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Reversible Photocontrol of Thrombin Activity by Replacing Loops of Thrombin Binding Aptamer using Azobenzene Derivatives Mengwu Mo, Dejia Kong, Heming Ji, Dao Lin, XinJing Tang, Zhenjun Yang, Yujian He, and Li Wu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00848 • Publication Date (Web): 24 Dec 2018 Downloaded from http://pubs.acs.org on December 26, 2018

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Bioconjugate Chemistry

Reversible Photocontrol of Thrombin Activity by Replacing Loops of Thrombin Binding Aptamer using Azobenzene Derivatives Mengwu Mo†, Dejia Kong†, Heming Ji†, Dao Lin†, Xinjing Tang‡, Zhenjun Yang‡, Yujian He*,†, Li Wu*,†,‡

†

School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing

100049, China ‡

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical

Sciences, Peking University, Beijing 100191, China

KEYWORDS: thrombin binding aptamer, reversible photocontrol, azobenzene, thrombin, loop

ABSTRACT：The photoisomerization of azobenzenes provides a general means for the photocontrol of many important biomolecular structure and organismal function. For

ACS Paragon Plus Environment

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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temporal and spatial control activity of thrombin binding aptamer (TBA) by light, azobenzene derivatives were carefully selected as light-triggered molecular switches to replace TT loops and TGT loop of TBA to reversibly control enzyme activity. These molecules interconverted between the trans and cis states under alternate UV and visible light irradiation, which consequently triggered reversible formation of Gquadruplex morphology. In addition, we investigated the impact of three azobenzene derivatives on stability, thrombin binding ability and anticoagulant properties. The result showed that 4,4’-bis(hydroxymethyl)azobenzene at TGT loop position significantly photoregulated affinity to thrombin and blood clotting in human plasma, which provided a successful strategy to control blood clotting in human plasma and a further evidence for design of TBA analogues with pivotal positions of modifications.

The regulation of enzymes is of great significance in biology and the field has attracted great attentions.

1, 2

Numerous investigations have been focused on controlling

enzymatic activity by altering their structure or morphology.

3, 4

catalytic activity, specificity of recognition and binding properties.

5

Enzymes have high This can be not only


2

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explored basic theoretical studies on the working mechanism of enzymes, further used in diagnostic and therapeutic applications. one of the most investigated class of proteases,

7

8, 9

6

but also

Thus far, thrombin has been due to its important role in

hemostasis, fibrinolysis, cell proliferation, and migration. 10 The thrombin binding aptamer (TBA) is a 15-mer DNA oligonucleotide (5’GGTTGGTGTGGTTGG-3’) that inhibits the formation of fibrin clots by binding to thrombin.

11, 12

The TBA folds in a distinctive antiparallel G-quadruplex structure

characterized by two overlapping G-tetrads and three lateral loops that contain a central TGT loop and two TT loops characterized by X-ray diffraction and nuclear magnetic resonance (NMR).

13, 14

Structural studies have shown that the TT loops form a pincer-

like structure which binds the protruding region of thrombin exosite I.

15

In addition, it’s

acknowledged that the stability and rigidity of TBA are important for the interaction between the aptamer and exosite I.

16

Therefore, many efforts have been made to

improve its thermal stability and biological activity via chemical and structural modifications, such as LNA (locked nucleic acid), acyclic nucleotide analogue,

20, 21

2’F-ANA/DNA,

22

17

UNA (unlocked nucleic acid),

18, 19

D-/L-isothymidine, 23 indole-modified


3


thymidine,

9

inversion of polarity sites

24

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and dibenzyl linker.

All these modifications

25

are irreversible control the activity of thrombin.

Figure 1. Quadruplex structures of azobenzene derivatives modified TBAs, and AZO represents azobenzene derivatives. Light as an external stimulus was reported to manipulate various objects' structural and functional properties at a desired time and space.

26-28

Optical control of TBA

conformation provides a simple biochemical tool for probing protein function in diverse systems.

29

For example, Heckel and Mayer have incorporated a photolabile group

directly into the thymidine nucleobases of TBA loop to mask of the function of thrombin, but the regulating process is usually irreversible.

30

Spada and co-workers have

introduced a photoactive moiety at the C8 in a lipophilic guanosine derivative, then the existence of G-quartets could be alternately switched on and off.

31

Ogasawara and co-

workers have developed a method of photoregulating G-quadruplexes by cis–trans


4

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photoisomerization of 8-fluorenylvinyl-2’-deoxyguanosine, incorporated in aptamers.

29

All these photocontrollers are modified nucleobases. Tan et al. have introduced azobenzene into the complementary chain of TBA via a PEG linker. Although the TBA aptamer is able to fold into the correct G-quadruplex structure that binds to thrombin, enzymatic activity and inhibiting coagulation are substantially low. On the contrary, visible light irradiation restores thrombin activity by stimulating the binding of the complementary strand and TBA. To effectively control hybridization between aptamer and complement, at least one azobenzene per two nucleotides is used in the complement. 32 Zhou et al. have developed a component comprised of enzyme, inhibitor and azobenzene derivative to control the conformations of telomere DNA thrombin activity upon photoirradiation.

