DNA Strand Replacement Mechanism in Molecular Beacons Encoded

Article pubs.acs.org/JPCB

DNA Strand Replacement Mechanism in Molecular Beacons Encoded for the Detection of Cancer Biomarkers Magdalena Stobiecka*,† and Agata Chalupa‡ †

Department of Biophysics, Warsaw University of Life Sciences (SGGW), 02776 Warsaw, Poland Institute of Nanoparticle Nanocarriers, 11010 Barczewo, Poland

‡

S Supporting Information *

ABSTRACT: Signaling properties of a fluorescent hairpin oligonucleotide molecular beacon (MB) encoded to recognize protein survivin (Sur) mRNA have been investigated. The process of complementary target binding to SurMB with 20mer loop sequence is spontaneous, as expected, and characterized by a high affinity constant (K = 2.51 × 1016 M−1). However, the slow kinetics at room temperature makes it highly irreversible. To understand the intricacies of target binding to MB, a detailed kinetic study has been performed to determine the rate constants and activation energy Ea for the reaction at physiological temperature (37 °C). Special attention has been paid to assess the value of Ea in view of reports of negative activation enthalpy for some nucleic acid reactions that would make the target binding even slower at increasing temperatures in a non-Arrhenius process. The targetbinding rate constant determined is k = 3.99 × 103 M−1 s−1 at 37 °C with Ea = 28.7 ± 2.3 kcal/mol (120.2 ± 9.6 kJ/mol) for the temperature range of 23 to 55 °C. The positive high value of Ea is consistent with a kinetically controlled classical Arrhenius process. We hypothesize that the likely contribution to the activation energy barrier comes from the SurMB stem melting (tm = 53.7 ± 0.2 °C), which is a necessary step in the completion of target strand hybridization with the SurMB loop. A low limit of detection (LOD = 2 nM) for target tDNA has been achieved. Small effects of conformational polymorphs of SurMB have been observed on melting curves. Although these polymorphs could potentially cause a negative Ea, their effect on kinetic transients for target binding is negligible. No toehold preceding steps in the mechanism of target binding were identified.

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INTRODUCTION The reactions of nucleic acids have been widely utilized in biomimetic recognition,1−5 DNA nanotechnology,6−10 and medical intervention in various diseases.11−15 However, the mechanisms of some of the simplest of these reactions, including hybridization, melting, and strand displacement, remain elusive.16−18 Some insights into the challenges in elucidating these mechanisms can be gained by looking at the various theories developed to explain the observed phenomena involving, for instance, toehold-mediated strand displacement,18,19 mismatched strand sliding,20,21 zippering,16 fraying,18 plectoneme tip bubbles and writhing, 17 and so forth. Furthermore, there are reports that the DNA strand association rate constants are either increasing22−24 or decreasing16,19,25,26 with temperature, with the latter calling into question the validity of the Arrhenius equation for biological systems. In this article, we provide the results of kinetic investigations of complementary strand binding to a hairpin oligonucleotide beacon and analyze both the thermodynamic and kinetic parameters of the processes involved, with the aim of better understanding these processes taking place at physiological temperature, in view of relaxations studied over a wider temperature range. © 2016 American Chemical Society

The molecular beacon probe investigated in this work has been designed to recognize protein survivin (Sur) mRNA. Sur is strongly expressed in cancer cells, boosting their proliferation and providing protection against anticancer drugs.13,27−31 There has been extensive literature published in the past decade on Sur, and we refer the reader to some of the excellent reviews on Sur relations to tumorigenesis, metastasis, drug resistance ,and cancer therapy.32−39 Survivin (encoded by BIRC5 gene40−42) is a small protein (IAP4:16 389 Da, 142 amino acids) of the group of apoptosisinhibiting proteins (IAP).43,44 The oligonucleotide molecular beacons encoded for Sur mRNA have recently been proposed for the indirect detection of Sur expression.45−53 This method is very fast and simple. However, the effectiveness of the clinical tumor screening using MBs, so far achieved, has been rather low, ranging from 21.1%54 to 61.3%55 for low-grade and highgrade tumors, respectively. Hence, the assays for Sur mRNA require further improvements in specificity and sensitivity, especially for low-grade cancer detection. These improvements Received: April 5, 2016 Revised: May 16, 2016 Published: May 17, 2016 4782

DOI: 10.1021/acs.jpcb.6b03475 J. Phys. Chem. B 2016, 120, 4782−4790

Article

The Journal of Physical Chemistry B

complete, increasing the chances of digestion of MB−target duplexes by endogenous nucleases. To protect MBs from this fate, PNA or locked DNA67,68 has been employed in constructing the MBs. Although it serves well for long-term monitoring, it further increases the MB melting temperature (even to over 95 °C67) and results in an even slower target binding rate. In this work, we have investigated a survivin mRNA-targeting molecular beacon with hairpin structure and a long 20-nt recognition sequence corresponding to the antisense sequence optimized for maximum efficiency in BIRC5 gene silencing.33 This sequence is longer than other sequences used in SurMBs reported in the literature (12−18 nt), and we have selected it for its high specificity. The longer recognition loop and longer complementary target sequence decrease the target-induced apparent stem melting temperature. However, with the increased length of the sequence, there is an increased probability of self-complementarity in the recognition sequence leading to the possibility of a more complex loop structure of the MB, including a multiloop formation, as illustrated in Figure 1B. Furthermore, the presence of hairpin intermediates has been implicated in the non-Arrhenius kinetics in strand displacement reactions,16 so they may influence the kinetics of target binding to MB.66 In this work, we have investigated these effects to evaluate the kinetic implications of isothermal Sur mRNA monitoring with MB.

will require a better understanding of the physicochemical nature of the processes involved in MB conformational transitions induced by target binding as well as the biochemical MB survivability in living environments. Molecular beacons were originally designed for the detection of genes1,2,4,56−60 and are composed of short oligonucleotide chains with a hairpin structure. Each MB consists of a selfhybridized stem and a single-stranded loop, as depicted in Figure 1A. The short stem, consisting of five to seven base pairs

