. 2) as well as that obtained from the equilibrium analysis (Supporting Information). Physically this means that the DNA requires no additional elongation in order to allow the ligand to unthread. These elongation values differ significantly from those for 4,4-P, where û[eq “ QP ZLWK xon = “ QP and xoff “ QP 30 4U4-P not only requires larger DNA elongations to thread, it also requires significant elongation to unthread from the duplex. These results confirm the conclusion that the sterically smaller 4,4-B deforms the DNA significantly less than the larger 4,4-P. To better visualize the differences in binding between the two ligands, the free energy landscape was approximated for both ligands )LJXUH 7KH VPDOOHU '1$ HORQJDWLRQ UHTXLUHG IRU 4,4-B to bind results in a smaller free energy barrier for binding and thus a faster association kinetics, while the extra elongation for unthreading required by 4,4-P results in a molecular lock mechanism. That is, the length change needed to unthread from the DNA serves to lock the bound 4,4-P lig~1.3 kBT ands into the DNA, decreasing the dissociation rate. For 4,4-B there is no step requiring elongation that limits the DNA unthreading rate, which accounts for the faster dissociation kinetics from DNA when compared to 4,4-P. However, these properties (xon = 0.26±0.01 nm, xoff -0.02±0.01 nm for 4,4-B) contrast strongly with conventional interca44-B “ QP DQG xoff -0.28±0.04 lators such as YO, for which xon = 44-P nm.38 This suggests that for association by 4,4-B (and also for 4,4-P), 4G0 the rate-limiting step requires a significant DNA length change, and this is not the case for YO. This result is consistent with the idea that basepair opening is the rate-limiting step for threading intercalation, while DNA base-pair unstacking, though required, is not the rate-limiting step for conventional intercalation. 0.34 nm 0.53 nm 0.60 nm 0.67 nm In this work, the effects of steric bulk on the kinetics and mechanism of DNA Deformation (nm) DNA threading intercalation were isolated from the equilibrium DNA binding interactions using single molecule force spectroscopy. The Figure 3. Comparison of the free energy landscape of ûû-B DNA-ligand binding kinetics measurements revealed the significantly binding to DNA (Blue) to the free energy landscape of its faster binding and unbinding of the smaller molecule, while also showparent molecule ûû-P (Violet). Solid line represents the ening that the two molecules have similar binding affinity. These measergy change while lengthening towards the insertion of ligurements provided complementary determination of the less extreme and and the broken blue line represents relaxing back to the DNA deformations needed for a sterically smaller molecule (4,4-B) to equilibrium lengthening indicating that ûû-B shows no lock bind to and dissociate from the DNA relative to 4,4-P. Thus the steric mechanism unlike the parent molecule. bulk of the threading moiety is responsible for modulating binding kinetics, as well as affecting the deformation of the duplex needed for this important intercalation mechanism.

Free Energy (kBT)

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ASSOCIATED CONTENT Supporting Information Experimental details: materials and methods, and table (PDF)

AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected]

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Notes The authors declare no competing financial interests.

FUNDING This work was funded by NSF grant MCBWR 0&: AGC was funded by a Northeastern University Lawrence Research Fellowship, a Physics Research Co-op Award, and an Altshuler Research Fellowship.

