Synthesis and structure investigation of stabilized aromatic oligoamides and their interaction with g quadruplex structures 7

Chapter 7 Colorimetric Screening of G-Quadruplex-Binding Ligands by Aptamer-Modified Gold Nanoparticles 7.1 Introduction Aptamers are short, single-stranded nuclic acid-based molecule

Chapter 7

Colorimetric Screening of G-Quadruplex-Binding Ligands by

Aptamer-Modified Gold Nanoparticles

7.1 Introduction

Aptamers are short, single-stranded nuclic acid-based molecules that can selectively recognize molecular targets (proteins, nucleic acids or small molecules) with high affinities They are selected from the pools of random sequences in vitro by systematic evolution of ligands by exponential enrichment (SELEX).1, 2 Because of their chemically stability and excellent controllability, they have been extensively applied in biological research,3 clinical therapeutics,4 sensing5 and molecular recognition.6

Aptamers possessing G-quadruplex structural motifs have been developed as useful biologics for binding or inhibiting specific proteins.7 Recently, some G-quadruplex aptamers have been proven to bind hemin to form a new class of DNAzyme with the peroxidase-like activity.8 An aptamer able to inhibit human Ribonuclease (RNase) H1 activity was also found to fold into a G-quadruplex.9 In addition, numerous viral and cellular proteins interact with quadruplex DNA in vivo.10 One of the most known aptamers capable of forming an intramolecular G-quadruplex motif is TBA (15-mer Thrombin Binding Aptamer) which can be used as an inhibitor of blood coagulation.11-14 Thrombin is coagulation protein that plays many roles in the

coagulation cascade - it converts soluble fibrinogen into insoluble strands of fibrin, and catalyze many other coagulation-related reactions Therefore, thrombin is usually considered as an important target when searching for anti-coagulants and antithrombotics to interfere with the blood coagulation process Consequently, TBA has been widely studied due to their efficiency in recognizing thrombin and acting as anticoauglants

However, the overdosing of anticoagulants may cause adverse effects To ensure TBA with controllable anticoagulating performance, TBA-based anticoagulant/antidote effector pairs have been studied.15-18 Hartig et al has confirmed the capability to antagonize the anticoagulant activity of TBA by cationic porphyrin 5,10,15,20‐tetra(N‐methyl‐4‐pyridyl)porphin (TmPyP4), a well-known quadruplex-binding compound.16 However, since then, few studies have been focused

on the development of novel TBA ligands, which could serve as antidote of the anticoagulant

Traditional methods such as circular dichroism (CD) and isothermal titration calorimetry (ITC) were used to identify TBAs.19 However, these techniques are both time- and DNA-consuming Recently, polymererase chain reaction (PCR) stop assay was widely used because of its high sensitivity and specificity.20 Nevertheless, most existing PCR-based protocols require complicated equipments and are not suitable for high-throughput screening Therefore, a new screening system which could quickly identify antidotes for anticoagulant needs to be developed

Herein, we developed a new AuNPs screening assay to detect the effects of small

molecules on the interaction between thrombin and its aptamers Furthermore, a new class of macromolecules was synthesized and evaluated as the stabilizers of G-quadruplex, which could also serve as antidote to TBA as evidence from our AuNPs screening assay

7.2 Result and Discussion

7.2.1 Design of Aptamer-Based AuNPs Screening Assay

AuNPs-based molecular recognition approaches are emerging as attractive colorimetric probes due to their high sensitivity and selectivity.5 Recently, a method based on enzymatic manipulation of DNA on AuNPs was reported for screening G-quadruplex ligands.21 Our strategy for detecting antidote of TBA was illustrated in Figure 7.1 Three key components are needed: gold nanoparticles (13nm diameter) functionalized with 3’-thiol-modified TBA, thrombin, and ligands able to bind G-quadruplex region of TBA Gold-nanoparticles were induced to form aggregation

by adding thrombin into the solution due to the interaction between protein and its aptamer However, AuNPs aggregation could dissemble after the introduction of small molecules into the above system This is attributed to the interaction between ligands and G-quadruplex aptamer could interfere with the binding of the aptamer toward thrombin In contrast, small molecules which are not G-quadruplex ligands will not disaggregate AuNPs since the association between protein and TBA is not disrupted

by small molecules

Figure 7.1 Illustration of AuNPs screening assay for identifying G-quadruplex ligands