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MATERIALS AND METHODS Chemicals. The following 30-mer SurMB and 20-mer target tDNA sequences have been used in experiments: SurMB: 5′-6-FAM-CCT GGC CCA GCC TTC CAG CTC CTT GCC AGG-Dabcyl-3′, tDNA: 5′-CAA GGA GCT GGA AGG CTG GG-3′ The underlining indicates the stem area bases, and boldface marks the antisense sequence. 6-FAM is fluorescent dye 6carboxyfluorescein, and Dabcyl is dark quencher 4,4-dimethylamino-azobenzene-4′-carboxylic acid. They were synthesized by the Laboratory of DNA Sequencing and Oligonucleotides Synthesis, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (IBB PAS, Warsaw, Poland). These oligonucleotides were purified and tested by HPLC. (For further information on reagents, see the Supporting Information (SI)). Instrumentation. The fluorescence spectra were recorded using spectrometer model LS55 (PerkinElmer, Waltham, MA, USA), with a 20 kW xenon light source and a photomultiplier tube detector. During the MB melting experiments, the temperature was scanned stepwise with a step height of 2°, and measurements were performed after 1 min of waiting at each temperature. (For further details concerning instrumentation, see the SI.) Calculations. Thermodynamic data for DNA structures were calculated using the UNAFold program developed on the basis of unified sequence-dependent base-pair stacking theory.69−74 The program was parametrized for hairpin and duplex DNA structures. The nonlinear curve fitting was performed using a standard two-state Boltzmann function or functions derived for the kinetic model developed in this work. The fitting was carried out using the simplex routine embedded in the Origin graphing software (OriginLab Corp., Northampton, MA, USA).

Figure 1. (A) Basic turn-on fluorescent molecular beacon for survivin mRNA recognition. (B) Conformational polymorphic structures 1−3 of SurMB at 25 °C and duplex with target tDNA 4 with their theoretical melting temperatures calculated using the UNAFold program. Fluorescent dye, FAM; quencher, Dabcyl; loop, 20-mer antisense oligonucleotide sequence targeting the BIRC5 gene of the survivin mRNA.

(bp), dehybridizes when a target oligonucleotide interacts with the longer sequence of the loop. The fluorophore and quencher groups attached to the ends of the beacon strand enable monitoring of the stem hybridization state. When the stem is hybridized, fluorescence is strongly quenched because of the Förster resonance energy transfer (FRET)2,61−64 from the fluorophore to the quencher being in close proximity. On the other hand, in the open MB conformation, with the dehybridized stem and the target bound to the MB loop (Figure 1A), the fluorescence emission is fully restored. The target binding by MBs is a slow reaction65,66 and requires raising the temperature, typically to 45−52 °C,2 to induce the conversion of MB from the OFF to ON state. This disadvantage is due to the relatively high MB stem melting temperature which is usually above 55 °C. Therefore, most of the rapid analyses utilizing MBs have been performed with extracts, lysates, body fluids, and so forth outside of a living organism. To carry out MB analyses in vivo, a temperature of 37 °C has to be maintained. Under these conditions, the target binding by MBs may take several hours to 4783


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RESULTS AND DISCUSSION Conformational Polymorphism of SurMB. The molecular beacon (SurMB) used in this study was a single-stranded oligonucleotide with a 6-FAM fluorophore and a Dabcyl quencher attached to the 5′ and 3′ ends of the strand, respectively. The SurMB recognition sequence consisted of a 20-mer antisense sequence: 5′-CCCAGCCTTCCAGCTCCTTG-3′ designed for targeting the survivin mRNA (the BIRC5 gene). The stem sequence was 5′-CCTGG···CCAGG-3′ plus one additional base pair originating from the antisense sequence. Dominant structures 1−3 of the SurMB sequence, calculated using the UNAFold program based on unified basepair stacking theory,69−74 for 100 nM SurMB in 50 mM NaCl solution at 25 °C, are presented in Figure 1B. All three structures have a negative Gibbs free energy of formation from the random coil (Table 1) and the most thermodynamically Table 1. Thermodynamic Data for the Formation of Conformational Polymorphic Structures of SurMB Calculated for 100 nM SurMB + 50 mM NaCl Solution at 25 °Ca

a

MB

ΔG° kcal/mol

ΔH°kcal/mol

ΔS° cal/(mol K)

tm °C

SurMB 1 SurMB 2 SurMB 3 duplex 4

−3.75 −3.44 −4.26 −26.63

−65.00 −58.40 −44.50 −161.4

−205.43 −184.34 −134.97 −452.1

43.3 43.7 56.6 58.4

Calculated using the UNAFold program.

stable is structure 3, with ΔG° = −4.26 kcal/mol. Using the dependence ΔG° = −RT ln K, the values of the melting equilibrium constants at 25 °C for structures 1−3 have been calculated, and the equilibrium concentrations of these conformers are determined to be c1 = 25.26 nM, c2 = 14.97 nM, and c3 = 59.73 nM, with the free random-coil form concentration of crc = 0.04 nM (calculation details provided in SI). In Figure 1B, there is also included duplex 4, which forms in the interaction of tDNA target with SurMB. Recent mechanisms proposed for hairpin structure formation,18,19 derived from the coarse-grained DNA model simulations, are based on the initial base-pair interaction, followed by fast consecutive bond formation. It has been indicated that because at higher temperatures the probability of successful bond formation is lower, then the formation of two or more bonds may be necessary to initiate complete hybridization. It is unclear to what extent the conformational polymorphism described above can influence the kinetics of target binding by MBs. Interactions of Antisense SurMB with Complementary tDNA Strands. At room temperature, SurMB assumes a closed conformation (the OFF state) with a low fluorescence emission intensity of Fmax = 89.8 ± 5.2 due to the close proximity of the fluorophore to the quencher (Figure 2A, curve 1). Figure 2C shows the temporal evolution of SurMB fluorescence after the addition of tDNA at different temperatures. It is seen that at higher temperatures the fluorescence emission of the 6-FAM dye of the MB increases faster (Figure 2B). The time responses of SurMB fluorescence emission indicate that the ON conformation of SurMB, represented by spectrum 2 in Figure 2A, is not completely open because the degree of opening is controlled by the slow kinetics of bindingstimulated dissociation. Nonetheless, the spectra in Figure 2A

