Hairpin-hairpin molecular beacon interactions for detection of survivin

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Biological and Medical Applications of Materials and Interfaces

Hairpin-hairpin molecular beacon interactions for detection of survivin mRNA in malignant SW480 cells Katarzyna Ratajczak,, Bartlomiej E. Krazinski, Anna E. Kowalczyk, Beata Dworakowska, Slawomir Jakiela, and Magdalena Stobiecka ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02342 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Applied Materials & Interfaces

Hairpin-hairpin molecular beacon interactions for detection of survivin mRNA in malignant SW480 cells Katarzyna Ratajczak1, Bartlomiej E. Krazinski2, Anna E. Kowalczyk2, Beata Dworakowska1, Slawomir Jakiela1*, Magdalena Stobiecka1* 1

Department of Biophysics, Warsaw University of Life Sciences (SGGW), 159 Nowoursynowska Street, 02776 Warsaw, Poland

2

Department of Human Histology and Embryology, University of Warmia and Mazury, 30 Warszawska Street, 10082 Olsztyn, Poland

KEYWORDS Cancer colorectal cells, SW480, normal colon epithelial cells, CCD 841 CoN, fluorescence offon survivin molecular beacon probe, Lipofectamine carrier

ACS Paragon Plus Environment

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ABSTRACT Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong anti-apoptotic and proliferationenhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for an effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur mRNA. Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the singleand double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB, with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore JoeTM (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy humancolon epithelial cells CCD841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism


2

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sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

1. INTRODUCTION Considerable progress has recently been achieved in reducing the cancer mortality-rate growth owing to the development of advanced screening and therapeutic procedures. Since the common feature of all cancers is the strong enhancement of proliferation with simultaneous inhibition of apoptosis,1-2 we have investigated the feasibility of down-regulating the proteins of the apoptosis-inhibiting protein group (AIP), concentrating on survivin (Sur), the smallest member of the group. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe and a novel hairpin-hairpin interaction principle, extending the broadly applied Tyagi-Kramer model.3 We propose the mechanism of the hairpinhairpin biorecognition process and show the proof-of-the-concept in a system designed for distinguishing cancer and normal cells for diagnostics of deadly colorectal cancer. In previous studies, we have developed new plasmonic and piezo-immunosensing methodologies for the detection of Sur protein 4-5 and Sur messenger RNA (mRNA) with molecular beacon probes.6-7 Among different malignancies, colorectal cancer (CRC) is the third most common cancer in males and second in females, despite of the well-characterized molecular background of CRC and improvements in diagnostic and therapeutic approaches.8 Survivin overexpression has been shown to significantly contribute to the CRC progression and low patients' survivability rates.9-10 The AIP proteins are strongly expressed in cancer cells

1-2, 7, 11

and have recently been also

implicated in the process of carcinogenesis.12 The members of the AIP group, especially survivin


3


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(Sur) 4-5, 13-14 and X-chromosome linked AIP (XAIP),12, 15 have been in the forefront of neoplasia diagnostics, serving as the molecular cancer biomarkers.16-19 Therefore, several groups proposed to utilize control over Sur and XAIP for tumor curing.

11, 20-23 24

However, the results of Sur

downregulation by short interfering RNA (siRNA), though leading to apoptosis of cancer cells,2527

were generally not as satisfactory as predicted, likely due to the inefficiency in delivery of the

genomic material to the tumor cells, complications in the biorecognition process, and the cancer cell adaptability reactions. The problems encountered in this respect have been analyzed by us 67, 28

and others.29-35 Recently, Alshamaileh et al.

36

have succeeded in applying an aptamer-

mediated survivin RNAi to alleviate the multidrug resistance and 5-fluorouracil to kill colorectal cancer stem cells. The antitumor effects of Sur siRNA delivered with lipid nanocarriers to colon cancer cells have also been reported by Wang et al.37. Recent progress in cancer therapy targeting survivin protein and silencing its gene BIRC5 by neutralizing the messenger RNA has been reviewed by Mazur et al.38. Liposomal nanoparticles have been utilized for drug delivery in cancer therapy owing to their biocompatibility and wide range of options for easy tunability to adjust to the given requirements, including targeted delivery of 3-bromopyruvate to suppress aerobic glycolysis and ATP production,39 delivery of chemotherapeutic drugs 40-41, and cancer cell detection 42-43. The oligonucleotide molecular beacons (MBs), introduced by Tyagi and Kramer

3

have been

widely applied as a biosensing platform to recognize single-stranded DNA. MBs consist of a ssDNA loop and a duplex stem formed by self-complementary ends of the strand, thus resembling a hairpin structure 28, 44. To a typical MB, a fluorophore and a quencher are attached at the ends of MB strand, so in the close conformation, no fluorescence is detected because of the efficient Forster resonance energy transfer (FRET)