7.2.2 Application of aptamer-based AuNPs screening assay

O

N

O

HN

O HN

O NH

NH

O

OMe

OMe

MeO

MeO OMe

1

O N

O

HN

O HN

O NH

NH O OH OMe

MeO

MeO OMe

9a

O N

O

HN

O HN

O NH

NH O OH OMe

MeO

HO OMe

10a

O

HN

O HN

O NH

NH

O

OH

O Me

HO

MeO OH

11a

O

HN

O HN

O NH

NH O OH OMe

MeO

HO OH

10b

Scheme 7.1 Circular pentamers as potential G-quadruplex binders

A new category of macrocylic molecules with an aromatic oligoamide backbone and different interior groups was synthesized in our lab (Scheme 7.1) According to our previous study, those oligomides could serve as potential ligands for binding

G-quadruplex In order to characterize their interaction with TBA, PCR stop assay was first used Based on the PAGE result, the double-stranded PCR product

disappeared when the concentration of 10a was higher than 1 μM, suggestive of the binding interaction between 10a and TBA (Figure 7.2a)

a)

b)

Figure 7.2 Effect of 10a on the double-stranded PCR product synthesized from (a) TBA template, (b)

Control DNA which can not form G-quadruplexes structure ds = double-stranded and ss = single-stranded

Next, the feasibility of AuNPs screening approach was tested against 10a As expected, by adding 10a into the system containing TBA-modified AuNPs (Au-TBA) and thrombin, AuNPs aggregation was inhibited (Figure 7.3) This indicates that 10a

could disrupt the thrombin-TBA interaction As in Figure 7.4, the shift of peak from

538 to 524 nm by the addition of 10a, which also indicates that 10a could hinder the AuNPs aggregation This result correlates well with PCR stop assay, suggesting 10a

could bind to TBA G-quadruplex region.

Lane

10a ( μM)

0

ds

ss

0 10a ( μM)

ds

ss

Figure 7.3 Gold nanoparticles-based colorimetric detection of G-quadruplex-binding molecule: (a)

Au-TBA only, (b) Au-TBA in the presence of thrombin, (c) Au-TBA in the presence of thrombin and

10a (2 μM)

Figure 7.4 UV-vis spectra of Au-TBA with both thrombin and 10a and with thrombin only

7.2.3 Sensitivity of Aptamer-Based AuNPs Screening Assay

To investigate the sensitivity of aptamer-based AuNPs assay, the minimum concentration that could effectively inhibit AuNPs aggregation was studied As in Figure 7.5, even though the aggregation of gold nanoparticles remained when the

concentration of 10a is lower than 1 μM, at 0.75 μM, partial disaggregation of AuNPs

is evidenced Coincidently, the lowest concentration that could inhibit the reaction of DNA G-quadruplex polymerase in PCR stop assay was also 1 μM for 10a in the previous study This result indicates that the sensitivity of this AuNPs screening assay

300 400 500 600 700 800 0.2

0.3 0.4 0.5 0.6 0.7

Wavelength (nm)

is as good as PCR stop assay

Figure 7.5 Solution color change involving AuNPs in the presence of 10a at different concentrations:

(a) 0.25 μM, (b) 0.5 μM, (c) 0.75 μM, (d) 1 μM, (e) 2 μM

7.2.4 Screening oligomers that binds TBA

To demonstrate its generality and utility, a series of different aromatic oligoamides were studied using the aptamer-based AuNPs screening system (Figure 7.6 and Figure