Figure 2. (A) Fluorescence spectra for a SurMB in (1) closed conformation (the OFF state) and (2) open conformation (the ON state) at 23 °C. (B) Temporal evolution of SurMB emission at different temperatures (°C): (1) 23, (2) 30, (3) 37, (4) 45, (5) 55. (C) Temporal evolution of SurMB emission in the presence of a complementary target oligonucleotide tDNA at different temperatures (°C): (1) 23, (2) 37, (3) 55. Conditions: CSurMB = 100 nM, CtDNA= 100 nM. Buffer: 10 mM MOPS + 50 mM NaNO3, pH 7.45. λex = 480 nm and λem = 516 nm.

show clearly the strong dependence of fluorescence emission on SurMB conformation and its high potential for the recognition of target oligonucleotide sequences. The dependence of the fluorescence emission of SurMB on the target tDNA concentration for temperature in the range of 23 to 55 °C is presented in Figure 3A. The widest dynamic 4784


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Figure 3. (A) Fluorescence emission spectra of SurMB recorded at 37 °C for increasing concentrations of target tDNA, CtDNA (nM): (1) 0, (2) 16.7, (3) 33, (4) 50, (5) 67, (6) 83, (7) 100. (B) Dependence of fluorescence intensity Fmax on CtDNA for different temperatures (°C): (1) 23, (2) 30, (3) 37, (4) 45, (5) 55. (C) Fluorescence spectra of SurMB at 37 °C for lower concentrations of tDNA, CtDNA (nM): (1) 0, (2) 3.3, (3) 6.7, (4) 10, (5) 13.3, (6) 16.7, (7) 20. (D) Calibration curve for the data in (C), showing the limit of detection for tDNA: LOD = 2 nM (S/N = 3σ). Conditions: incubation time after tDNA injection τhold = 60 s, CSurMB = 100 nM for data in (A) and (B), CSurMB = 20 nM for data in (C) and (D). Buffer: 10 mM MOPS + 50 mM NaNO3, pH 7.45; λex = 480 nm and λem = 516 nm.

range (from 16.7 to 100 nM) was obtained for a SurMB concentration of 100 nM at 55 °C (Figure 3B). A high sensitivity of SurMB was also achieved at 37 °C by lowering the SurMB concentration to 20 nM, with the limit of detection LOD = 2 nM (S/N = 3σ), as illustrated in Figure 3C,D. The residual fluorescence (∼75 au) creates a small background observed at CtDNA = 0. The complex behavior of SurMB, illustrated in Figures 2 and 3, requires further elucidation. It follows from the presented data that the SurMB response to the temperature change is much faster in the absence than in the presence of target tDNA, although the addition of tDNA should accelerate the conformational transition of MB. Also, after the injection of tDNA at 37 °C or at a lower temperature, the time needed for the SurMB fluorescence response to stabilize is very long (2 h or more). On the other hand, at 55 °C, a stable fluorescence emission is observed within 10 min of the injection of tDNA. These results have profound significance for Sur mRNA analysis. For the analysis carried out in sampled solutions (outside the cells or tissue), the temperature can be raised to 55 °C for quick and efficient analysis. However, for the analysis carried out in vitro or in vivo, the temperature should be maintained at 37 °C to avoid any temperature influence on Sur expression, mRNA generation, and other intracellular processes. Therefore, in the next sections, a detailed analysis of

SurMB melting behavior is presented and a kinetic model for the interaction of SurMB with tDNA discussed. SurMB Melting Characteristics. To characterize the fluorescence ON−OFF behavior of an MB, the temperature dependence of its fluorescence emission is investigated. These characteristics are related to the MB melting behavior and, for a reversible system, can be represented by a two-state Boltzmann distribution function. Here, we present the SurMB melting characteristic (Figure 4A) for the sake of comparison with the behavior of SurMB during tDNA target binding (Figure 4B,C). The experimental data were fitted with a two-state Boltzmann function F=

F1 − F2

( t −s t )

1 + exp

m

+ F2 (1)

where F1 and F2 are the fluorescence intensities for SurMB in closed and open conformations, respectively, t is the temperature (°C), tm is the midpoint melting temperature (°C), and s is the slope parameter (°C). The two-state Boltzmann function describes the experimental data very well, with the regression coefficient R2 = 0.9986 indicating that the influence of the conformational polymorphs on the SurMB melting process may be negligible. However, the value of tm,f for the forward scan, determined by the simplex fitting algorithm, is tm,f = 53.7 ± 0.2 °C (other parameters: F1 = 144.1 ± 15.8 °C, F2 = 2759.6 4785


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± 17.6 °C, s = 4.01 ± 0.09 °C). This means that the experimental apparent melting temperature tm,f is slightly lower than theoretically predicted for SurMB structure 3 (tm = 56.6 °C), which is likely to be due to the lower melting temperatures of structures 1 and 2 (43.3 and 43.7 °C, respectively), shifting the lower portion of the fitting curve to produce a slightly lower net melting temperature. The backward temperature scan shows a little hysteresis, with the melting characteristic shifted by −3.9 °C (as measured at the inflection point), but it is evident that SurMB undergoes complete back hybridization of the stem to the OFF state on returning to room temperature. Small deviations of the experimental points from the two-state function in the temperature range from 25 to 45 °C confirms the melting of less-stable conformers 1 and 2. Interestingly, the analysis of the initial slope of the melting transients for SurMB alone indicates that the melting rate increases with temperature. Target Binding-Induced Dissociation of SurMB Stem. In the presence of a target, the temperature dependence of SurMB fluorescence changes dramatically, as illustrated in Figure 4B. At the starting point of the temperature scan, at 23 °C, immediately after mixing SurMB with tDNA, the fluorescence intensity is low (F = 132), indicating that the hybridization of the target with the MB loop has not commenced because of the slow dissociation of the MB stem at low temperature. When the temperature increases to beyond ca. 40 °C, the stem dissociation, accelerated by tDNA hybridization with the MB loop, becomes evident. This melting process synchronized with tDNA binding is completed when the temperature reaches t = tmax, where tmax corresponds to the fluorescence maximum (Fmax = 3249). At a temperature scan rate of 2 deg/min, tmax = 59.7 ± 0.3 °C. Interestingly, upon further temperature increase, the fluorescence does not remain constant but decreases slightly to the level of F = 2927. This fluorescence decrease can be due only to the self-quenching of dehybridized SurMB strands that assume dynamically random conformations, with some of them a exhibiting FAM-Dabcyl distance that is lower than that for a straightened oligonucleotide chain in a duplex with tDNA. Hence, we can ascribe the temperature range of 60−81 °C to the dissociation of the SurMB-tDNA complex, represented by two-state Boltzmann line 5 in Figure 4B. The melting temperature is higher for the SurMB-tDNA complex than for the SurMB stem because of the longer duplex length of the complex. It is seen from Figure 4B that in the presence of the tDNA strand, complementary to the SurMB loop, stem melting occurs at a temperature lower than that for SurMB alone by 8.0 °C. This downward shift of tm,f is due to the targetstimulated stem dissociation process. The entire melting characteristics for the system SurMB − tDNA for a temperature scan from 23 to 81 °C can no longer be described by a twostate model because the system clearly passes through three states and some of the transitions are not reversible. The first transition is observed from SurMB in the OFF conformation at 23 °C (state A) with respect to the intermediate state of duplex SurMB-tDNA in the ON conformation at t = 59.7 °C (state M), and the second transition is from state M to the random open structure of molten SurMB above 70 °C in the ON conformation (state B). However, unlike the system of SurMB alone, which is highly reversible, the system SurMB − tDNA is not reversible, as will be shown in the next section. On the Reversibility of the SurMB Interaction with Target tDNA. In the presence of the tDNA complement to the SurMB loop, the temperature dependence of SurMB