34, 35

33

and

It is reported that the conversion of

azobenzene from trans to cis could trigger TBA linked with telomere to bind the thrombin, while the conversion of azobenzene from cis to trans prevents the TBA to bind thrombin. However, this regulating process requires high concentrations of azobenzene, which usually causes potential toxic side effects. Therefore, we propose to


5


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replace the TBA loop by photoactivatable azobenzene residue to operate the thrombin enzyme activity We

have

previously

designed

a

photo-responsive

linker

unit

(4,4’-

bis(hydroxymethyl)azobenzene)to replace loop nucleotides in a DNA hairpin, which enhanced the oligonucleotide stability.

36

In addition, we successfully photoregulated

RNA digestion by using azobenzene linked dumbbell antisense oligodeoxynucleotides. 37

Moreover, Heckel and Schwalbe have recently developed a photoswitchable G-

quadruplex module via the azobenzene and its analogues.

38

In this study, we

investigated TBA analogues containing different azobenzene derivatives on loops of the quadruplex, with the aim of reversible regulation of enzymatic activity. The TBA structure has been proposed to remain after binding to thrombin.

39

Firstly, we

synthesized series of azobenzene derivatives as linker units and further incorporated them into modified TBA (Figure 1). Then, we examined the influence of single and/or multiple introductions of azobenzene derivatives within the TBA moietyand their thermodynamic stability with light irradiation. Next, we characterized the photoinduced changes of interaction between thrombin and modified TBA. Finally, we analyzed the


6

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photoregulation of anticoagulant potential of the novel, chemically modified TBA variants. This finding provided a potential strategy for design and selection of compounds with the necessary control of biological properties for therapeutic applications. RESULTS Design and synthesis of azobenzene-modified TBAs. We had previously reported that there existed the most dramatic change in hairpin stability based on 4,4’bis(hydroxymethyl)-azobenzene photoswitching and found the potential effects of subtle changes in spacer length between the DNA and the switch.

36, 37

Recently, Heckel et al.

presented a tetrameric G-quadruplex consisting of two dimeric units enabling the formation of an intermolecular and conformationally well-defined G-quadruplex structure with 4,4’-bis(hydroxymethyl)-azobenzene upon UV/Vis irradiation.

38

Herein, we extend

the study by introduction of the same azobenzene or its analogues to be developed a photoswitchable G-quadruplex-forming aptamer which was conducted to reversibly regulate

of

enzymatic

activity

upon

light

irradiation.

Thus,

4,4’-

bis(hydroxymethyl)azobenzene (a), 4,4’-bis(hydroxyethyl)azobenzene (b) and 3,3’-


7


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bis(hydroxymethyl)azobenzene (c) were synthesized from 4-nitrobenzylalcohol in one step as previously reported in the literature

36, 37

(Figure S1). These azobenzene linkers

were further introduced to a series of TBA G-quadruplex sequences via synthetic method of phosphoramidite using DNA solid-phase synthesizer. Previous investigations concerning TBA- thrombin interaction have shown that loop regions of TBA are crucial for the interaction between the aptamer and thrombin.

15, 40

To investigate the effect of three different loop regions, a series of modified TBA sequences was designed and synthesized with azobenzene a, b and c modifications on varying three loop regions (Figure 2 ). With simple prediction, the substitution of an azobenzene derivative at its suitable position would remain a pincer-like structure without affecting the stability and rigidity of TBA, with the goal of improving the binding interactions with thrombin. On the other hand, the other isomer should destroy or affect the formation of a G-quadruplex. Clearly, both the azobenzene linker and substitution pattern should be considered to be a suitable balance between the rigidity and flexibility of the overall structure. Moreover, we expect that minor changes in the structure of the azobenezene linker as well as substitution in different loop position result in significant


8

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changes of photoswitching G-quadruplex with light irradiation, thus this will be able to effectively regulate thrombin activity.

Figure

2.

Structures

of

4,4’-bis(hydroxymethyl)azobenzene

(a),

4,4’-

bis(hydroxyethyl)azobenzene (b), 3,3’-bis(hydroxymethyl)-azobenzene (c) and ODN sequences used in the study. 1, 2 and 3 represented TGT loop position, Structural analysis of azobenzene-modified TBAs. The folding of TBA into a typical 5’-end TT loop and 3’-end TT loop position, respectively. chair type antiparallel G-quadruplex causes a well-known CD profile characterized by three positive bands at 210, 247 and 295 nm and one negative absorption peak around 270 nm

41, 42.