28, 45

to the quencher, being in close


4

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proximity to fluorophore. Fluorescence is restored when a target ssDNA strand binds to the MB loop causing dehybridization of the stem and separation of the fluorophore-quencher couple. A detailed analysis of MB melting characteristics allows one to evaluate the binding energies (stem and hybridization with the target) and the investigations of turn-on kinetics enable to elucidate the mechanism of the processes involved and to determine the activation energy.6 Tsourkas et al. 46

have examined in detail the effects of target affinity, MB stem stability, loop sequence length,

and binding kinetics rates. Other modalities of fluorescence switching sensors include nanoflares with plasmonic quenching 47, an on-chip cell capture with FRET analysis, 48 and graphene oxidebound MBs that are dissociatively captured by target nucleotides. 7, 49 An electrochemistry based switching sensor has also been developed for mRNA analysis in captured cells.50 In this work, we have studied the turn-on fluorescence oligonucleotide probe encoded for recognition of Sur mRNA under the terms of the proposed new biorecognition model based on hairpin-hairpin interactions which is an extension of the Tyagi-Kramer model 3 of the molecular beacon principle. The published methods of Sur mRNA detection are listed in Table S2. The SurMB-Joe probes have been delivered via liposomal endocytosis to SW480 cells – an in vitro model of human colorectal cancer. The mechanism of hairpin-hairpin interactions has been elucidated and the limit of detection for target oligonucleotides complementary to SurMB probe has been determined. The control experiments for transfection of human normal colon epithelial cells CCD 841 CoN with SurMB-Joe probe have also been carried out to evaluate the feasibility of distinguishing the colorectal cancer cells from the normal cells using the proposed hairpinhairpin biorecognition principle.


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2. MATERIAL AND METHODS 2.1. Chemicals Survivin molecular beacon sequence (SurMB-Joe) encoded for survivin mRNA with sequence provided in Table 1 has been 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). The oligonucleotides complementary to SurMB (St), those with 1 and 2 mismatches (St-1 and St-2, respectively), as well as the non-complementary DNA strands (St-nc), were synthesized by FutureSynthesis (Poznan, Poland). The purity of these oligonucleotides was tested by HPLC. Sodium phosphate dibasic (Na2HPO4), potassium phosphate monobasic (KH2PO4), and other essential chemicals were obtained from SigmaAldrich

Chemical

Company (St.

Louis,

MO,

USA).

Fetal

bovine serum

(FBS),

penicillin/streptomycin and Dulbecco’s modified Eagle’s Medium (DMEM) were obtained from cell culture company PAA (Immuniq, Warsaw, Poland). Eagle's Minimum Essential Medium (EMEM) was obtained from ATCC (LGC Standards Sp. z.o.o., Lomianki, Poland). All chemicals were of analytical grade purity. Aqueous solutions were prepared with freshly deionized water with 18.2 MΩ cm resistivity (Hydrolab, Wiślina, Poland). All concentrations of added reagents cited in this paper are final concentrations obtained after mixing.

2.2. Apparatus The fluorescence spectra were recorded using Spectrometer model LS55 (Perkin Elmer, Waltham, MA, USA), with 20 kW pulsed Xenon light source and a photomultiplier tube detector. The excitation and emission slit widths were set to 5.0 nm with scan speed 500 nm/min. The measurements were performed in 10 mM PBS buffer, pH 7.4. During the MB melting


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experiments, the temperature was scanned stepwise, with step height of 2 deg, and measurements were performed after 1 min waiting at each temperature. The excitation and emission wavelength were set to λex = 520 nm and λem = 548 nm, respectively. The fluorescence cell images were acquired with a Nikon Eclipse TE300 inverted light microscope with a blue B-2A fluorescence filter with a 30-50 nm bandwidth excitation filter, long-pass dichromatic mirror and long-pass barrier filter. Images were recorded digitally using a Canon Power Shot A640 scope-mounted camera. All images were made with exactly the same exposure. The images were then imported into Photoshop Elements and the brightness and contrast of all images were adjusted using the Levels function for green channel by shifting the black limit from 0 to 26 of the green luminosity scale which effectively enabled removing the trace background luminosity. The histograms were composed using the original fluorescence images and represent the overall distribution of the pixel counts over the luminosity within the green channel.

2.3. Cell Culture The human colon cancer cell line SW480 and human normal colon epithelial CCD 841 CoN cell lines were purchased from ATCC (LGC Standards Sp. z.o.o., Lomianki, Poland) and were cultured in culture medium containing Dulbecco’s modified Eagle’s Medium (DMEM) and Eagle's Minimum Essential Medium (EMEM) supplemented with 10 % FSB, in a humidified atmosphere of 5% CO2 in the air at 37°C using a Shell Lab Model 2123-TC CO2 Incubator. The SW480 cells were subcultured every 2–3 days. After experiments, the used cells were collected and disposed appropriately.

