AGAROSE DROPLET MICROFLUIDIC APPROACH FOR MOLECULAR

EVOLUTION OF APTAMERS Wenhua Zhang, Weiyun Zhang, Zhiyuan Liu, Cong Li, Gareth Jenkins, Chaoyong James Yang *

Department of Chemical Biology, Xiamen University, Xiamen 361005, P. R. China

ABSTRACT

We have developed a novel method for efficient screening of affinity ligands (aptamers) from a complex single-stranded

DNA (ssDNA) library by employing single molecule emulsion PCR based on agarose droplet microfluidic technology . Statisti-

cally diluted ssDNA of a pre-enriched library was encapsulated into individual uniform agarose droplets for highly efficient

single molecule emulsion PCR to generate clonal agarose beads. The binding affinity of amplified ssDNA from each clonal

agarose bead was tested via high-throughput flow cytometry. DNA clones with high binding affinity and low dissociation

constant (Kd) were chosen as potential aptamers and can be directly used for downstream biomedical applications.

KEYWORDS: Aptamer, Molecular Evolution, Microfluidics, Droplet

INTRODUCTION

System evolution of ligands by exponential enrichment (SELEX) [1] is an in vitro conventional aptamer screening tech-

nique, which involves progressive selection of aptamers by repeated rounds of partitioning and amplification from a combina-

torial oligonucleotide library. In a typical SELEX process, a pre-enriched library is sequenced first, and tens to hundreds of ap-

tamer candidates are screened via a bioinformatic approach. Possible candidates are then chemically synthesized, and their

binding affinities are measured individually. Such a process is time consuming, labor-intensive, inefficient, and expensive. To

address these problems, we have developed a novel method for the efficient screening of affinity ligands (aptamers) from a

complex single-stranded DNA (ssDNA) library by employing single molecule emulsion PCR based on agarose dro plet micro-

fluidic technology. To illustrate the principle of this new method, selection cycle is performed using the cancer biomarker pro-

tein Shp2 [2] as target to obtain a pre-enriched ssDNA library. Compared to a conventional sequencing-chemical synthesis-

evaluation work flow, this novel aptamer screening approach not only avoided repeat expensive chemical synthesis and s e-

quencing, but also reduced the probability of possible aptamer loss as far as possible. In this regard, it will find its wide appli-

cation in molecular evolution technologies including mRNA display, phage display and so on.

DESIGN

The principal steps of our agarose droplet microfluidic approach for aptamer screening was schematically depicted in Fig-

ure 1. Our proposed method capitalized on the unique thermoresponsive sol-gel switching property of agarose for highly effi-

cient ssDNA amplification and amplicon trapping [3]. ssDNA of pre-enriched library as template is introduced into the PCR mix

in a statistically diluted concentration according to Poisson distribution so that on average there will be no more than one

template in one droplet. High throughput of uniform agarose droplets generated via a microfluidic chip offers the advantage of

massively parallel individual DNA template. After encapsulation and amplification of DNA templates, binding affinity and Kd

of droplets containing individual DNA were determined by means of flow cytometry. Only the specific positive clones con-

taining DNA template with high binding affinity and low Kd values which are potential aptamers were sequenced.

Fig. 1 Schematics of aptamer selection by agarose droplet microfluidics technology. Statistically diluted ssDNA of the

pre-enriched library were encapsulated into individual droplet displaying Poisson distribution and single molecule emul-

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 64 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

sion PCR in picolitre-volume droplet was performed. The binding affinity of amplified ssDNA with Shp2 protein was deter-

mined by flow cytometry. ssDNA with high binding affinity and low Kd value were selected as aptamers for sequencing.

EXPERIMENTAL

The emulsion generator was constructed from two glass wafers. The glass channels were rendered hydrophobic with octa-

decyltrichlorosilane (OTS) treatment. The PCR cocktail was prepared at 65 °C by mixing 2% agarose, 5 U/μl EasyTaq DNA po-

lymerase, 10 × EasyTaq buffer, 2.5 mM dNTPs each, 10 μM primers (5’-AGC GTC GAA TAC CAC TAC AG -3’ , 5’-CTG ACC

ACG AGC TCC ATT AG-3’) amplifying a 80bp sequence of pre-enriched ssDNA library and specified amounts of template.

The thermal cycling conditions were as follows: 94°C for 3 min, 25 cycles of 94°C for 30 sec, 53°C for 30 sec, and 72°C for 30

sec, followed by a single final extension for 5 min at 72°C in a peltier thermal cycler. Agarose beads carrying amplicons were

solidified by cooling to 4 °C for 1 h. After removing the oil phase in the supernatant by pipette, agarose beads were stained in

60 μl oil phase with 3 μl 20-fold SYBR Green.

Agarose beads were diluted with oil and bright beads in which amplified ssDNA were encapsulated were picked out ma-

nually using a capillary tube. One hundred beads obtained as templates were then amplified with primers by conventional PCR.

Consequently, the PCR products were amplified with FAM-labeled sense primer and biotin-labeled antisense primer and then

incubated with streptavidin-coated Sepharose beads . After denatured in NaOH (0.1 M), FAM-labeled ssDNA was separated

from biotinylated antisense ssDNA strand on streptavidin-coated Sepharose beads by a homemade filter column and purified

by NAP-5 desalting column. Consequently, concentration of the FAM-labeled ssDNA was determined using a Nanodrop and

concentrated by vacuum freeze drying.