The CD analysis of the modified TBAs showed that the TBA variants

maintain a good anti-parallel G-quadruplex which in aaccordance with that of TBA (Figure 3). Neverthless, the intensity of corresponding peaks of the azobenzenemodified TBAs were all decreased, indicating that the modifications indeed perturbed


9


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the regular formation of the chair type antiparallel G-quadruplex. Notably, when 4,4’bis(hydroxymethyl)azobenzene replaces TGT loop, the G-quadruplex T1a will display stronger

peak

signals

at

295

nm

than

T1b

and

T1c,

with

4,4’-

bis(hydroxyethyl)azobenzene and 3,3’-bis(hydroxymethyl)azobenzene (Figure 3a). This indicates that the azobenzene derivative a in TGT loop is more favaroble for forming Gquadruplex than other two azobenzene. However, the G-quadruplexes (T2b and T2c), with

substitution

of

4,4’-bis(hydroxyethyl)azobenzene

and

3,3’-

bis(hydroxymethyl)azobenzene in 5’-end TT loop, exhibited a stronger signal intensity than that of corresponding T2a (Figure 3b). For T3a, T3b and T3c with modification in 3’-end TT loop, the CD signals showed the same changing trend as that in the 5’-end TT loop (Figure 3c).

Figure 3. Comparison of circular dichroism spectra and structural characteristics between different azobenzne-modified TBAs and TBA without UV irradiation. The concentration of TBA is 25 μM. ACS Paragon Plus Environment

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The structural changes upon photoswitching of the azobenzene units with UV irradiation were investigated (Figure S4). UV/Vis spectra showed the differential absorption signal typical for the azobenzene from trans to cis structure. Change of CD spectra could be obtained upon UV irradiation as shown in Figure 4. After irradiation with UV light, the CD signal of T1a at 295 nm and 270 nm dropped significantly and an induced CD signal at 445 nm was observed, and the system reached structural equilibrium within 9 min. A similar variation tendency occurred for T1b and T1c with UV irradiation, respectively. Irradiation with visible light for a short period of time (4 min) led to the almost complete recovery of the initial CD signal (Figure S5). This clearly indicated that the TGT loop can be a key location, which easily regulated TBA conformation by trans-cis isomerization of azobenzene units. Differently, when the azobenzene linkers replaced TT loops, T2a and T3a even turned out a slightly stronger CD signal due to trans to cis isomerization, whereas there existed little or no obvious change for T2b, T3b, T2c and T3c under UV light irradiation.


11


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Figure 4. Circular dichroism spectra and structural characteristics of TBA variants under UV irradiation for different times. Experiment was performed in phosphate buffered saline (138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4, 100 mM KCl ) by using a handy UV lamp (Shanghai Baoshan, ZF-7A, 8 We have also provided further evidence that the strategy of photoswitching TBA GW, λ=365 nm ). The concentrations of TBA variants are 25 μM. quadruplex can be determined by modification of multiple azobenzene linkers in loop positions. As shown in Figure S3, multiple 3,3’-bis(hydroxymethyl)azobenzene units were introduced on loop positions of TBA and still remained chair type antiparallel Gquadruplex structures. However, intensity of CD bands for all the modified TBAs with


12

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two or three azobenzene linkers (T1c2c, T2c3c and T1c2c3c) became obviously weak with UV irradiation, even including substitution of two TT loops, although a single c have no influence on structural change on either TT loop by light irradiation. Thermal analysis of azobenzene-modified TBAs. In order to evaluate the structural stability when the azobenzene units occupied each loop position in comparison with the original TBA, CD melting curves for each variant were measured and the Tm values were provided in Table 1 and Supplementary Figure S6. Based on the result, it can be concluded that substitution of three azobenzene derivatives in TGT loop resulted in significant destabilization of the quadruplex structure comparing with TBA and the magnitude of destabilization increased for these three TBA variants when shifting from a towards b or c. For example, Tm values decreased by 11.8 oC for T1a, 22.9 oC for T1b and 19.3 oC for T1c. T2a, T2b, T3a and T3b with a or b in TT loops displayed decreased thermodynamic stability by 16.0 oC for T2a, 3.8 oC for T2b, 10.8 oC for T3a, and 3.8 oC for T3b, relative to TBA, whereas the TBA variants modified with c in two terminal TT loops appeared to be more stable (5.7 oC for T2c and 4.1 oC for T3c higher than TBA). It was found that the thermodynamic stabilities of these TBA variants


13


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increased when three azobenzene units shifted from a to c in either TT loop, and the changing trend showed in opposite direction comparing with those in TGT loop. This was consistent with the changed trend from the CD spectra (Figure 3). In addition, when two cs units located in two terminal TT loops, thermodynamic stability of T2c3c was higher than that of T2c and T3c with unit in either TT loop, far higher than that of TBA, while TBA variants replaced in TGT loop by c were thermodynamically

unfavorable

for


14

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Table 1. Melting temperatures for TBAs with azobenzene modification Tm (°C) ΔTm1 a(°C)

ΔTm2 b(°C)