REFERENCES [1] Muller, W., and Crothers, D. M. (1968) Studies of the binding of actinomycin and related compounds to DNA, J Mol Biol 35, 251-290. [2] Hurley, L. H. (2002) DNA and its associated processes as targets for cancer therapy, Nat Rev Cancer 2, 188-200. > @ &KDXUDVL\D K. R., Paramanathan, T., McCauley, M. J., and Williams, M. C. (2010) Biophysical characterization of DNA binding from single molecule force measurements, Phys Life Rev 7, 299[4] Johnson, N. P., Mazard, A. M., Escalier, J., and Macquet, J. P. (1985) Mechanism of the Reaction between Cis->3WFO 1K @ DQG '1$ Invitro, J Am Chem Soc 107 [5] Kopka, M. L., Yoon, C., Goodsell, D., Pjura, P., and Dickerson, R. E. (1985) Binding of an antitumor drug to DNA, Netropsin and CG-C-G-A-A-T-T-BrC-G-C-G, J Mol Biol 183 [6] Goodsell, D., and Dickerson, R. E. (1986) Isohelical Analysis of DNA Groove-Binding Drugs, J. Med. Chem. 29 > @ $OGHUGHQ 5 $ 0HOORU + 5 0RGRN 6 +DPEOH\ 7 : DQG &DOODJKDQ 5 &\WRWR[LF HIILFDF\ RI DQ DQWKUDTXLQRQe linked platinum anticancer drug, Biochem Pharmacol 71 -1145. [8] Paramanathan, T., Vladescu, I., McCauley, M. J., Rouzina, I., and Williams, M. C. (2012) Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D, Nucleic Acids Res 40, 4925[9] Yasbin, R. E., Matthews, C. R., and Clarke, M. J. (1980) Mutagenic and toxic effects of ruthenium, Chem Biol Interact 31 > @ -LDQJ * % ;LH < < /LQ * - +XDQJ + / /LDQJ = + DQG /LX < 6\QWKHVLV FKDUDFWHUL]DWLRQ '1$ interaction, antioxidant and anticancer activity studies of ruthenium(II) polypyridyl complexes, J Photochem Photobiol B 129, 48-56. [11] Clarke, M. J., Zhu, F., and Frasca, D. R. (1999) Non-platinum chemotherapeutic metallopharmaceuticals, Chem Rev 99, 2511[12] Allardyce, C. S., and Dyson, P. J. (2001) Ruthenium in Medicine: Current Clinical Uses and Future Prospects, Platinum Metals Rev. 45, 62-69. > @ 5DGHPDNHU-Lakhai, J. M., van den Bongard, D., Pluim, D., Beijnen, J. H., and Schellens, J. H. (2004) A Phase I and pharmacological study with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a novel ruthenium anticancer agent, Clin Cancer Res 10, [14] Bergamo, A., Masi, A., Jakupec, M. A., Keppler, B. K., and Sava, G. (2009) Inhibitory Effects of the Ruthenium Complex KP1019 in Models of Mammary Cancer Cell Migration and Invasion, Met Based Drugs 2009 [15] Barton, J. K., Danishefsky, A. T., and Goldberg, J. M. (1984) Tris(Phenanthroline)Ruthenium(Ii) - Stereoselectivity in Binding to DNA, J Am Chem Soc 106 [16] Lincoln, P., and Norden, B. (1996) Binuclear ruthenium(II) phenanthroline compounds with extreme binding affinity for DNA, Chem. Comm., 2145-2146. > @ 2QIHOW % /LQFROQ 3 DQG 1RUGHQ % (QDQWLRVHOHFWLYH '1$ WKUHDGLQJ G\QDPLFV E\ SKHQD]LQH-linked, J Am Chem Soc 123, [18] Wilhelmsson, L. M., Westerlund, F., Lincoln, P., and Norden, B. (2002) DNA-binding of semirigid binuclear ruthenium complex delta,delta-[mu-(11,11'-bidppz)(phen)(4)ru(2)](4+): extremely slow intercalation kinetics, J Am Chem Soc 124, 12092[19] WeVWHUOXQG ) (QJ 0 3 :LQWHUV 0 8 DQG /LQFROQ 3 %LQGLQJ JHRPHWU\ DQG SKRWRSK\VLFDO SURSHUWLHV RI '1$-threading binuclear ruthenium complexes, J Phys Chem B 111 > @ :HVWHUOXQG ) 1RUGHOO 3 1RUGHQ % DQG /LQFROQ 3 .LQHWLF FKDUDFWHUL]DWLRQ RI DQ H[WUHPHO\ VORZ '1$ ELQGLQg equilibrium, J Phys Chem B 111 [21] Almaqwashi, A. A., Paramanathan, T., Rouzina, I., and Williams, M. C. (2016) Mechanisms of small molecule-DNA interactions probed by single-molecule force spectroscopy, Nucleic Acids Res 44 > @ $URUD 6 . 0ROHFXODU-Structure, Absolute Stereochemistry, and Interactions of Nogalamycin, a DNA-Binding Anthracycline Anti-Tumor Antibiotic, J Am Chem Soc 105 > @ %DKLUD 0 0F&DXOH\ 0 - $OPDTZDVKL $ $ /LQFROQ 3 :HVWHUOXQG ) 5RX]LQD , DQG :LOOLDPV 0 & $ Uuthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps, Nucleic Acids Res 43, 8856[24] Westerlund, F., Nordell, P., Blechinger, J., Santos, T. M., Norden, B., and Lincoln, P. (2008) Complex DNA binding kinetics resolved by combined circular dichroism and luminescence analysis, J Phys Chem B 112, 6688-6694. > @ $QGHUVVRQ - )RUQDQGHU / + $EUDKDPVVRQ 0 7XLWH ( 1RUGHOO 3 DQG /LQFROQ 3 /LIHWLPH KHWHURJHQHLW\ of DNAbound dppz complexes originates from distinct intercalation geometries determined by complex-complex interactions, Inorg Chem 52, 1151-1159. [26] Andersson, J., Li, M., and Lincoln, P. (2010) AT-specific DNA binding of binuclear ruthenium complexes at the border of threading intercalation, Chemistry 16 -11046. > @ $OPDTZDVKL $ $ $QGHUVVRQ - , Lincoln, P., Rouzina, I., Westerlund, F., and Williams, M. C. (2016) Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation, Biophys J 110, 1255[28] Vladescu, I. D., McCauley, M. J., Nunez, M. E., Rouzina, I., and Williams, 0 & 4XDQWLI\LQJ IRUFH-dependent and zero-force DNA intercalation by single-molecule stretching, Nat Methods 4 -522.