2.4. Cell Transfection


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Transfection experiments were conducted with SW480 cells and CCD 841 CoN cells using commercial transfer agents Lipofectamine® 3000 (Life Technologies, USA) loaded with SurMB-Joe (SurMB-Joe@Lip). In solution A, 195 µL of DMEM solution was mixed with 5 µL of Lipofecatmine3000 Reagent. Then, in solution B, 195 µL of DMEM was mixed with 4 µL of P3000 Reagent Solution and 1 µL of 100 µM SurMB-Joe. Next, solutions A nad B were mixed together and incubated for 5 min at room temperature. The SW480 cells were washed with fresh solution of DMEM and next the SurMB-Joe-liposome complexes were added to the cells and incubated for 4h at 37°C. Table 1. Oligonucleotide sequences used for survivin mRNA molecular beacon and targets Oligonucleotide * SurMB-Joe

Sequence ** 5’-Joe - CCTGGC CCA GCC TTC CAG CTC CTT GCCAGG - Dabcyl-3’

St

5’-CAA GGA GCT GGA AGG CTG GG-3’

St-1

5’-CAA GGA GCT GCA AGG CTG GG-3’

St-2

5’-CAA GGA GCT CCA AGG CTG GG-3’

St-nc

5’-CCC AGC CTT CCA GCT CCT TG-3’

* Target strands: St – target complementary to SurMB loop; St-1 – strand St with 1 mismatch; St-2 – strand St with 2 mismatches; St-nc – non-complementary target; ** SurMB stem is marked in italics and underlined, mismatches marked in black bold.

3. RESULTS 3.1. Molecular beacon SurMB-Joe The survivin molecular beacon used in this study was a 30-mer single-stranded oligonucleotide with a fluorescence dye Joe attached to the 5’ end and a quencher Dabcyl attached to the 3’ end


8

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of the strand (SurMB-Joe). The stem of the molecular beacon was formed by 6 nucleotides at both ends of the strand which are complementary to each other. The loop of the SurMB-Joe probe consisting of 18 nucleotides was targeting the survivin mRNA.14 Sequences of the molecular beacon probes and the target oligonucleotide are presented in Table 1. The general scheme of the principle of molecular beacon operation according to the Tyagi-Kramer model is depicted in Figure 1A. At room temperature, the SurMB-Joe shows a low fluorescence emission intensity IFL,max = 26 (Figure 1B, curve 1). It is due to the close proximity of the Joe fluorophore to the Dabcyl quencher and closed conformation of the molecular beacon (the "OFF" state). After interactions of SurMB-Joe with target oligonucleotide complementary to the loop sequence, the increase of fluorescence of Joe fluorophore dye is observed (IFL,max = 561.2). It indicates that hybridization of the molecular beacon loop and target oligonucleotide has occurred and a conformational change in the structure of SurMB-Joe to the "ON" state is observed. Figure 1C shows the temporal evolution of SurMB-Joe molecular beacon fluorescence at different temperatures in the absence of the target oligonucleotide. It is seen, that the intensity of fluorescence emission of the Joe dye at higher temperatures (30 - 50 °C) increases rapidly and stabilizes after ca. 9 min. The fluorescence signal changes from IFL,1 = 26 a.u. at 23°C to IFL,2 = 69.5 a.u. at 50°C after 21 min (Figure 1C, curves 1 and 6, respectively). It indicates that changes in conformation of SurMB-Joe molecular beacon and opening of its structure are taking place upon the temperature increase but the fluorescence signal is still very low.


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Figure 1. (A) Scheme of the principle of molecular beacon SurMB-Joe operation according to the Tyagi-Kramer model. (B) Fluorescence spectra for a SurMB-Joe in: (1) closed conformation (the "OFF" state) and (2) open conformation (the "ON" state), at 23 °C, after addition of a complementary target and intermittent heating to 70 °C. (C) Temporal evolution of SurMB-Joe emission at different temperatures t [°C]: (1) 23, (2) 30, (3) 35, (4) 40, (5) 45, (6) 50. Conditions: CSurMB-Joe = 100 nM, CtDNA= 100 nM, buffer: 10 mM PBS, pH 7.4; λex = 520 nm, λem = 548 nm.

3.2. Interactions of SurMB-Joe with complementary tDNA strands Firstly, the investigations of SurMB-Joe with fully complementary to loop oligonucleotides were made. In Figure 2A, the dependence of fluorescence emission of SurMB-Joe on concentration of a complementary target tDNA (St) is presented. It is seen that additions of tDNA cause an


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increase of the fluorescence emission signal of molecular beacon by 1.8-, 3.0-, 5.4-, 6.6- and 7.9fold after addition of St target to a concentration of 17, 33, 50, 67, 83 and 100 nM, respectively. It indicates that more SurMB-Joe molecular beacons change their conformation from hairpin shape to open structure upon hybridization process between the molecular beacon loop and the target oligonucleotide. The fluorescence signal of SurMB-Joe in the new conformation is restored since the Joe dye is located far from the Dabcyl quencher in this conformation. This detection system can specifically detect the complementary St strands in a broad linear detection range from 16.7 to 100 nM with a limit of detection LOD = 26 nM (S/N = 3σ), as illustrated in Figure 2B.

Figure 2. (A) Fluorescence emission spectra of SurMB-Joe recorded at 23 °C for increasing concentrations of complementary target tDNA, CtDNA [nM]: (1) 0, (2) 17, (3) 33, (4) 50, (5) 67, (6) 83, (7) 100. (B) Dependence of the SurMB-Joe emission maximim IFL,max vs. CtDNA. Other conditions: CSurMB-Joe = 100 nM, t = 23 °C, buffer: 10 mM PBS, pH 7.4; λex = 520 nm.

According to Li at al.