The FAM-labeled ssDNA (100 nM) was dissolved in 200 µl binding buffer, (100 mg tRNA and 5 ml of 1 M MgCl2 were add-

ed into 1 liter of phosphate buffered saline), denatured by heating at 95 °C for 5 min, then quickly cooled on ice for 10 min, and

subsequently incubated with Shp2 beads , which were prepared according to reference [4], at 37 °C for 45 min in the dark. Next,

beads were washed 3 times with binding buffer by means of filtration and re-suspended in 400 µl binding buffer. Finally, the

fluorescence was determined using a flow cytometer by counting 10000 events. The FAM-labeled ssDNA with high binding

affinity was further studied to determine its Kd value with Shp2 protein interaction. Potential aptamers with low Kd value were

then amplified, cloned and sequenced.

RESULTS AND DISCUSSION

A microfabricated agarose droplet generator that produces uniform, nanoliter-volume agarose emulsion droplets for apta-

mer screening was developed and evaluated. Highly uniform monodisperse droplets which are large enough to pick out were

observed in a bright-field microscope. Images of agarose beads after PCR amplification from template concentration of 0

copy/bead (Fig. 2C) and 0.3 copy/bead (Fig. 2D) were stained by SYBR Green and captured by fluorescence microscope. The

bright beads in Fig. 2D indicated that DNA template was successfully encapsulated and amplified, while the dark beads ind i-

cated that no DNA template existed. As a result, bright beads were picked out using a capillary tube for further analysis.

Fig. 2 Individual amplification of ssDNA in agarose droplet. Generation of agarose droplet on chip (A). Uniform drop-

lets in oil (B). Fluorescence microscope images of agarose beads after PCR amplification from template concentration of 0

copy/bead (C) and 0.3 copy/bead (D). The bright bead indicated that ssDNA was encapsulated and amplified. The scale

bar is 50 μm.

To assess the binding affinity of individual ssDNA amplification with Shp2 protein, flow cytometry was applied. As we

can see from Fig. 3, the transition of signal strength from 0 pool to 10 pool showed a detectable onset of enrichment. Co m-

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pared with 10 pool, there was a clear fluorescence signal shift of the 21st positive clone, 15th positive clone and 18th pos itive

clone, which were DNA amplifications from three of one hundred bright beads. However, DNA amplifications from the other

picked beads did not exhibit obvious fluorescence shift, compared with 10 pool. Hence, their Kd values with Shp2 protein inte-

raction were calculated using SigmaPlot software. After calculation, Kd value of the 21st positive clone and 15th positive

clone are 68.7 ± 43.9 nM and 354.0 ± 359.9 nM, while Kd values of the 18th positive clone was beyond the nanomolar range.

Accordingly, the 21st positive clone was selected for the further investigation. The sequence of the 21st positive clone was

synthesized and its Kd value was re-determined. In the dissociation curve of aptamer-Shp2 protein interaction as analyzed by

SigmaPlot software, the 21st positive clone did not show high binding affinity with GST protein, unselected library (0 pool)

non-specifically bound to Shp2 protein, while the 21st positive clone specifically bound to Shp2 protein with a Kd value of

24.9 ± 14.6 nM (Fig. 4), indicating that it is an aptamer against Shp2 protein.

Fig. 3 A. Binding assay of positive clones with Shp2 protein monitored by flow cytometry. B. Kd determination of 21st

positive clone by flow cytometry using SigmaPlot software. Kd of 21st positive clone is 24.9 ± 14.6 nM. The mean fluores-

cence intensity of each concentration for 21st positive clone-Shp2 protein interaction was examined with 3 replicates.

CONCLUSION

Taking advantage of the unique thermoresponsive sol-gel switching property of ultra-low gelling agarose, highly efficient

individual ssDNA amplification of pre-enriched library and amplicon trapping were achieved. After single molecule emulsion

PCR in agarose droplets which serve as microreactors, the binding affinity and Kd value of amplified individual ssDNA was

determined by flow cytometry. Only the amplified ssDNA with high binding affinity and low Kd was sequenced, therefore, ap-

tamer selection was successfully achieved in an efficient, cost effective and sensitive manner. Based on the improvement in

evaluation time and reduction in reagent consumption, our proposed agarose droplet microfluidic approach could be widely

utilized for SELEX and molecular evolution technologies including mRNA display, phage display and so on.

ACKNOWLEDGEMENTS

This work was supported by the Natural Science Foundation of Fujian Province, China (2008J0107, 2010J06004), the Na-

tional Scientific Foundation of China (20805038, 21075104, 30772546, 20620130427) and the National Basic Research Program of

China (2007CB935603, 2010CB732402, 2010CB945004). We thank Dr. Gensheng Feng at the University of California San Diego

for the gift of the plasmid pGEX4T1-Shp2.

REFERENCES

[1] A. D. Ellington, J.W. Szostak, “In vitro selection of RNA molecules that bind specific ligands ,” Nature,Vol. 346, PP.818-822.

Aug.1990

[2] J.M. Cunnick, S. Meng, Y. Ren, C. Desponts, H.G. Wang, J.Y. Djeu, J. Wu, “Regulation of the mitogen-activated protein

kinase signaling pathway by SHP2,” J Biol Chem, Vol. 277, PP.9498-9504. Jan. 2002

[3] X. F. Leng, W. H. Zhang, L. Cui, C. J. Yang, “Agarose droplet microfluidics for highly parallel and efficient single molecule

emulsion PCR,” Lab Chip, Vol. 10, pp. 2841-2843, Aug. 2010

[4] J. Hu, J. Wu, C. Li, L. Zhu, W. Y. Zhang, G. Kong, Z. Lu, C. J. Yang, “A G-quadruplex aptamer inhibits the phosphatase ac-

tivity of oncogenic protein Shp2 in vitro,” Chembiochem,Vol. 12, PP. 424-430, Feb. 2011

CONTACT

*C.Y. Yang, tel: +86 5922187601; cyyang@xmu.edu.cn

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