Figure 4. (A) Dependence of the SurMB fluorescence on temperature for (1) the forward scan (filled squares) and (2) the backward scan (open circles), showing the system reversibility; the forward and backward scan half-melting points are tm,f = 53.7 ± 0.2 °C and tm,b = 49.8 ± 0.2 °C. Solid lines: two-state fitting function. (B) Comparison of the forward-scan temperature dependences of fluorescence emission for (1) SurMB alone (squares) and (2) SurMB + tDNA (triangles); Δtm = −8 °C. Theoretical lines 3−5 represent transfers: (3) from state A to B, (4) from state A to C, and (5) from state D to B (see the text). (C) Dependence of the SurMB fluorescence on temperature in the presence of complementary target tDNA for (1) the forward scan (filled squares) and (2) the backward scan (open circles), showing the system irreversibility; tm,f = 53.7 ± 0.2 °C. Theoretical lines 3−5 represent transfers: (3) from state A to B, (4) from state C to D, and (5) from state B to E (see the text). Conditions: CSurMB = 100 nM and CtDNA= 100 nM. Buffer: 10 mM MOPS + 50 mM NaNO3, pH 7.45. λex = 480 nm and λem = 516 nm. 4786


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The Journal of Physical Chemistry B fluorescence for the backward temperature scan (Figure 4C) shows a dramatic departure from SurMB behavior in the absence of tDNA. It is seen that the high fluorescence level achieved at the end of the forward scan is maintained during the backward scan. A more detailed inspection of curve 2 indicates that for the initial backward temperature scan from 81 °C to ca. 60 °C the fluorescence increases to the value of the fluorescence maximum (Fmax = 3283) close to that observed during the forward temperature scan, only the peak is slightly shifted toward lower temperatures (tmax = 59.0 °C), in comparison to that observed during the forward scan (tmax = 59.7 °C). For temperatures below ca. 50 °C, the fluorescence remains constant: F = 3030 ± 51. These temperature scan experiments indicate that the same states B and M are observed during the forward and backward scans. Therefore, the transitions between these states are reversible. However, the system does not return to state A during the backward scan. Therefore, state A, observed initially before temperature scanning, was a metastable state that existed only because of the very slow kinetics of the target binding at lower temperatures. State D corresponding to the pure, fully straightened SurMB-tDNA duplex is not achieved either, as evidenced by the experimental data that follow the transition path from state M to state E (theoretical line 5 rather than the transition from state M to D (theoretical line 4)). State E, with slightly lower fluorescence than the fluorescence maximum M, is likely to be due to the minor interbeacon interactions forming dimeric (SurMB-tDNA)2 complexes. In summary, the transitions among states B, M, and E appear to be reversible, whereas the transition from A to M is not. The Gibbs free energy ΔG°b for the target binding by SurMB at 25 °C can be calculated using the equation ΔG° b = ΔG° f,4 − ΔG° f,3

Deq =

λ=

D

kb

v=

⎛ Dt D ⎞ = exp{−Δk f τ }⎜⎜1 − λ t ⎟⎟ Deq Deq ⎠ ⎝

(B0 + T0 + K )2 − 4B0 T0

⎛ ∂D ⎞ ⎜ ⎟ = k(B − D)(T − D) 0 0 ⎝ ∂τ ⎠T

(10)

(11)

where c0 = B0 = T0. Hence, the following expression for fluorescence emission intensity as a function of SurMB-tDNA complex concentration has been assumed

(2)

F = F1 + F2 = εoff (c0 − D) + εonD

(12)

where the first term (F1) corresponds to the residual fluorescence from SurMB in the closed conformation and the second term (F2) corresponds to the fluorescence of the SurMB-tDNA duplex; εi is the fluorescence coefficient for species i:

⎛ ∂F ⎞ εi = ⎜ ⎟ ⎝ ∂ci ⎠

(13)

By combining eqs 11 and 12 and rearranging, one obtains

(3)

⎡ ⎤ 1 F = Fini + (Fmax − Fini)⎢1 − ⎥ (1 + c0kτ ) ⎦ ⎣ (4)

(14)

where Fini is the initial fluorescence and Fmax is the fluorescence of the pure SurMB-tDNA duplex at concentration c0. By fitting eq 14 to the kinetic data using simplex iterations (solid lines in Figure 5A), the rate constants k for different temperatures have been obtained. The values of k range from 7.30 × 102 (for 23 °C) to 7.64 × 104 M−1 s−1 (for 55 °C). The high values of the regression coefficient (R2 = 0.994−0.997) indicate that the kinetic model that is developed is well suited to describe the target-stimulated SurMB dissociation, and the reaction mechanism does not involve multiple steps. The Gibbs free energies and equilibrium constants for the formation of a tDNA-MB duplex and the forward and backward reaction rate constants for duplex dissociation in 100 nM SurMB + 50 mM

(5)

where Dt and Deq are the concentrations of the duplex at time τ and at equilibrium, respectively. Δ=

(9)