48

, the typical concentration of survivin mRNA measured directly in

cytosol of cancer cells is ca. 4 µM, which is more than two orders of magnitude higher than the


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LOD of our sensing platform at MB concentration of 100 nM. Moreover, we have shown previously

6

that the LOD of SurMB detection can be lowered to 2 nM by decreasing the

concentration of MB to 20 nM. Hence, this molecular beacon platform is a suitable method for detecting the Sur mRNA expression in cancer cells.

3.3. Interactions of SurMB-Joe with mismatched and non-complementary tDNA strands Next, the investigations of interactions of the molecular beacon with mismatched and noncomplementary oligonucleotides were carried out (Table 1). Figure 3 presents spectra of SurMBJoe in the presence of a target with 1 mismatch (St-1), 2 mismatches (St-2) and a noncomplementary target (St-nc). It is seen that mismatched target oligonucleotides, St-1 and two St-2, cause a considerable decrease of the fluorescence signal of molecular beacon-target oligonucleotide duplex at temperature 23°C (Figure 3, curve 1) as compared to fully complementary target (Figure 2 curve 6). The fluorescence emission maximum was IFL,St-1 = 181.4 a.u. and IFL,St-2 = 147.4 a.u. for target strands with single and double mismatch, respectively. The oligonucleotides non-complementary to SurMB loop have caused no changes to the native fluorescence signal of SurMB-Joe after interaction with the probe (Figure 1B curve 1). These results indicate that the detection system is very selective and sensitive.


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Figure 3. (A) Fluorescence emission spectra for a SurMB-Joe probe interacting with different target oligonucleotides: (1) complementary, (2) with a single-mismatch (St-1), (3) with a doublemismatch (St-2), and (4) non-complementary target (St-nc), at 23 °C. (B) Dependence of IFL,max vs. CtDNA for target oligonucleotides: (1) with a single-mismatch, (2) with a double-mismatch, and (3) non-complementary. Conditions: CSurMB-Joe = 100 nM, CtDNA = 100 nM, buffer: 10 mM PBS, pH 7.4, λex = 520 nm.

3.4. SurMB-Joe melting characteristics To further explore the interactions of SurMB-Joe with complementary and mismatched tDNA, the dynamic melting characteristics have been investigated. Where appropriate, the experimental data were fitted with a quasi-reversible two-state Boltzmann function:

, ,

, ,

(1)

where IFL,1 and IFL,2 are the fluorescence intensities for SurMB-Joe in closed and open conformations, respectively, t is the temperature (oC), tm is the midpoint melting temperature (oC), and s is the slope parameter (oC). The forward (tm,f) and backward (tm,b) melting


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temperatures for SurMB-Joe in the presence of different target oligonucleotides are collected in Table 2. The dynamic melting/hybridization curves are presented in Figure 4, as follows: (i) SurMB-Joe alone. The dynamic melting characteristic for SurMB-Joe alone has been presented in Figure 4A. The value of the forward-scan melting temperature tm,f determined by the Simplex fitting algorithm is: tm,f = 71.24±0.21 ºC (other parameters: IFL,1 = 41.28, IFL,2 = 1992.2, s = 3.520 ºC, R2 = 0.9971). The reverse temperature scan shows a hysteresis loop. The hysteresis width at the melting points is 6.1 ºC (tm,b = 65.14±0.22 ºC; IFL,1 = 74,2 IFL,2 = 2109.8, s = 3.902 ºC, R2 = 0.9968). These results confirm that SurMB-Joe undergoes a complete melting of the stem and conformation change from the OFF state to the ON state upon temperature scanning to 91 °C and then returns back to the self-hybridized OFF state upon scanning back to the room temperature. (ii) SurMB-Joe + complementary target. In the presence of the oligonucleotide complementary to the SurMB-Joe loop, the temperature scan from 23 ºC to 91 ºC produces an irreversible three-state melting characteristic. The first transition is observed from SurMB-Joe in the OFF conformation at 23 ºC to the intermediate state consisting of a fraction of intact SurMBJoe in the OFF conformation and a fraction of SurMB-Joe in the ON conformation with a molten stem and hybridized loop-St, at ca. t ≅ 61 ºC. The second transition is observed during the dissociation of St from the duplex with SurMB-Joe and formation of a random structure of molten SurMB-Joe. The third state is formed during the backward temperature scan which shows the completion of hybridization of SurMB-Joe-St and further increase of fluorescence emission. Here, evidently the two processes taking place simultaneously, i.e. melting of SurMB-Joe stem and hybridization of SurMB-Joe with complementary tDNA strand St, cannot be separated and two dynamic constants tm,f and tm,b for the forward and backward temperature scans are used to