48.9±0.2

/

/

37.1±1.6

33.4±0.5

-11.8

-3.6

T1b

26.0±2.0

29.0±1.5

-22.9

3.0

T1c

29.6±3.1

23.3±1.2

-19.3

-6.3

T2a

32.9±1.3

39.1±1.2

-16.0

6.2

T2b

45.1±0.5

47.1±3.0

-3.8

2.0

T2c

54.6±3.5

47.6±2.0

5.7

-7.0

T3a

38.1±1.6

37.0±2.1

-10.8

-1.1

T3b

45.1±2.5

43.1±0.5

-3.8

-2.0

T3c

53.0±1.0

43.3±1.6

4.1

-10.0

T1c2c

35.2±0.5

38.4±1.5

-13.7

3.2

T2c3c

67.4±2.5

40.8+0.2

18.5

-26.6

T1c2c3c

43.0±0.3

39.8±0.8

-5.9

-3.2

Name

−UV

+UV

TBA

48.9±0.5

T1a

aΔT

= Tm (−UV) − Tm (TBA),

bΔT

= Tm (+UV) − Tm (−UV)

m1 m2

quadruplex formation. For example, T1c2c and T1c2c3c decreased Tm values by 13.7 oC

and 5.9 oC relative to TBA, respectively. Aromatic ring of c in TT loop stack over the

quartet, and apparently substitution of c may show optimal orientation for the type of interaction.


15


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A change of thermodynamic stability for TBA variants due to isomerization of azobenzene may be a factor for photomodulation of bioactive activity. Thus, we measured the Tm of the TBA variants after UV irradiation under the same conditions. The results displayed that less conclusive correlation was observed for the relatively unstable T1a, T1b, T1c, T2a, T2b, T3a and T3b aptamers. With replacement of TGT loop, T1a and T1c showed a decrease of the thermodynamic stabilities under UV irradiation, while T1b displayed a slight increase. T2a, with a in TT loop, became more stable after UV irradiation than before UV irradiation, while T2b, T3a and T3b showed no obvious change. This may be due to different linkers used at the position of different loops, as it has been found not only size of linker but also flexibility impact quadruplex thermodynamic stability. Gratifyingly, the more thermodynamically stable TBA variant (T2c and T3c) with c in TT loop showed obviously decreased stability after UV irradiation than before UV irradiation. Especially for T2c3c with two cs in TT loops, UV radiation decreased Tm value by up to 26.6

oC.

In general, changes in the

thermodynamic structure for the TBA variants were quite a bit different from the abovementioned assay involving structural folding.


16

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Effect of azobenzene-modified TBAs on affinity to thrombin. Surface plasmon resonance (SPR) assay was performed to characterize the affinity of azobenzenemodified TBAs to thrombin in molecular level

43.

With thrombin fixed on the chip,

different concentrations of TBA solutions were used to determine the dissociation constant (KD) (Figure 5)

23.

As expected, the kinetic profile of the unmodified TBA

revealed a KD value of 39.8 nM (Figure S7 and Table S1), reflecting high affinity to thrombin.

Figure 5. Characterization of the affinity between thrombin and TBA variants without UV illumination. Their KD values were labelled in the corresponding curves. ND, no data.


17


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Higher KD value was measured for T1a containing a in TGT loop (32.5 nM), while a slight decrease in the strength of thrombin-TBA interactions was observed for T1b and T1c, and the resulting KD values were 61.1 nM and 73.6 nM, respectively. In contrast, T2b displayed a significant reduction of affinity (KD = 2.4 μM), while the rest of the TBA variants (T2a, T2c, T3a, T3b and T3c) showed no KD values were detected, suggesting lack of affinity towards thrombin after incorporation of azobenzene linkers in TT loops, although these substitutions maintain similar anti-parallel G-quadruplex structure and comparable thermal stability.These results indicated that the two TT loops played a key role in the binding of TBA to thrombin, and their lack could easily hinder this combination, which stayed in accordance with previously published studies by crystallographic and NMR.

14, 44, 45

Based on CD spectra and thermal melting assay, photoswitching behavious of the TBA variants were also evident. The azobenzene isomerization induced upon UV illumination indeed perturbed G-quadruplex structure and thermal stability when the TGT loop was replaced by a, b and c, respectively. Similarly, T1a, T1b and T1c showed much lower affinities to thrombin after UV illumination than before UV illumination


18

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(Figure

6

and

Table

S1).

Figure 6. Characterization of the affinity between thrombin and TBA variants after UV illumination, Their KD values were labelled in the corresponding curves. For instance, KD values of T1a, T1b and T1c were 32.5 nM, 61.1 nM and 73.6 nM in ND, no data. their trans states and 76.3 nM, 85.2 nM and 105.6 nM in their cis states after UV illumination, respectively. The result showed much better photomodulation effect (2.3fold) for T1a under current SPR assay conditions. And T1a exhibited difficult dissociation curves due to the strong specific binding. However, T2a, T2c, T3a, T3b and T3c in their trans state did not show binding to thrombin, and still no thrombin binding


19


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activity was detected for their cis isomers. The KD values of T2b is a 2.2-fold decrease from 2.4 μM to 1.1 μM upon UV illumination due to conformational changes, although the interaction of T2b with thrombin was very weak in both its trans state and cis state. In comparison with these single azobenzene linked TBAs, the modified TBA (T1c2c) with two cs linkers in TGT loop and 5’-end TT loop, showed much lower affinity (Figure S7), which was evidenced by the KD values of 116.5 nM. Interaction between aptamer and thrombin was not detected for T2c3c with substitution of c on the two TT loop positions. Unexpectedly, with substitution of three cs, the T1c2c3c

exhibited a slightly

stronger binding activity (KD = 86.8 nM). In addition, UV irradiation induced the trans to cis transformation of azobenzene loops, which led to about 2.5-fold and 2.0-fold decrease in affinity to thrombin for T1c2c and T1c2c3c, respectively, while T2c3c still remained no thrombin binding activity with UV illumination. Photoregulation of the thrombin-dependent blood clotting time using azobenzenemodified TBAs. To investigate the inhibition activities of azobenzene-modified TBAs, some TBA variants were tested in a thrombin time assay. According to the results of CD, thermostability and SPR, several TBA variants were chosen based mainly on their