⎞ ⎛ 1 D = c 0 ⎜1 − ⎟ (1 + c0kτ ) ⎠ ⎝

has been solved by Tsourkas et al.75 In this equation, B, T, and D are the concentrations of MB, target tDNA, and duplex MB· tDNA, respectively; kf and kb are the forward and backward rate constants, and τ is the time. The solution to differential eq 4 is 1−

(8)

After integration, one obtains a simple function describing the progression of the target binding by SurMB

and kinetic equation dD = k f BT − k bD dτ

B0 T0

where Ft, Feq, and F0 are the fluorescence intensities at time τ, at equilibrium, and initially, respectively, the experimental function (Ft − F0)/(Feq − F0) can be fitted directly by eq 5. Because the separation of Δ and kf values from their product and further parameter extraction using eqs 6−8 is not straightforward, we have adopted an abbreviated solution to the kinetic system by analyzing only the initial part of the transients and neglecting the backward reaction. This is justified by the fact that K is large and thus kf ≫ kb. This leads to an abridged second-order kinetic equation of the form

MB + tDNA →MB·tDNA ←

Deq 2

Dt F − F0 = t Deq Feq − F0

kf

T

(7)

where B0, T0, and D0 are the initial concentration of reactants and K = kf/kb. Because

on the basis of the data for the free energy of formation for compounds 4 and 3 from Table 1. The obtained value of ΔG°b = −22.37 kcal/mol leads to the equilibrium binding constant: Kb = 2.51 × 1016 M−1. This high value indicates the high binding affinity of the target to SurMB, despite the slow kinetics. Interestingly, K decreases with temperature while the reaction rate increases. Kinetics of Target Binding by SurMB. The kinetics of a target binding to an MB with the general reaction B

1 (B0 + T0 + K − Δ) 2

(6) 4787


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Ea RT

{ }

k = k 0 exp −

(15)

in the form log k = log k 0 −

Ea 2.303RT

(16)

where R is the gas constant (R = 1.987 cal mol−1 K−1), T is the absolute temperature, and k0 is the experimental preexponential term. The value of Ea = 28.7 ± 2.3 kcal mol−1 (or 120.2 ± 9.6 kJ mol−1) is obtained. This value is consistent with kinetic control of the target-binding reaction in a classical Arrhenius-type process. In this investigation, NaCl was used in solutions because it is a major component of cytosol and body fluids. From this perspective, the reaction rates can be increased by increasing the concentration of NaCl (or KCl), which enhances the electrostatic screening of DNA strands and enables a closer approach of the target to an MB. The reaction rates can also be increased by introducing divalent metal ions, such as Mg2+, that are able to form interstrand bridges, thus neutralizing the electrostatic repulsions between ssDNA strands and adding points of attractive interactions. However, in the latter case, the reaction has a different mechanism (with bridge formation), and thus it should be considered separately.

NaCl solution at temperatures from 23 to 55 °C are presented in Table 2. Table 2. Thermodynamic and Kinetic Parameters for the Formation of a tDNA-MB Duplex in 100 nM SurMB + 50 mM NaCl Solution at Temperature in the Range from 23 to 55 °C ΔGo kcal/mola

K, M−1b

kf, M−1 s−1c

23 30 37 45 55

−23.00 −20.78 −18.56 −16.03 −12.86

× × × × ×

× × × × ×

9.50 9.66 1.21 1.02 3.66

16

10 1014 1013 1011 108

7.30 2.24 3.99 2.60 7.64

2

10 103 103 104 104

kb, s−1d 7.69 2.32 3.29 2.54 2.09

× × × × ×

CONCLUSIONS

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ASSOCIATED CONTENT

We have demonstrated that the target binding by antisense survivin MB in the temperature range of 23 to 55 °C is kinetically controlled with a positive activation energy of Ea = 28.7 ± 2.3 kcal/mol in a classical Arrhenius-type process. This finding is in contrast to some strand exchange processes for which a negative activation enthalpy has been observed, leading to the decrease in the rate constants with increasing temperature. In the case of SurMB studied here, the target binding rate constant kf has been found to increase with increasing temperature from 7.30 × 102 to 7.64 × 104 M−1 s−1 over the temperature range investigated. Backward rate constant kb also increased with increasing temperature, from 7.69 × 10−15 to 2.09 × 10−4 s−1. The rates of increases of kf and kb with temperature are different to accommodate the decrease in the affinity constant with temperature. Although the nature of the non-Arrhenius processes remains to be elucidated, this study shows clearly that nucleic acid processes can also conform to the classical Arrhenius kinetics. In conclusion, we can say that in the process of target binding to SurMB studied in this work, the fast preceding step or steps are either absent or produce too little energy to generate a negative activation enthalpy.

Figure 5. (A) Fitting of experimental kinetic transients (marks) with eq 7 (solid lines) to determine the rate constants of target binding for SurMB at different temperatures, from 23 to 55 °C, marked on the curves. (B) Arrhenius plot of log k versus 1/T with the LSQ fitting line for the determination of the activation energy Ea. Conditions: CSurMB = 100 nM, CtDNA = 100 nM. Buffer: 10 mM MOPS + 50 mM NaNO3, pH 7.45. λex = 480 nm and λem = 516 nm.

t, °C

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10−15 10−12 10−10 10−7 10−4

a

Gibbs free energy of tDNA-MB duplex formation. bEquilibrium constant. cForward reaction rate constant. dBackward reaction rate constant.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b03475. Additional information about chemicals, instrumentation, calculation of equilibrium concentrations of conformational polymorphs of SurMB, and kinetics of targetbinding (PDF)

The rate constants are plotted versus 1/T in a semilogarithmic Arrhenius plot in Figure 5B. The value of the activation energy Ea has been obtained by fitting the Arrhenius equation 4788