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describe quantitatively the system behavior. The values of these constants are: tm,f = 50.7 °C and tm,b = 53.6 °C. The dynamic stem melting and loop hybridization processes observed here show clearly how important is the temperature for applications of SurMB-Joe for the detection and binding to the survivin mRNA in cancer cells and to inactivate the BIRC5 gene. (iii) SurMB-Joe + single- and double-mismatched targets. The melting hybridization characteristics for SurMB-Joe with mismatched targets are presented in Figures 4C,D. It is seen that in both cases, much weaker interaction of SurMB-Joe with targets is observed leading to a single two-state transition from the OFF state to the ON state upon heating and return to lowfluorescence OFF-state upon cooling back to the room temperature. The forward-scan melting temperature tm,f for single mismatch target St-1 is: tm,f = 72.02±0.31 ºC (other parameters: IFL,1 = 114.51, IFL,2 = 2138.0, s = 4.212 ºC, R2 = 0.9966) and the backward-scan melting temperature is: tm,b = 61.74±0.50 ºC (other parameters: IFL,1 = 116.61, IFL,2 = 2070.6, s = 4.8573 ºC, R2 = 0.9929). Thus, the hysteresis width is ∆tm = 10.28 ºC. The forward-scan melting temperature for double-mismatch target St-2 is: tm,f = 73.82±0.26 ºC (other parameters: IFL,1 = 15.26, IFL,2 = 2142.7, s = 4.373 ºC, R2 = 0.9976) and the backward-scan melting temperature is: tm,b = 61.84±0.35 ºC (other parameters: IFL,1 = 25.14, IFL,2 = 2087.8, s = 4.4896 ºC, R2 = 0.9958). Thus, the hysteresis width is ∆tm = 11.98ºC. Unlike in the case of the complementary target, the mismatched targets do not accelerate the SurMB-Joe stem melting and act rather to slow it down. Also, they do not form the ON conformation of SurMB-Joe upon cooling during the backward temperature scan.


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(iv) SurMB-Joe + non-complementary target. The effect of a non-complementary target on SurMB-Joe melting is negligible, as expected. The results of melting characteristics are presented in Figure 4E. The melting curves are similar to those of SurMB-Joe alone (Figure 4A). In summary, the data presented above indicate that SurMB-Joe under study exhibits singlenucleotide polymorphism (SNP) sensitivity. They also show that the fluorescence signal of SurMB-Joe probe depends strongly on temperature changes. While SurMB-Joe alone undergoes a complete melting of the stem and conformation change from the OFF state at room temperature to the ON state upon temperature scanning to 91 °C, the cooling brings SurMB-Joe back to the self-hybridized OFF state, as indicated in Figure 4A. However, in the presence of a complementary target strand, a duplex SurMB-target begins to form upon heating, but it melts at higher temperatures. During the backward cooling temperature scan, the duplex forms again and remains the stable form down to room temperature, with the SurMB switched to the ON state. Thus, in the presence of a complementary strand, the SurMB-Joe does not return to the original OFF state upon cooling, but is switched to a high ON state characteristic of the MB duplex with the target strand.


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Figure 4. Dynamic SurMB-Joe fluorescence melting curves in the absence (A) and the presence (B-E) of target oligonucleotides: (B) complementary strand (St-c); (C) with a single mismatch (St1);

(D) with a double mismatch (St-2); (E) non-complementary strand (St-nc); Conditions: CSurMB-

Joe

= 100 nM, CtDNA = 100 nM, buffer: 10 mM PBS, pH 7.4, λex = 520 nm, λem = 548 nm.


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Table 2. Forward (tm,f) and backward (tm,b) melting temperatures for SurMB-Joe in the presence of different target oligonucleotides*. Target

Fluorescence upon cooling back

tmf, °C

tmb, °C

none (SurMB alone)

71.24

65.14

low

single mismatch

72.02

61.74

low

double mismatch

73.82

61.84

low

non-complementary

69.94

62.97

low

complementary

53.63

50.79

high

*Conditions: 10 mM PBS buffer, pH 7.4; CSurMB-Joe = 100 nM, Ctarget = 100 nM.

3.5. Calculation of the structures of target sequences To gain deeper insights into the mechanism of interactions of target oligonucleotides with SurMB-Joe hairpin probe, theoretical calculations of target structures and their thermodynamic data (Table S1) were carried out using UNAFold 3.9 program with Quikfold application at the DINAMelt web server based on unified base-pair theory

51-52

. In Figure 5, the obtained stable

structures of target ssDNA strands at 25 °C in 50 mM NaCl are presented. It is shown, that the target oligonucleotides form small-loop hairpin structures. All structures have a negative Gibbs free energies of formation: ∆G = - 2.33 kcal/mol; ∆G = - 2.83 kcal/mol;

∆G = - 1.38

kcal/mol; ∆G = - 0.51 kcal/mol, for complementary, single- and double-mismatch, and non-

complementary targets, respectively. Note that these calculated Gibbs free energies for mismatched and perfect targets concern the formation of bulged structures from the random conformations. While the small differences in stability of these bulges will affect target interactions with MB, there is basically no reason for the bulge on the perfect target to have the lowest Gibbs free energy. This energy depends on the form and size of the bulge that is formed


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on a target strand. Therefore, any of the target strands considered may form a bulge of lower or higher stability. Interestingly, the formation of bulges on target strands may inhibit the gene recognition. However, generally, these bulges are relatively small and thus, their melting temperatures are lower than or close to the melting temperature of the MB itself. The calculated melting temperatures of the targets investigated in this work are: 60.5, 68.1, 43.2, and 34.1 °C (Table S1), for complementary, single- and double-mismatch, and non-complementary targets, respectively. These calculations predisposed us to develop a mechanism for SurMB-Joe recognition of mRNA, based on hairpin-hairpin interactions, as shown in Figure 6.