20

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ability to form G-quadruplex structure and bind strongly to thrombin, and the results were shown in Table 2. As expected, TBA significantly inhibited the blood clotting formation relative to the blank group without TBA. Among the TBA variants, T1a, T1b and T1c showed significantly enhanced ability to inhibit thrombin activity relative to the blank group (the clotting times were 80.5 s, 77.4 s and 45.0 s for T1a, T1b and T1c, respectively), although they couldn't compete with the unmodified TBA. However, T1c2c and T1c2c3c showed poor inhibitory activity against thrombin on fibrin-clot formation, and T2b and T2c3c showed almost no anticoagulant properties. Considering effective photoregulation of in vitro TBA-thrombin interaction, changes of thrombin activity in human plasma samples were investigated upon photoswitching of azobenzene units. The result demonstrated that the clotting time of T1a with UV irradiation (45.2 s) was much shorter than that of the samples with no irradiation (80.5 s), with a 1.8-fold decrease, reflecting the fact that T1a succeed in photoregulating blood plasma clotting. After the above sample were further irradiated with visible light, the blood clotting time (79.0 s) was similar to that of the untreated samples, suggesting the efficient recovery of thrombin activity. In contrast, with reduced ability to inhibit thrombin


21


T1b

activity,

and

T1c

Page 22 of 51

showed

slightly

Table 2. The biological effects of TBA and azobenzene-modified TBAs T-time (s) a Name

−UV

+UV

UV+VIS

Blank

19.4±1.4

20.6+1.2

/

TBA

135.0±1.6

136.0±1.3

/

T1a

80.5±1.8

45.2±2.5

79.0±2.6

T1b

77.4±2.1

57.6±2.2

79.6±3.2

T1c

45.0±2.0

40.8±0.8

46.6±1.2

decreasedT2control of inhibitory efficiency when exposed to 24.0±1.5 UV light, indicating that b 21.8±0.5 25.0±1.6 introductions ofc b and c 32.2±1.8 in TGT loop have25.4±2.2 a negative influence on photoregulating T1c2 31.4±1.9 anticoagulant due to different-sized linkers. Similarly,24.6±1.1 the anticoagulant effect T2c3properties c 24.2±2.7 26.8±1.9 poorer after 27.0±1.3 UV irradiation (32.2 s vs 25.4 s for T1c2c, of T1c2c and T1c2T1 c3cc2c3c became 31.0±0.5 29.4±2.3 31.0 s vs a27.0 s forisT1 c2time c3c),required and no for anticoagulant effectin was observed for T2b and ‘T-time’ the a clot formation the plasma from a blood sample. ACS Paragon Plus Environment

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T2c3c both before and after light activation. Thus, T1a had the potential to achieve reversible regulation of thrombin activity to control coagulation. DISCUSSION The control of inhibitory function of TBA quadruplex is a challenging issue in spite of lots of modification having been studied,

46-48

but only few studies exist in which nucleic

acid are switched. Considering the loops as the key players, we studied the TBA variants by replacing one loop of the TBA with the azobenzene linker to control over the aptamer’s inhibitory function using light (Figure 1). Indeed, trans-cis isomerization of the azobenzene unit was effectively performed in the well-defined G-quadruplex structures (Figure S4). CD studies suggested the structures of all the variants resembled that of the TBA because of an aromatic portion, to mimic the aptitude of some loop residues in the TBA G-quadruplex to stack on the adjacent G-quartet; 49 Neverthless, the level of similarity is not the same: As for three linkers a, b and c, the CD signal became more and more weak in TGT loop while the opposite trend appeared in the TT loop (Figure 3). Furthermore, UV irradiation rendered the signal of CD much weaker for modification in


23


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TGT loop (Figure 4). In line with CD data which arranged a, b and c in TGT loop, the CD melting temperatures of the T1a, T1b and T1c were sequentially decreased. Differently, substantial stabilization of the G-quadruplex occurred when c occupied the TT loop position (ΔTm1 (T2c) = +5.7 oC and ΔTm1 (T3c) = +4.1 oC, compared to that of TBA; Table 1). Interestingly, changes of thermal melting temperatures were observed after UV irradiation than before UV irradiation, as for the TBA variants, with the exception of T2b, T3a and T3b (Table 1). The results from SPR assay (Figures 5 and 6) assessed the involvement of the azobenzene linker in the formation of TBA-thrombin complex, while those from the thrombin time assay (Table 2) showed the influence of the modification on the anticoagulant properties. The most relevant outcomes emerged from the TBA variants modified in the TGT loop. By modifying the TGT loop, it resulted that T1a, T1b and T1c that folded into the a bit unstable G-quadruplex, still displayed relatively high affinity towards thrombin in SPR assay (Figure 5), and showed the apparently anticoagulant effects in the anticoagulant test although with reduced efficiency relative to TBA (Table 2). Especially, among these three TBA variants, the best-structured T1a was also the