7.7) Both the colorimetric assay and UV-via spectra suggest 10a, 11a and 10b could

serve as antidotes of TBA by binding TBA and so disrupting the protein-TBA interaction On the other hand, aggregation of gold nanoparticle still occurs in the

presence of either 1 or 9a, suggesting that either 1 or 9a display weak binding toward

TBA

Figure 7.6 Colorimetric assay of Au-TBA with thrombin and different oligomers: Photographs of

interaction between thrombin and aptamer inhibiting by (a) 10a (b) 11a, (c) 10b, (d) 9a, (e) 1

Figure 7.7 UV-vis spectrum of Au-TBA with both thrombin and different oligomers

PCR stop assays were also performed for all the circular pentamers As shown in

Figure 7.8, 10a, 11a, 10b showed the ability to inhibit double-stranded PCR product, whereas double-stranded DNA could still be detected with the addition of 1 or 9a

This PCR stop assay result correlates well with the colorimetric result of

AuNPs-based screening assay, indicating that only 10a, 11a, 10b could bind to TBA

These results were in accordance with our previous study, showing that the presence

of more interior hydroxyl groups results in higher binding affinity of aromatic oligoamides toward thrombin aptamer

Figure 7.8 PCR stop assay for identifying TBA-binding molecules at 2 μM In the absence of pentagon-shaped molecules, PCR stop assay produced ds DNA product (lane 1); addition of pentamers

10a (lane 2), 11a (lane 3), and 10b (lane 4) completely suppressed the production of ds DNAs while

the production of ds DNAs was largely suppressed in the presence of pentamers 1 (lane 5) and 9a (lane

6); shows the position of ss DNA (lane 7)

ds

ss

300 400 500 600 700 800 0.05

0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75

Wavelength (nm)

10a 11a 11b 9a 1

Detailed PAGE studies for PCR stop assay were shown in Figure 7.9 For 10a, 11a,

10b, the detection limits were all about 1 μM

a)

b)

Figure 7.9 Effect of 10b and 11a on the double-stranded PCR product synthesized from TBA

template

As shown in Figure 7.10, when the concentrations of 10a, 11a, 10b were lower than

1 μM, double-stranded DNA PCR product could be detected by PAGE This is consistent with the phenomenon of colorimetric assay in Figure 7.5, which showed the aggregation of gold nanoparticles when the concentration of 10a was lower than 1

μM

Figure 7.10 Effect of 0.25 μM and 0.5 μM of 10a, 10b and 11a on the double-stranded PCR product

synthesized from TBA template

Acylic oligomers were also tested by this AuNPs screening assay and were found to

Lane

11a (μM)

0

ds

ss

Lane

10b ( μM)

0

ds

ss

Lane

Conc ( μM)

0.5 0.25 0.5 0.25 Oligomer 10a 10a 11a 11a 10b 10b

0.5 0.25 ds

ss

be unable to disintegrate the aggregated AuNPs (Figure 7.11 and Figure 7.12) This result reinforces our previous finding, demonstrating that helically folded acylic oligomers do not lead to the stabilization of G-quadruplex structure

NO 2

N O H O

O O

O

OC 8 H 1 7

(H 3 C) 2 HCO

N H O

O

NO 2

N O H O

O O

O

OCH(CH 3 ) 2

OC 8 H 1 7

N H O

O N N

O H O

O O

O

O N H O

O 2 N O

OCH(CH 3 ) 2

5d

Scheme 7.2 Acyclic aromatic oligoamides

Figure 7.11 PCR stop assay illustrating the production of ds DNA product in the presence of no small

molecule (lane 1), 2c (lane 2), 3f (lane 3) and 5b (lane 4)

Figure 7.12 Colorimetric assay of acyclic oligomers showing (a) the disaggregation of AuNPs in the

absence of acyclic oligomers and the aggregation of AuNPs induced by (b) thrombin, (c) thrombin + 2c, (d) thrombin + 3c, (e) thrombin + 5d