Figure 5. Stable structures of target ssDNA strands at 25 °C, 50 mM NaCl, pH 7.4 PBS.


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Figure 6. Mechanism of hairpin-hairpin interactions for SurMB-Joe with ssDNA targets. (A) Schematic view of the probe and target oligonucleotides; (B) hybridization of one leg of the target strand with MB loop; (C) melting of the stem of target; (D) partial hybridization of target with MB loop; (E) hybridization of the entire target with MB loop; (F) melting of the MB stem to form a free duplex MB-target.

Figure 6 presents a plausible mechanism of the hairpin-hairpin interactions which has been designed on the basis of theoretical calculations of target structures (Figure 5) and their thermodynamic data (Table S1), in particular the calculated Gibbs free energies of: (i) target-MB binding, (ii) target bulge formation, and (iii) MB hairpin self-hybridization from a random conformation, as well as our earlier considerations of the strand replacement model,

6

and the

Tyagi-Kramer molecular beacon model principles 3. Figure 4 confirms some of the assumptions


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of the proposed model, such as the roles of bulge melting and MB stem melting on the MB state switching between the OFF and ON states. The mechanism of target recognition by an MB in the hairpin-hairpin interaction model proceeds as follows. After the addition of a hairpin target St, complementary to the loop of a hairpin MB probe (Figure 6, A), a hybridization of one leg of the target strand with MB loop is taking place (Figure 6, B), followed by melting of the target (Figure 6, C) and its randomization (Figure 6, D), finally leading to the hybridization of the entire target with MB loop (Figure 6, E) and melting of the MB stem to form a free duplex (Figure 6, F). As seen, there are certain critical steps in the target recognition mechanism which may control the rate of the recognition process and even prevent the completion of the recognition event. In particular, the target melting step (Figure 6, B-C) may slow down or even prevent the recognition process, depending on the target-stem size, its base-pair composition and the size of the target loop. Thus, the target melting temperatures are of primary importance for the completion of the target hybridization with the MB. All the remaining dependencies concerning the MB opening efficiency upon full hybridization of MB with target, determined by the Tyagi-Kramer model still apply. While the proposed mechanism of hairpin-hairpin binding process of Figure 6 has been rationally derived by giving preference to steps requiring lower temperature and expected to have lower activation energy, there is solid experimental evidence that the process follows the proposed pathway: (a) The rate of target binding at lower temperatures (23-30 °C) is very slow, indicating that the target bulge melting (C) is hindering the progress of target binding beyond the hybridization of the target leg to the MB loop (B). However, a discernible increase in fluorescence for the system SurMB-St in comparison to that for SurMB alone, shown in Figures 4A and 4B, indicates that


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the hybridization of the target leg to the MB loop (B) occurs already at these low temperatures. Melting of the target bulge (C) is not occurring yet in this temperature range, as confirmed by calculations (Table S1). Note that the initial interaction (B) may start by binding of either of the two free legs of the hairpin target to the MB. However, the leg with longer sequence and higher number of G-C and G-G stacking pairs will be more likely to bind to the MB loop first. This is basically a matter of the competition of Gibbs free energies of hybridization and thus, the leg with more negative ∆G0B will be involved in the main pathway, while the interactions of the other leg with MB loop may lead to a parallel, less efficient, binding path. The analysis of the kinetics of this initial step of target binding to MB loop is identical to the analysis of the strand replacement mechanism discussed earlier 6. (b) The decrease of experimental melting temperature of the system SurMB-St in comparison to that for SurMB alone, illustrated in Figure 4 and detailed in Table 2, is due to the onset of target bulge melting (C) which enables further progress in target binding to the MB loop (D,E) which exerts pressure on MB loop to straighten up and weakens the binding of the stem (E,F). The melting of the target bulge requires some energy and this step is likely to contribute to the activation energy of the binding process. This also means that processes discussed for the step (B) are less likely to control the overall binding kinetics. After the bulge melting, a randomization and formation of a flexible arm of the unbound part of the target strand will occur. (c) Further increase in temperature results in melting of all system components: target bulge, MB stem, and the duplex MB-St, as evidenced by relatively high fluorescence. These processes are overlapped because in this system, the target bulge melting temperature is close to the MB stem melting temperature (theoretical values of tm: 60.5 and 58.4 °C, for target bulge and SurMB, respectively). After complete target binding to the MB loop, the melting of MB stem is expected.


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Again, this process requires energy to break the hydrogen bonds holding the stem together and thus, it will likely contribute to the activation energy of the binding process. Note that melting of all system components at high temperatures is not included in the pathway mechanism since in the analytical determinations, the heating is restricted to intermediate temperatures (ca. 50-60 °C), sufficient to hybridize MB with the target strand and melt the MB stem. (d) Upon cooling, fluorescence increases further due to the linear duplex formation of MB-St, (F) which is much stiffer than the random-coil MB and is able to more efficiently separate the fluorophore-quencher pair. This step concludes the proposed pathway. Therefore, there are two critical steps in the proposed pathway that will likely control the binding kinetics: the melting of the target bulge and the melting of MB stem. This means that the melting temperatures of the target bulge and MB stem will be ones of the primary parameters controlling the binding kinetics. Note that the steps of the target binding to an MB, which are listed separately in Figure 6, may overlap with each other making the analyses a more difficult task. The proposed model of hairpin-hairpin binding differs from the model of the so-called hairpin kissing complexes studied recently by Ouldridge and coworkers 53 using the coarse-grained DNA simulations, in which two complementary hairpin oligonucleotides interact with each other at sufficiently low temperature preventing melting of their stems. Under these conditions, the binding of the loops of the kissing complexes is constrained due to topological effects. In our model, the topological constrains are relaxed due to the presence of unbounded legs in the target hairpins and allowance for temperature increase which promotes target stem melting.