24

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strongest inhibitor of coagulation and has stable TBA-thrombin interaction. On the other hand, after UV irradiation, both the binding interaction and anticoagulant activity of these three TBA variants resulted in a decline, and T1a showed the most potent photoswitching effect. To the best of our knowledge, this is the first evidence that loop structure is a further parameter capable to photoswitch the anticoagulant efficiency of the TBA G-quadruplex. However, modifications of b and c in TT loops of TBA negatively interfere with its thrombin affinity and anticoagulant activity without affecting the G-quadruplex stability. These results could reflect no tight correlation between the structural stability of Gquadruplex and the antithrombin efficiency. Such incoherence between the activity of thrombin binding aptamer and thermostability data was previously observed by Anna Pasternak et al. and Satoru Nagatoishi et al. for other TBA variants.

18, 49

In the study,

T2c and T3c, which showed much higher thermodynamic stability than TBA, resulted totally unable to form TBA-thrombin complex (Figures 5 and 6). The overall results evidenced that, despite the TBA variants well sustained the replacement of the azobenzene linker in either TT loop, the TBA-thrombin complex did not tolerate these


25


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modifications, suggesting that TT loop could also play a crucial role in determining the anticoagulant effect of such type of TBA derivatives. Consequently, their anticoagulant activities for modifications in either TT loop were not further investigated. However, these TBA variants didn’t show a cooperative effect with respect to the two or more functional effects, and thus T1c2c and T1c2c3c still displayed weak binding interaction between the aptamers and thrombin, and relatively poor anticoagulant activity. In order to get deeper insight into the regulation of structure and function of TBA by the different azobenzene derivatives, we performed Gaussian optimization for the three linkers (Figure S9), and analyzed how the change in the length of the azobenzene structure upon isomerization matched the TGT loop of TBA by using Hyperchem program. Seeing that two benzene rings of azobenzene occupied the TGT loop region may provide for the similar stacking interaction with the nucleobases on the adjacent quartet for all three linkers, thus the linked spacers between azobenzene and nucleotide are key for different regulation efficiency in addition to stacking interaction. According to our calculated results, we found that the distance between the two outer oxygen atoms for trans-a is 13.43 Å and shrinks to 10.26 Å in its cis form, with a difference of 3.17 Å.


26

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The molecular distance of a in the trans form should well match the TGT loop of TBA, resulting in a strongly similar structure to TBA and relatively high stability, while the photoinduced distance change by a isomerization may interrupt this type of balance and thus regulated the functional change. For para-para substitution pattern of longer linker and meta-meta substitution pattern of shorter linker, b has the distance change from 16.10 Å in trans-b to 11.14 Å in cis-b, and c ranges from 11.33 Å in trans-c to 7.43 Å in cis-c, respectively. A longer or shorter linker might hinder optimal adaption for TGT loop, isomerization causes unfavorable structure relaxation, which is manifested in regulating TBA anticoagulant properties. In addition, as for three linkers, the modification of c at TT loop position did not provoke substantial alteration of the resulting G-quadruplex, even resulted in higher stability relative to TBA. Furthermore, photoswitching properties in thermodynamics were also very manifest. This is explained by the fact that c in trans form may stably capped in TT loop position, while the size of its cis form gravely deviated from that of normal TT loop.

36

However, the central loop is not directly involved in specific

interactions with thrombin, and the inhibitory effect is a consequence of blocking active


27


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site of thrombin by the two TT loops. Consequently, almost all the above mentioned contacts between TBA and thrombin for TBA variants with replacement at TT loop position was significantly prevented and anticoagulant properties could be completely inhibited. CONCLUSION In conclusion, we reported the replacement of TBA loop by azobenzene derivatives to reversibly photocontrol the thrombin activity. By screening all TBA variants, the substitution of the TGT loop with the azobenzene linkers was able to fold into wellknown TBA G-quadruplex topology and realized reversibly photocontrol thrombin activity. In particular, T1a, with 4,4’-bis(hydroxymethyl)-azobenzene in TGT loop, showed the best photomodulation of anticoagulant activity and light-induced trans to cis transformation caused 1.8-fold decrease. Neverthless, substitution of 3,3’bis(hydroxymethyl)-azobenzene in TT loop, although formed stable G-quadruplex structure, didn’t significantly inhibit the anticoagulant activity before or after UV irradiation. The presented data demonstrated that more complex phenomena among G-quadruplex topology, stability, TBA-thrombin interaction and thrombin activity, but


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herein described a potential means to obtain photoswitching G-quadruplex tools for the understanding of the molecular factors that contribute to the anticoagulant activity of TBA. In addition, we expected that the aptamers demonstrated in this work can trigger the use of photoresponse bioswitches in future biomedical and pharmaceutical applications.