7.2.4 Specificity of Aptamer-based AuNPs Screening Assay

To provide evidence that the inhibition of AuNPs aggregation in the designed screening assay was caused by the interaction between the aptamer and small molecules, lysozyme was chosen as the control protein In the study carried out in

chapter 6, we observed that lysozyme could also induce the AuNPs aggregation due to the nonspecific interactions between lysozyme and DNAs attached to the AuNPs With the addition of various pentamers into the solution containing DNA-modified AuNPs and lysozyme, AuNPs aggregation was still observed (Figure 7.13) This result indicates that those pentamers can not disrupt the nonspecific interactions between lysozyme and DNAs In contrast, binding between specific protein and its aptamer such as TBA could be specifically disrupted by small molecule capable of strong binding toward TBA Thus, the developed AuNPs screening assay allows the efficient and specific identification of G-quadruplex-binding ligands

Figure 7.13 Aggregation states of Au-TBA in the presence of both lysozyme and pentamers: (a) 10a,

(b) 11a , (c) 10b, (d) 9a, and (e) 1

Figure 7.14 UV-Vis spectra of Au-TBA containing 10a and lysozyme or 10a and thrombin

7.2.5 Aromatic pentamers can also bind to the 29-mer thrombin aptamer

350 400 450 500 550 600 650 700 750 800 0.1

0.2 0.3 0.4 0.5 0.6 0.7

Wavelength (nm)

Lysozyme with 10a Thrombin with 10a

The interaction between circular pentamers and 29-mer thrombin aptmer TBA2 were also studied using the developed AuNPs screening assay, which combines with

UV-vis spectra to demonstrate that 10a, 11a and 10b could effectively bind TBA2,

presumably through its G-quadruplex region akin to their binding toward TBA

Figure 7.15 Visualizing the interactions between circular oligomers and TBA2: Au-TBA2 with (a)

thrombin only, (b) with both thrombin and 1, (c) with both thrombin and 9a, (d) with both thrombin and 10b, (e) with both thrombin and 11a, (f) with thrombin and 10a

7.3 Conclusion

In summary, aptamer-based AuNPs screening assay was proven to be an efficient way to detect antidote of anticoagulant TBA In addition, our new approach could also be applied as a general method for identifying G-quadruplex-binding ligands, such as aptamers of PDGF AuNPs is better than PCR stop assays considering about time (instant detection), expense (requiring no detection device) and detection accuracy (slightly higher than PCR stop assay)

7.4 Experiment Section

Performance of Taq Polymerase Stop Assay:

Thrombin aptamer and the corresponding complementary sequence 5'-TCTCTGTCACCAACCACA-3' were used The chain-extension reaction was

performed in PCR buffer containing 0.2 mM dNTP, 2.5 U Taq polymerase, 7.5 pmol oligonucleotides, 50 mM KCl or NaCl, and various concentrations of cyclic pentamers The mixtures were incubated in a thermocycler with the following cycling conditions: 94 oC for 30 s, 47 oC for 30s and 72 oC for 30 s PCR products were resolved on 20% native polyacrylamides gels in 1*TBE buffer and stained with Sybe Gold The intensity of Sybe Gold by measured and quantified

Preparation of Aptamer-based AuNPs screening assay:

The 3’-terminal disulfide groups of the Thrombin binding apatamer strands were first cleaved by soaking them in a 0.1 M dithiothreitol (DTT) phosphate buffer solution (0.1 M phosphate, pH 8.0) for 2 hours and subsequently purified on a NAP-5 column (GE Healthcare) To 800 μL of gold colloid solution was added 3 nmol of the purified oligonucleotide The solution was brought to 0.3 M NaCl, 10 mM NaH2PO4/Na2HPO4, pH 7.0 buffer (0.3 M PBS) gradually by adding aliquots of 3 M NaCl and 0.1 M NaH2PO4/Na2HPO4, pH 7.0 buffer solutions every 12 hours After 48 hours, the nanoparticle solutions were centrifuged and re-dispersed in 750 μL buffer (0.3 M PBS) The final concentrations of the DNA on the probe were estimated to 0.3

μM