A plug-and-play aptamer diagnostic platform based on linear dichroism spectroscopy

A plug-and-play sandwich assay platform for the aptamer-based detection of molecular targets using linear dichroism (LD) spectroscopy as a read-out method has been demonstrated. A 21-mer DNA strand comprising the plug-and-play linker was bioconjugated onto the backbone of the filamentous bacteriophage M13, which gives a strong LD signal due to its ready alignment in linear flow. Extended DNA strands containing aptamer sequences that bind the protein thrombin, TBA and HD22, were then bound to the plug-and-play linker strand via complementary base pairing to generate aptamer-functionalised M13 bacteriophages. The secondary structure of the extended aptameric sequences required to bind to thrombin was checked using circular dichroism spectroscopy, with the binding confirmed using fluorescence anisotropy measurements. LD studies revealed that this sandwich sensor design is very effective at detecting thrombin down to pM levels, indicating the potential of this plug-and-play assay system as a new label-free homogenous detection system based on aptamer recognition.


DNA strands
All DNA strands were bought from commercial suppliers except for PPL, which had been prepared and characterized previously. [S1]. The dye 6-carboxyfluorescein dye (6-FAM) and disulfide reagents used for the synthesis of PPL are shown below.

Synthesis
The general procedure for M13 bacteriophage isolation from bacterial culture, the two-step conjugation procedure to DNA, and the subsequent purification and quantification were all performed as described previously. [S1, S2, S3]. A batch of the DNA-functionalised bacteriophage, M13-PPL that had been prepared for earlier work [S1] was used again here. For convenience, a summary of the synthetic procedure used is outlined below.

M13-PPL Purification and Characterisation
The conjugated phage M13-PPL was purified by SEC ( Figure 2a) using an ÄKTA Explorer 10 purification system. The SEC column (Superdex S200 HiLoad 16/60) was first washed with 1.2 column volumes of MilliQ® water and then equilibrated with 1.2 column volumes of 100 mM potassium phosphate buffer, 150 mM NaCl, pH 7.2. The flow rate was set at 1.0 mL/min for 1.2 column volumes with 100 mM potassium phosphate buffer, 150 mM NaCl, pH 7.2. Three absorbances were detected A269, A280 and A495. The eluent collected as 2 mL fractions with a Frac-950 auto collector (Amersham Pharmacia Biotech). Fractions of interest containing the DNAconjugated phage were combined and concentrated using MWCO spin concentrators by centrifugation at 4000 rpm at 4 °C. Each sample was washed with 1.0 mL of 100 mM potassium phosphate buffer, 150 mM NaCl, pH 7.2 and reduced to 200 µL and the sample removed. The spin concentrator was washed with 3 x 200 µL of 100 mM potassium phosphate buffer, 150 mM NaCl, pH 7.2 and added to the sample. As described previously [S1, S2], the average number of DNA strands appended to each chassis in M13-PPL was estimated as 15 from the UV/vis spectrum ( Figure 2b) by knowing the molar extinction coefficients of the M13 phage at 269 nm and 6-FAM at 495 nm.

CD studies
Circular dichroism (CD) experiments were carried out using a Jasco J-1500 spectropolarimeter (Jasco, Japan) at 293 K using a 1 cm pathlength 6Q quartz cuvette. A baseline was recorded using the corresponding buffer to the sample and subtracted from the sample spectra. All measurements were conducted with the following parameters: Range 190-350 nm, Response 1s, Data pitch 0.5 nm, Scanning mode Continuous, Scan speed 50 nm/min, Band width 1 nm, and Accumulation 3. The CD spectra (Fig. S1) of the original (TBA and HD22) and extended (TBA15 and HD22T5) aptamers were very similar in three different solvent conditions (water alone, water with KCl added to induce folding and thrombin binding (TB) buffer). S 5

EMSA Gel Studies
Each sample was prepared in 20 mM Tris-HCl, 1 mM MgCl2, 120 mM NaCl, 10 mM KCl, 2 mM CaCl2, pH 7.4 and 3% glycerol. All aptamer strands were denatured at 90 °C for 5 minutes and allowed to cool at RT slowly before addition to thrombin or BSA. These mixtures were incubated at 37 °C for 30 minutes to ensure equilibration. Gel EMSAs were performed using 12% acrylamide 0.8% bisacrylamide with 10 mM potassium chloride, 1 x TBE, 10% APS (140 µL) and TEMED (70 µL) in a 1 x TBE + 10 mM potassium chloride, pH 8.3 electrophoresis buffer. The gels were performed at 100 V for 120 min and visualized with SYBR GOLD® nucleic acid stain (15 min) using an ultraviolet transilluminator fitted with a camera. Post DNA staining was conducted with InstantBlue™Ultrafast Protein Stain for 15 minutes, with subsequent treatment with water (15 min). A protein stain of the same gel used for DNA visualisation (main text Figure 1) indicated a series of protein bands for the thrombin sample alone ( Figure S2).

LD Studies
LD spectroscopy was carried out according to previously reported procedures [S1, S2, S3]. For this this work, a Jasco J-715 spectropolarimeter (Jasco, Japan) was used, which had been modified for linear dichroism spectroscopy. Samples (60 µL) were placed into a micro-couette cell apparatus within the LD sample chamber. A baseline was recorded (with a non-rotating capillary) and subtracted from the sample with a couette rotating at 3 V and the signal zeroed at 800 nm. All measurements were conducted with the following parameters: Range 190-800 nm, Sensitivity 0.1 dOD, Data pitch 1.0 nm, Scanning mode continuous, Scan speed 200 nm/min, Band width 2 nm, and Accumulation 3. Initial samples were incubated for 30 min before addition of target aliquots, with an additional incubation time of 10 minutes after each addition.
The LD spectrum of M13-PPL was very similar to that of unfunctionalised M13, as reported previously [S1].