For

example, the

ability

to activate the coagulation reaction

leading to alleviate selective blockage of blood vessels, and the ability to control angiogenesis at specific positions minimizing nutrition delivery to the cancerous site, opens new therapeutic avenues by potentially allowing a physician to locally target therapeutic effects. METHODS Synthesis of phosphoramidite monomers derived from azobenzene derivatives and the azobenzene-modified TBAs. The synthesis of phosphoramidite monomers of azobenzene derivatives was performed according to our previous reports.

36

Synthetic

details were described in the Supplemental Information. The TBA and the azobenzenemodified TBAs were synthesized on an ABI 394 automated RNA/DNA synthesizer. All oligonucleotides were synthesized using standard solid phase DNA chemistry on a


29


controlled pore glass (CPG) by using solid-phase phosphoramidite method.

Page 30 of 51

50, 51

The

coupling time of every modified monomer was prolonged from 2 min to 15 min. The oligomers were detached from the solid support and deprotected by treatment with concentrated ammonia solution (33%) at 50 °C for 8 hours. The combined filtrates and washings were concentrated in vacuo, dissolved in H2O, and purified by HPLC using reverse high performance liquid chromatography (ZORBAX Eclipse XDB-C18, 5 μm, 9.4 × 250 mm) with a linear gradient (from 0% to 100% A in 50 min) of TEAA at pH 7.4 (A: 0.05 M TEAA; B: CH3CN, elution time 37 min). Then the isolated DMT-on oligonucleotides were detritylated with 80% acetic acid for 15 min at room temperature. After the solution was spun dry with a concentrator, dissolved the solid pellet in 50-100 μL of water and then concentration measured with NanoDrop (Thermo scientific). The composition of all oligonucleotides was confirmed by by ESI-MS. CD measurement. Circular dichroism (CD) spectra of modified TBAs were obtained with a Jasco J815 spectrometer (Japan) using 0.5-mL quartz cuvettes with a 2-mm path length. Oligonucleotide concentrations were measured by UV absorbance at 260 nm using NanoDrop. The concentrations of all oligonucleotides were 25 μM. The samples


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dissolved in 1 ×PBS buffer, which contained 100 mM KCl, 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4, submitted to the annealing procedure (heating at 95°C and slowly cooling at room temperature). The measurements were taken at 25°C and the wavelengths ranged from 200 nm to 600 nm. To further confirm that the G-quadruplex structure of modified TBAs will be changed after UV-visible irradiation. We also measured the CD data of the sample after exposure to 365 nm UV light for different time. Data were levelled using the system software. UV irradiation in vitro experiments were carried out using handy UV lamp (Shanghai Baoshan, ZF-7A, 8 W, λ =365 nm). Visible irradiation was performed using general energy-efficient lamp (>400 nm, 9 W). Melting curve analysis of azobenzene-modified TBAs. Oligonucleotides were prepared at a concentration of 10 μM in the same buffer containing 100 mM KCl, pH 7.4. The samples were denatured by heating at 95 °C for 5 min and then slowly cooled to room temperature. Absorbance versus temperature curves were obtained by the melting method detected at 295 nm in the temperature range of 20-90 °C on a Jasco J815 spectrometer (Japan) equipped with temperature controller by using 0.5-mL quartz


31


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cuvettes with a 2-mm path length. A rate of 0.2 °C·min −1 was fixed because under it the melting and annealing curves were reproducible and strictly superimposable. The lack of hysteresis phenomena implied that the dissociation and association process of the quadruplex was in a state of thermodynamic equilibrium. Moreover, in the absence of visible light, the CD profile of the oligonucleotides was also measured after exposure to 365 nm UV light (20min). Surface plasmon resonance analysis. Real-time measurements of the interaction between thrombin with either TBA or the modified TBAs were performed using a Biacore T200 system. Thrombin (T6884, Sigma-Aldrich) was diluted in 5 mM sodium acetate (pH 5.0), and 1670 response units were immobilized via amine coupling to CM5 sensor chips were from GE Healthcare. Briefly, proteins were mixed with equal volumes of

freshly

prepared

N-hydroxysuccinimide

(NHS)

and

N-ethyl-

N’(dimethylaminopropyl)carbodiimide (EDC), and capping of unreacted carboxymethyl sites was achieved by injection of ethanolamine (pH 7.0). The composition of the running buffer and the sample analysis buffer was a PBS buffer (138 mM NaCl, 2.7 mM


32

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KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4, 100 mM KCl, 0.05% P20) at 25 °C. All the buffers were filtered and degassed before use. After loading CM5, PBS was used to wash the chip surface at 10 μL/min, the mixture of EDC and NHS was circulated through the surface at 5 μL/min for 10 min to activate the carboxyl. The system was balanced with PBS buffer for 3 h. The oligonucleotides were dissolved in PBS buffer at 2.0 μM, annealed (as described in the CD measurement section), and injected over the surfaces at different concentrations. All solutions were sequentially injected over the sensor surface for 3 min at 30 μL/min with a 5 min dissociation time beginning with the lowest concentration. What’s more, the sample after exposure to 365 nm UV light (20 min) was also measured and this measurement remains in the absence of visible light. The raw data were analyzed to determine the binding constant for each oligonucleotide. To correct for refractive index changes and instrument noise, responses from the control surface were subtracted from the responses obtained from the reaction surface using biospecific interaction analysis evaluation 4.1. The data were analyzed with Biacore evaluation software (T200 Version 2.0) by curve fitting using a 1:1 binding model.