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the Presence of Copper(II) Ions. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2012, 735, 1−11. (16) Ouldridge, T. E.; Šulc, P.; Romano, F.; Doye, J. P. K.; Louis, A. A. DNA Hybridization Kinetics: Zippering, Internal Displacement and Sequence Dependence. Nucleic Acids Res. 2013, 41, 8886−8895. (17) Matek, C.; Ouldridge, T. E.; Doye, J. P. K.; Louis, A. A. Plectoneme Tip Bubbles: Coupled Denaturation and Writhing in Supercoiled DNA. Sci. Rep. 2015, 5, 7655. (18) Srinivas, N.; Ouldridge, T. E.; Šulc, P.; Schaeffer, J. M.; Yurke, B.; Louis, A. A.; Doye, J. P. K.; Winfree, E. On the Biophysics and Kinetics of Toehold-Mediated DNA Strand Displacement. Nucleic Acids Res. 2013, 41, 10641−10658. (19) Sulc, P.; Ouldridge, T. E.; Romano, F.; Doye, J. P. K.; Louis, A. A. Modelling Toehold-Mediated RNA Strand Displacement. Biophys. J. 2015, 108, 1238−1247. (20) Schmitt, T. J.; K, T. A., IV Thermodynamics of DNA Hybridization on Surfaces. J. Chem. Phys. 2011, 134, 205105. (21) Schmitt, T. J.; Rogers, C.; K, T. A., IV Exploring the Mechanisms of DNA Hybridization on a Surface. J. Chem. Phys. 2013, 138, 035102. (22) Morrison, L. E.; Stols, L. M. Sensitive Fluorescence-Based Thermodynamic and Kinetic Measurements of DNA Hybridization in Solution. Biochemistry 1993, 32, 3095−3104. (23) Porschke, D.; Uhlenbeck, O. C.; Martin, F. H. Thermodynamics and Kinetics of the Helix-Coil Transition of Oligomers Containing GC Base Pairs. Biopolymers 1973, 12, 1313−1335. (24) Sikorav, J. L.; Orland, H.; Braslau, A. Mechanism of Thermal Renaturation and Hybridization of Nucleic Acids: Kramers’ Process and Universality in Watson−Crick Base Pairing. J. Phys. Chem. B 2009, 113, 3715−3725. (25) Craig, M. E.; Crothers, D. M.; Doty, P. Relaxation Kinetics of Dimer Formation by Self Complementary Oligonucleotides. J. Mol. Biol. 1971, 62, 383−392. (26) Porschke, D.; Eigen, M. Co-Operative Non-Enzymatic Base Recognition. J. Mol. Biol. 1971, 62, 361−381. (27) Cao, W.; Fan, R.; Wang, L.; Cheng, S.; Li, H.; Jiang, J.; Geng, M.; Jin, Y.; Wu, Y. Expression and Regulatory Function of miRNA-34a in Targeting Survivin in Gastric Cancer Cells. Tumor Biol. 2013, 34, 963−971. (28) Aliabadi, H. M.; Mahdipoor, P.; Uludağ, H. Polymeric Delivery of siRNA for Dual Silencing of Mcl-1 and P-Glycoprotein and Apoptosis Induction in Drug-Resistant Breast Cancer Cells. Cancer Gene Ther. 2013, 20, 169−177. (29) Olsen, A. L.; Davies, J. M.; Medley, L.; Breen, D.; Talbot, D. C.; McHugh, P. J. Quantitative Analysis of Survivin Protein Expression and its Therapeutic Depletion by an Antisense Oligonucleotide in Human Lung Tumors. Mol. Ther.–Nucleic Acids 2012, 1, e30. (30) Kappler, M.; Kotzsch, M.; Bartel, F.; Fussel, S.; Lautenschlager, C.; Schmidt, U.; Wurl, P.; Bache, M.; Schmidt, H.; Taubert, H.; Meye, A. Elevated Expression Level of Survivin Protein in Soft-Tissue Sarcomas Is a Strong Independent Predictor of Survival1. Clin. Cancer Res. 2003, 9, 1098−1104. (31) Konopka, K.; Spain, C.; Yen, A.; Overlid, N.; Gebremedhin, S.; Duzgunes, N. Correlation Between the Level of Survivin and Survivin Promoter-Driven Gene Expression in Cancer and Non-Cancer Cells. Cell. Mol. Biol. Lett. 2009, 14, 70−89. (32) Huang, J.; Lyu, H.; Wang, J.; Liu, B. MicroRNA Regulation and Therapeutic Targeting of Survivin in Cancer. Am. J. Cancer Res. 2015, 5, 20−31. (33) Olie, R. A.; Simoes-Wust, A. P.; Baumann, B.; Leech, S. H.; Fabbro, D.; Stahel, R. A.; Zangemeister-Wittke, U. A Novel Antisense Oligonucleotide Targeting Survivin Expression Induces Apoptosis and Sensitizes Lung Cancer Cells to Chemotherapy1. Cancer Res. 2000, 60, 2805−2809. (34) Arora, R.; Shuda, M.; Guastafierro, A.; Feng, H.; Toptan, T.; Tolstov, Y.; Normolle, D.; Vollmer, L. L.; Vogt, A.; Dömling, A.; Brodsky, J. L.; Chang, Y.; Moore, P. S. Survivin is a Therapeutic Target in Merkel Cell Carcinoma. Sci. Transl. Med. 2012, 4, 133ra156.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +48.22.593.8621. Fax: +48.22.593.8619. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by funding provided by Program SONATA of the National Science Center, grant no. DEC2012/05/D/ST4/00320, and partially by Grant Iuventus Plus, no. IP2012058072, of the Ministry of Science and Higher Education.

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ABBREVIATIONS SurMB, survivin molecular beacon; Ea, activation energy; tDNA, target DNA; bp, base pairs; K, equilibrium constant; kf, forward reaction rate constant; kb, backward reaction rate constant; tm, melting temperature