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Interestingly, among the target strands used in molecular beacon determinations of Sur mRNA, there are many bulged structures which would conform to the mechanism proposed here.

3.6. Testing of SurMB delivery to colon cancer cells - transfection of SW480 cell line In order to assess the capability of SurMB-Joe as a sensitive probe for the survivin mRNA detection in living cells of human colon cancer cell lines SW480, the optical and fluorescence microscopic images were analyzed. The cellular uptake of LipofectamineTM Lip carriers with SurMB-Joe (SurMB-Joe@Lip) was monitored using inverted light microscope with fluorescence filters (B-2A blue excitation filter). The images obtained are presented in Figures 7A,B. As a negative control, the transfection of SW480 cells with empty LipofectamineTM carriers (Lip) was also performed (Figure 7C,D). It is seen in Figure 7 that after 4h incubation of the SW480 cells with SurMB-Joe@Lip, the SurMB-Joe probes have entered the cells. The intensity of green emission signal from transfected cells is related to the hybridization of SurMB-Joe with survivin mRNA present in cytosol of the cells, opening of the molecular beacon structure, and restoring the fluorescence signal of the Joe dye. After introducing empty Lipofectamine carriers without survivin molecular beacon, only very weak residual fluorescence signal is observed (Figure 7C,D). This result demonstrates the high effectiveness of the transfection of human colon cancer SW480 cells with SurMB-Joe@Lip and the expression of survivin mRNA in the SW480 cells. Transfection with non-complementary oligonucleotide molecular beacon has shown no fluorescence signal, as expected (Figure 7E,F). The expression of survivin mRNA in SW480 cells was also detected by Sarela et al. using RT-PCR method 54. Similar results were reported in the study by Chu et al.

55

, which confirmed the presence of survivin mRNA and protein in

colorectal cancer (CRC) cell lines (e.g. SW480) by RT-PCR and Western blot assays.


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Figure 7. Optical (left panel) and fluorescence (right panel) microscopic images of malignant colorectal SW480 cells: (A,B) cells transfected with LipofectamineTM carriers loaded with SurMB-Joe (SurMB-Joe@Lip), (C,D) cells transfected with empty LipofectamineTM carriers (Lip) and (E,F) cells transfected with non-complementary oligonucleotide (MBn-cJoe@Lip). Conditions: blue B-2A excitation filter; the uptake of SurMB-Joe was visualized after 4 h transfection by green fluorescence from Joe dye upon hybridization with Sur mRNA in cytosol of SW480 cells.

The control experiments were also performed using human normal colon epithelial cells CCD 841 CoN. The obtained results are shown in Figure 8. It is seen that the fluorescence emitted


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from healthy colon cells transfected with SurMB-Joe is negligible, as expected for cells that do not overexpress survivin. Kawasaki et al. have also found that normal colon epithelium did not express survivin

56

. According to Ambrosini et al., the expression of survivin mRNA in the

adjacent normal colon gland epithelium by in situ hybridization was also absent

57

. Chu et al.

has shown that no expression of survivin mRNA and protein was detected in normal intestinal epithelial cells (HIEC) using RT-PCR and Western blot assay 55.

Figure 8. Optical (left panel) and fluorescence (right panel) microscopic images of human normal colon epithelial CCD 841 CoN cells: (A,B) cells transfected with LipofectamineTM carriers loaded with SurMB-Joe (SurMB-Joe@Lip) and (C,D) cells transfected with empty LipofectamineTM carriers (empty liposomes, Lip). Conditions: blue B-2A excitation filter; the uptake of SurMB-Joe was visualized after 4 h transfection by green fluorescence from Joe dye upon hybridization with Sur mRNA in cytosol of CCD 841 CoN cells.