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[29] Mihailovic, A., Vladescu, I., McCauley, M., Ly, E., Williams, M. C., Spain, E. M., and Nunez, M. E. (2006) Exploring the interaction of ruthenium(II) polypyridyl complexes with DNA using single-molecule techniques, Langmuir 22, 4699> @ $OPDTZDVKL $ $ 3DUDPDQDWKDQ 7 /LQFROQ 3 5RX]LQD , :HVWHUOXQG ) DQG :LOOLDPV 0 & 6WURQJ '1$ GHIormation required for extremely slow DNA threading intercalation by a binuclear ruthenium complex, Nucleic Acids Res 42 11641. > @ :LOKHOPVVRQ / 0 (VEMRUQHU ( . :HVWHUOXQG ) 1RUGHQ % DQG /LQFROQ 3 0HVR VWHUHRLVRPHU DV D SUREH RI enantioselective threading intercalation of semirigid ruthenium complex [mu-(11,11'-bidppz)(phen)(4)Ru-2](4+), J. Phys. Chem B 107 > @ 3DUDPDQDWKDQ 7 :HVWHUOXQG ) 0F&DXOH\ 0 - 5RX]LQD , /LQFROQ 3 DQG :LOOLDPV 0 & 0HFKDQLFDOO\ PDQipulating the DNA threading intercalation rate, J Am Chem Soc 130 > @ %RHU ' 5 :X / 6 /LQFROn, P., and Coll, M. (2014) Thread Insertion of a Bis(dipyridophenazine) Diruthenium Complex into the DNA Double Helix by the Extrusion of AT Base Pairs and Cross-Linking of DNA Duplexes, Angewandte Chemie-International Edition 53, 1949-1952. > @ 1L\D]L +., Hall, J. P., O'Sullivan, K., Winter, G., Sorensen, T., Kelly, J. M., and Cardin, C. J. (2012) Crystal structures of Lambda[Ru(phen)(2)dppz](2+) with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes, Nature Chem. 4, 621-628. > @ 6RQJ + .DLVHU - 7 DQG %DUWRQ - . &U\VWDO VWUXFWXUH RI 'HOWD-[Ru(bpy)(2)dppz](2+) bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation, Nature Chem. 4, 615-620. > @ 1RUGHOO 3 :HVWHUOXQG ) :LOKHOPVVRQ / 0 1RUGHQ % DQG /LQFROQ 3 .LQHWLF UHFRJQLWLRQ RI $7-rich DNA by ruthenium complexes, Angew Chem Int Ed Engl 46 -2206. > @ $OPDTZDVKL $ $ $QGHUVVRQ - /LQFROQ 3 5RX]LQD , :HVWHUOXQG ) DQG :LOOLDPV 0 & '1$ LQWercalation optimized by two-step molecular lock mechanism, Sci Rep 6 > @ %LHEULFKHU $ 6 +HOOHU , 5RLMPDQV 5 ) +RHNVWUD 7 3 3HWHUPDQ ( - DQG :XLWH * 7KH LPSDFW RI DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics, Nat Commun 6

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