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REFERENCES

(1) Stobiecka, M.; Chałupa, A. Biosensors Based on Molecular Beacons. Chem. Pap. 2015, 69, 62−76. (2) Stobiecka, M.; Molinero, A. A.; Chalupa, A.; Hepel, M. Mercury/ Homocysteine Ligation-Induced ON/OFF-Switching of a T-T Mismatch-Based Oligonucleotide Molecular Beacon. Anal. Chem. 2012, 84, 4970−4978. (3) Han, H.; Valle, V.; Maye, M. M. Probing Resonance Energy Transfer and Inner Filter Effects in Qdot − Large Metal Nanoparticle Clusters Using a DNA-Mediated Quench and Release Mechanism. J. Phys. Chem. C 2012, 116, 22996−23003. (4) Tyagi, S.; Kramer, F. R. Molecular Beacons: Probes that Fluoresce Upon Hybridization. Nat. Biotechnol. 1996, 14, 303−308. (5) Grossmann, T. N.; Roglin, L.; Seitz, O. Triplex Molecular Beacons as Modular Probes for DNA Detection. Angew. Chem., Int. Ed. 2007, 46, 5223−5225. (6) Pinheiro, A. V.; Han, D.; Shih, W. M.; Yan, H. Challenges and Opportunities for Structural DNA Nanotechnology. Nat. Nanotechnol. 2011, 6, 763−772. (7) Ouldridge, T. E.; Louis, A. A.; Doye, J. P. K. DNA Nanotweezers Studied with a Coarse-Grained Model of DNA. Phys. Rev. Lett. 2010, 104, 178101. (8) Šulc, P.; Ouldridge, T. E.; Romano, F.; Doye, J. P. K.; Louis, A. A. Simulating a Burnt-Bridges DNA Motor with a Coarse-Grained DNA Model. Nat. Comput. 2014, 13, 535−547. (9) Ouldridge, T. E.; Johnston, I. G.; Louis, A. A.; Doye, J. P. K. The Self-Assembly of DNA Holliday Junctions Studied with a Minimal Model. J. Chem. Phys. 2009, 130, 065101. (10) Ouldridge, T. E.; Hoare, R. L.; Louis, A. A.; Doye, J. P. K.; Bath, J.; Turberfield, A. J. Optimizing DNA Nanotechnology Through Coarse-Grained Modeling: A Two-Footed DNA Walker. ACS Nano 2013, 7, 2479−2490. (11) High, K. A. Gene Therapy-the Moving Finger. Nature 2005, 435, 577−579. (12) Ratcliff, F.; Harrison, B. D.; Baulcombe, D. C. A Similarity Between Viral Defense and Gene Silencing in Plants. Science 1997, 276, 1558−1560. (13) Altieri, D. C. Survivin, Cancer Networks and Pathway-Directed Drug Discovery. Nat. Rev. Cancer 2008, 8, 61−70. (14) Park, J. H.; Maltzahn, G. v.; Xu, M. J.; Fogal, V.; Kotamraju, V. R.; Ruoslahti, E.; et al. Cooperative Nanomaterial System to Sensitize, Target, and Treat Tumors. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 981−986. (15) Hepel, M.; Stobiecka, M.; Peachey, J.; Miller, J. Intervention of Glutathione in Pre-Mutagenic Catechol-Mediated DNA Damage in 4789


Article


Bladder Cancer in the Prospective Study UroScreen. PLoS One 2012, 7, e35363. (55) Xue, Y.; An, R.; Zhang, D.; Zhao, J.; Wang, X.; Yang, L.; He, D. Detection of Survivin Expression in Cervical Cancer Cells Using Molecular Beacon Imaging: New Strategy for the Diagnosis of Cervical Cancer. Eur. J. Obstet. Gynecol. Reprod. Biol. 2011, 159, 204−208. (56) Marras, S. A.; Tyagi, S.; Kramer, F. R. Real-Time Assays with Molecular Beacons and Other Fluorescent Nucleic Acid Hybridization Probes. Clin. Chim. Acta 2006, 363, 48−60. (57) Santangelo, P. J.; Nix, B.; Tsourkas, A.; Bao, G. Dual FRET molecular Beacons for mRNA Detection in Living Cells. Nucleic Acids Res. 2004, 32, e57. (58) Huang, K.; Martí, A. A. Recent Trends in Molecular Beacon Design and Applications. Anal. Bioanal. Chem. 2012, 402, 3091−3102. (59) Guo, J.; Ju, J.; Turro, N. J. Fluorescent Hybridization Probes for Nucleic Acid Detection. Anal. Bioanal. Chem. 2012, 402, 3115−3125. (60) Wu, C.; Wang, C.; Yan, L.; Yang, C. J. Pyrene Excimer Nucleic Acid Probes for Biomolecule Signaling. J. Biomed. Nanotechnol. 2009, 5, 495−504. (61) Stobiecka, M. Novel Plasmonic Field-Enhanced Nanoassay for Trace Detection of Proteins. Biosens. Bioelectron. 2014, 55, 379−385. (62) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (63) Stobiecka, M.; Hepel, M. Multimodal Coupling of Optical Transitions and Plasmonic Oscillations in Rhodamine B Modified Gold Nanoparticles. Phys. Chem. Chem. Phys. 2011, 13, 1131−1139. (64) Stobiecka, M.; Chalupa, A. Modulation of Plasmon-Enhanced Resonance Energy Transfer to Gold Nanoparticles by Protein Survivin Channeled-Shell Gating. J. Phys. Chem. B 2015, 119, 13227−13235. (65) Gao, Y.; Wolf, L. K.; Georgiadis, R. M. Secondary Structure Effects on DNA Hybridization Kinetics: a Solution Versus Surface Comparison. Nucleic Acids Res. 2006, 34, 3370−3377. (66) Romano, F.; Chakraborty, D.; Doye, J. P.; Ouldridge, T. E.; Louis, A. A. Coarse-Grained Simulations of DNA Overstretching. J. Chem. Phys. 2013, 138, 085101−085101. (67) Wang, L.; Yang, C. J.; Medley, C. D.; Benner, S. A.; Tan, W. Locked Nucleic Acid Molecular Beacons. J. Am. Chem. Soc. 2005, 127, 15664−15665. (68) Tsourkas, A.; Behlke, M.; Bao, G. Hybridization of 2′-O-Methyl and 2′-Deoxy Molecular Beacons to RNA and DNA Targets. Nucleic Acids Res. 2002, 30, 5168−5174. (69) Markham, N. R.; Zuker, M. UNAFold: Software for Nucleic Acid Folding and Hybridization. In Bioinformatics; Keith, J. M., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2008; Vol. II, pp 3−31. (70) Markham, N. R.; Zuker, M. DINAMelt Web Server for Nucleic Acid Melting Prediction. Nucleic Acids Res. 2005, 33, W577−W581. (71) Dimitrov, R. A.; Zuker, M. Prediction of Hybridization and Melting for Double-Stranded Nucleic Acids. Biophys. J. 2004, 87, 215− 226. (72) Mathews, D. H.; Sabina, J.; Zuker, M.; Turner, D. H. Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of RNA Secondary Structure. J. Mol. Biol. 1999, 288, 911−940. (73) SantaLucia, J. A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA Nearest-Neighbor Thermodynamics. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 1460−1465. (74) Walter, A. E.; Turner, D. H.; Kim, J.; Lyttle, M. H.; Müller, P.; Mathews, D. H.; Zuker, M. Coaxial Stacking of Helixes Enhances Binding of Oligoribonucleotides and Improves Predictions of RNA Folding. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 9218−9222. (75) Tsourkas, A.; Behlke, M. A.; Rose, S. D.; Bao, G. Hybridization Kinetics and Thermodynamics of Molecular Beacons. Nucleic Acids Res. 2003, 31, 1319−1330.