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4. CONCLUSIONS We have designed a hairpin-hairpin biorecognition model as an extension of the original TyagiKramer model of loop hybridization-induced FRET-deactivation for oligonucleotide molecular beacons. The detailed mechanism of hairpin-hairpin interactions has been proposed. The model differs from the so-called hairpin kissing complexes studied by Ouldridge and coworkers 53 using the coarse-grained DNA simulations, in which two complementary hairpin oligonucleotides interact with each other at sufficiently low temperature preventing melting of their stems. Under these conditions, the binding of the loops of the kissing complexes is strained due to topological effects. In our model, the topological constrains are relaxed due to the presence of unbounded legs in the target hairpins and allowance for temperature increase which promotes target stem melting. We have demonstrated for the first time that small-loop hairpin-structured nucleotide targets, mimicking survivin mRNA, can be detected using a simple fluorescence turn-on molecular beacon probe, SurMB-Joe. Transfection of human colon cancer cells SW480, used as the model system, with SurMB-Joe probe beacons using Lipofectamine nanocarriers, enabled fluorescence monitoring of hybridization process with survivin mRNA in cytosol. The probes showed single-nucleotide polymorphism sensitivity and the achieved limit of detection (LOD) for 20-mer target oligonucleotide was 26 nM (which can be further reduced if lower concentration of MB is practical). The strong fluorescence emission of SurMB-Joe in malignant SW480 cells contrasts well with negligible signal generated in transfected healthy epithelial colon cells CCD 841. Thus, the proposed fluorescence turn-on platform can distinguish readily between the cancer and normal cell lines and has a potential for utilization in the detection of colorectal tumors and help in future in gene silencing during the time of chemotherapeutic treatment by neutralizing the overexpressed Sur mRNA.


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Supporting Information Additional information about the thermodynamic data for the stable hairpin structures of target ssDNA strands at 25 °C, 50 mM NaCl and the published methods of Sur mRNA detection.

Corresponding Author * Magdalena Stobiecka, e-mail: [email protected]; ORCID number: 0000-0002-8827-5601 Phone: +48.22.593.8614 Fax:

+48.22.593.8619

*Slawomir Jakiela, e-mail: [email protected] ORCID number: 0000-0003-1557-1650 Phone: +48.22.593.8626

ACKNOWLEDGMENTS This research was supported by funding provided by the Program OPUS of the National Science Centre, Poland, Grant No. 2017/25/B/ST4/01362 and the statutory grant of the School of Medicine, Collegium Medicum, University of Warmia and Mazury in Olsztyn, Poland.

ABBREVIATIONS


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AIP, apoptosis-inhibiting protein; Sur, survivin protein; SurMB-Joe, survivin molecular beacon with fluorescence dye Joe; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s Medium; EMEM, Eagle's Minimum Essential Medium; IFL, fluorescence intensity; LOD, limit of detection; tm,f, melting forward temperature; tm,b, melting backward temperature; St, target tDNA; St-c, complementary target strand; St-1, target strand with single mismatch; St-2, target strand with double mismatch; St-nc, non-complementary target strand; SNP, single-nucleotide polymorphism; SurMB-Joe@Lip, survivin molecular beacon with Lipofectamine; Lip, Lipofectamine carriers. REFERENCES 1. Altieri, D. C., Survivin, Cancer Networks and Pathway-Directed Drug Discovery. Nat. Rev. Cancer 2008, 8, 61-70. 2. Andersen, M. H.; Svane, I. M.; Becker, J. C.; Straten, P. T., The Universal Character of the Tumor-Associated Antigen Survivin. Clin. Cancer Res. 2007, 13 (20), 5991-5994. 3. Tyagi, S.; Kramer, F. R., Molecular Beacons: Probes That Fluoresce Upon Hybridization. Nat. Biotechnol. 1996, 14 (3), 303–308. 4. Stobiecka, M.; Chalupa, A.; Dworakowska, B., Piezometric Biosensors for Anti-Apoptotic Protein Survivin Based on Buried Positive-Potential Barrier and Immobilized Monoclonal Antibodies. Biosens. Bioelectron. 2016, 84, 37-43. 5. 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 (41), 13227–13235. 6. Stobiecka, M.; Chalupa, A., DNA Strand Replacement Mechanism in Molecular Beacons Encoded for the Detection of Cancer Biomarkers. J. Phys. Chem. B 2016, 120, 4782-4790. 7. Stobiecka, M.; Dworakowska, B.; Jakiela, S.; Lukasiak, A.; Chalupa, A.; Zembrzycki, K., Sensing of Survivin mRNA in Malignant Astrocytes Using Graphene Oxide NanocarrierSupported Oligonucleotide Molecular Beacons. Sens. Actuat. B 2016, 235, 136-145. 8. M. Maida; Macaluso, F.; Ianiro, G.; Mangiola, F.; Sinagra, E.; Hold, G.; Maida, C.; Cammarota, G.; Gasbarrini, A.; Scarpulla, G., Screening of Colorectal Cancer: Present and Future. Expert Rev. Anticancer Ther. 2017, 17 (12), 1131-1146. 9. Jakubowska, K.; Pryczynicz, A.; Dymicka-Piekarska, V.; Famulski, W.; GuzińskaUstymowicz, K., Immunohistochemical Expression and Serum Level of Survivin Protein in Colorectal Cancer Patients. Oncol. Lett. 2016, 12 (5), 3591-3597.


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Fig 0 TOC 35x15mm (600 x 600 DPI)



Fig 1 98x76mm (600 x 600 DPI)


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Fig 2. 61x26mm (600 x 600 DPI)



Fig 3. 59x25mm (600 x 600 DPI)


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Fig.4 153x184mm (600 x 600 DPI)



Fig 5 74x34mm (600 x 600 DPI)


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Fig 6 105x88mm (600 x 600 DPI)



Fig 7. 86x92mm (600 x 600 DPI)


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Fig 8 91x65mm (600 x 600 DPI)


Hairpin-hairpin molecular beacon interactions for detection of survivin

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