33


Page 34 of 51

Blood clotting assay of Human Plasma. The inhibitory effect of TBA and the azobenzene-modified TBAs on the thrombin time was measured with a Pun-2048b Two Channel Blood Coagulation Analyzer (Beijing Poulenc Medical Science and Technology Co., Ltd, China). Venous blood was obtained in a tube containing 109 mM sodium citrate as the anticoagulant. The blood was subsequently centrifuged for 12 minutes at 3500 rpm at room temperature to remove blood cells, yielding plasma that was ready for analysis. The thrombin reagent (10 U/mL) was preincubated with TBA or the modified TBAs (2.2 μM) at 37 °C for 5 min before adding to the plasma for the measurement of thrombin clotting time in 1×PBS buffer. The anti-thrombin effect was assessed by the extra time required for clotting in the presence of aptamers as compared with the blank sample. In order to achieve our experimental design purposes, we used trans- and cisazobenzene modified TBAs to measure of thrombin clotting time, which was reversibly controlled via UV and visible light irradiation. ASSOCIATED CONTENT Supporting Information


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Supplementary Figure S1-S9, Table S1-S2. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Dr. Jing Wang (Peking University, CN) for assistance with SPR experiments. This work was supported by National Natural Science Foundation of China (Nos. 21778054, 51772289), National Key Research and Development Plan of China (No. 2016YFF0203703), Fusion Project of Molecular Science and Education for Institute of Chemistry

(No.

Y82901NED2),

UCAS

Students’

Entrepreneurship

Research

(No.118900EA12), Open Project Fund of State Key Laboratory of Natural and Biomimetic Drugs (No. K20150204). REFERENCES


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(50). Asanuma, H., Liang, X., Nishioka, H., Matsunaga, D., Liu, M., and Komiyama, M. (2007) Synthesis of azobenzene-tethered DNA for reversible photo-regulation of DNA functions: hybridization and transcription, Nat Protoc. 2, 203-212. (51). Chen, J., Zhang, L. R., Min, J. M., and Zhang, L. H. (2002) Studies on the synthesis of a G-rich octaoligoisonucleotide (isoT)2(isoG)4(isoT)2 by the phosphotriester approach and its formation of G-quartet structure, Nucleic Acids

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For Table of Contents Use Only

Reversible Photocontrol of Thrombin Activity by Replacing Loops of Thrombin Binding Aptamer using Azobenzene Derivatives

Mengwu Mo†, Dejia Kong†, Heming Ji†, Dao Lin†, Xinjing Tang‡, Zhenjun Yang‡, Yujian He*,†, Li Wu*,†,‡

Three

azobenzene

derivatives,

4,4’-bis(hydroxymethyl)azobenzene,

4,4’-

bis(hydroxyethyl)azobenzene and 3,3’-bis(hydroxymethyl)azobenzene, were introduced to replace loops of TBA G-quadruplex sequences. The impact of three azobenzene derivatives on G-quadruplex topology, thermodynamic stability, thrombin binding ability and thrombin activity, anticoagulant properties were investigated. Suitable azobenzene linker, capable to photoswitch the anticoagulant efficiency, was identified, and key loop positions were also suggested as an explanation for previous observations.


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Figure 2. Structures of 4,4’-bis(hydroxymethyl)azobenzene (a), 4,4’-bis(hydroxyethyl)azobenzene (b), 3,3’bis(hydroxymethyl)-azobenzene (c) and ODN sequences used in the study. 1, 2 and 3 represented TGT loop position, 5’-end TT loop and 3’-end TT loop position, respectively. 139x40mm (300 x 300 DPI)


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Figure 1. Quadruplex structures of azobenzene derivatives modified TBAs, and AZO represents azobenzene derivatives. 66x44mm (600 x 600 DPI)



Figure 3. Comparison of circular dichroism spectra and structural characteristics between different azobenzne-modified TBAs and TBA without UV irradiation. The concentration of TBA is 25 μM.


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Figure 4. Circular dichroism spectra and structural characteristics of TBA variants under UV irradiation for different times. Experiment was performed in phosphate buffered saline (138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4, 100 mM KCl ) by using a handy UV lamp (Shanghai Baoshan, ZF-7A, 8 W, λ=365 nm ). The concentrations of TBA variants are 25 μM.



Figure 5. Characterization of the affinity between thrombin and TBA variants without UV illumination. Their KD values were labelled in the corresponding curves. ND, no data.


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Figure 6. Characterization of the affinity between thrombin and TBA variants after UV illumination, Their KD values were labelled in the corresponding curves. ND, no data.


Reversible Photocontrol of Thrombin Activity by ... - ACS Publications

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