(35) Ghadimi, M. P.; Young, E. D.; Belousov, R.; Zhang, Y.; Lopez, G.; Lusby, K.; Kivlin, C.; Demicco, E. G.; Creighton, C. J.; Lazar, A. J.; Pollock, R. E.; Lev, D. Survivin is a Viable Target for the Treatment of Malignant Peripheral Nerve Sheath Tumors. Clin. Cancer Res. 2012, 18, 2545−2557. (36) Fukuda, S.; Pelus, L. M. Survivin, a Cancer Target with an Emerging Role in Normal Adult Tissues. Mol. Cancer Ther. 2006, 5, 1087−1098. (37) Wang, Q.; Wu, P. C.; Roberson, R. S.; Luk, B. V.; Ivanova, I.; Chu, E.; Wu, D. Y. Survivin and Escaping in Therapy-Induced Cellular Senescence. Int. J. Cancer 2011, 128, 1546−1558. (38) Zhang, K. J.; Zhang, J.; Wu, Y. M.; Qian, J.; Liu, X. J.; Yan, L. C.; Zhou, X. M.; Xiao, R. J.; Wang, Y. G.; Cao, X.; et al. Complete Eradication of Hepatomas Using an Oncolytic Adenovirus Containing AFP Promoter Controlling E1A and an E1B Deletion to Drive IL-24 Expression. Cancer Gene Ther. 2012, 19, 619−629. (39) Choi, I. K.; Yun, C. O. Recent Developments in Oncolytic Adenovirus-Based Immunotherapeutic Agents for Use Against Metastatic Cancers. Cancer Gene Ther. 2013, 20, 70−76. (40) Hunter, A. M.; LaCasse, E. C.; Korneluk, R. G. The Inhibitor of Apoptosis (IAPs) as Cancer Targets. Apoptosis 2007, 12, 1543−1568. (41) Fulda, S. Inhibitor of Apoptosis Proteins in Hematological Malignancies. Leukemia 2009, 23, 467−476. (42) Ambrosini, G.; Adida, D.; Altieri, D. C. A Novel Anti-Apoptosis Gene, Survivin, Expressed in Cancer and Lymphoma. Nat. Med. 1997, 3, 917−921. (43) Shi, Y. Survivin Structure: Crystal Unclear. Nat. Struct. Biol. 2000, 7, 620−623. (44) Noton, E. A.; Colnaghi, R.; Tate, S.; Starck, C.; Carvalho, A.; Ferrigno, P. K.; Wheatley, S. P. Molecular Analysis of Survivin Isoforms. Evidence that Alternatively Spliced Variants do not Play a Role in Mitosis. J. Biol. Chem. 2006, 281, 1286−1295. (45) Yang, L.; Cao, Z.; Lin, Y.; Wood, W. C.; Staley, C. A. Molecular Beacon Imaging of Tumor Marker Gene Expression in Pankreatic Cancer Cellls. Cancer Biol. Ther. 2005, 4, 561−570. (46) Peng, X. H.; Cao, Z. H.; Xia, J. T.; Carlson, G. W.; Lewis, M. M.; Wood, W. C.; Yang, L. Real-time Detection of Gene Expression in Cancer Cells Using Molecular Beacon Imaging: New Strategies for Cancer Research. Cancer Res. 2005, 65, 1909−1917. (47) Wang, Z. Q.; Zhao, J.; Zeng, J.; Wu, K. J.; Chen, Y. l.; Wang, X. Y.; Chang, L. S.; He, D. L. Specific Survivin Dual Fluorescence Resonance Energy Transfer Molecular Beacons for Detection of Human Bladder Cancer Cells. Acta Pharmacol. Sin. 2011, 32, 1522− 1528. (48) Nitin, N.; Santangelo, P. J.; Kim, G.; Nie, S.; Bao, G. PeptideLinked Molecular Beacons for Efficient Delivery and Rapid mRNA Detection in Living Cells. Nucleic Acids Res. 2004, 32, e58. (49) Piao, Y.; Liu, F.; Seo, T. S. A Novel Molecular Beacon Bearing a Graphite Nanoparticle as a Nanoquencher for In Situ mRNA Detection in Cancer Cells. ACS Appl. Mater. Interfaces 2012, 4, 6785−6789. (50) Liu, J.; Zhou, H.; Xu, J. J.; Chen, H. Y. Switchable ″On-Off-On″ Electrochemical Technique for Direct Detection of Survivin mRNA in Living Cells. Analyst 2012, 137, 3940−3945. (51) Adinolfi, B.; Pellegrino, M.; Baldini, F. Human Dermal Fibroblasts HDFa Can be Used as an Appropriate Healthy Control for PMMA Nanoparticles-Survivin Molecular Beacon Cellular Uptake Studies. Biomed. Pharmacother. 2015, 69, 228−232. (52) Carpi, S.; Fogli, S.; Giannetti, A.; Adinolfi, B.; Tombelli, S.; Pozzo, E. D.; Vanni, A.; Martinotti, E.; Martini, C.; Breschi.; et al. Theranostic Properties of a Survivin-Directed Molecular Beacon in Human Melanoma Cells. PLoS One 2014, 9, e114588. (53) Li, X. L.; Shan, S.; Xiong, M.; Xia, X. H.; Xu, J. J.; Chen, H. Y. On-Chip Selective Capture of Cancer Cells and Ultrasensitive Fluorescence Detection of Survivvin mRNA in a Single Living Cell. Lab Chip 2013, 13, 3868−3875. (54) Johnen, G.; Gawrych, K.; Bontrup, H.; Pesch, B.; Taeger, D.; Banek, S.; Kluckert, M.; Wellhaußer, H.; Eberle, F.; Nasterlack, M.; Bruning, T. Performance of Survivin mRNA as a Biomarker for 4790


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