Before each SELEX cycle, fresh aliquots of TCS D and E matrices were washed three times with 200 µL of 1X BWB and individually resuspended in 200 µL of Blotto solution for two hrs at RT to reduce the likelihood of non-specific binding by the oligonucleotides to the matrix. The amount of TCS-conjugated matrices was adjusted for each cycle of selection depending on the amount of selection pressure desired for the cycle.
2.4.2 Heat-denaturing of ssDNA aptamer library
The synthetic N40 library (3 nmol) was resuspended in 200 µL 1X BWB. Thereafter, the library solution was heat-denatured for 10 mins at 90 °C and then cooled at 4 °C for 15 mins before further incubation at RT for 10 mins. The heat-denaturing step was also utilised to denature aptamer libraries (each ~ 50 pmol) from subsequent SELEX rounds. 2.4.3 Co-incubation of aptamer library with affinity matrices
Following heat-denaturing step, the aptamer library was added into each TCS-conjugated matrix solution (100 µL per matrix) and the resulting solutions were then incubated at 4 °C with gentle shaking. The incubation time was adjusted for each cycle of selection depending on the amount of selection pressure required.
2.4.4 Removal of unbound oligonucleotides
Following incubation, each aptamer-bound matrix was transferred into a filtered syringe column. Unbound oligonucleotides were removed by thoroughly washing aptamer- bound matrices using 1X BWB. The washing volume was increased for each cycle of selection in order to increase the selection pressure. The resulting matrices were each resuspended in 65 µL of DNAse free water and were used as templates for subsequent PCR amplification.
2.4.5 PCR amplification
PCR was undertaken to amplify aptamer-bound matrices for selection, dot blot assay, and cloning and sequencing procedures using different primer combinations (Table 2.2). A PCR sample was prepared at a final reaction volume of 50 µL using HotMasterTM Taq kit.
The PCR buffer was composed of 10 µM dNTPs, 1.1 µM of each primer, 2.5 mM Mg2+, and
2 units of Taq DNA polymerase. PCR cycles were carried out using the following steps: an initial denaturing step of 95 °C for 2 mins to ensure complete denaturation of the template; followed by 35 cycles of 94 °C for 40 sec (denaturing), 55 °C for 20 sec (annealing), and 72 °C for 30 sec (extension); and a final extension step at 72 °C for 2 mins.
2.4.6 Agarose gel electrophoresis
Agarose gel electrophoresis was used to separate and analyse PCR products following amplification. The PCR products were first incubated with 1.6 µL of 100X SYBR green dye for 8 mins at RT. Then, 6X DNA loading dye was added to the products to give a final concentration of 1X before loading the products into the wells of a 3 % (w/v) agarose gel prepared using 1X TAE buffer. The gel was run in a Mini-Sub Cell GT Gel Tank (Bio-Rad, Hercules, California, USA) submerged in 1X TAE buffer for 75 mins at 95 volts. Thereafter, the gel was visualised using a UVsolo TS stand-alone gel documentation system (CORE Life Sciences, Laguna Niguel, California, USA), and the presence of 75 mer dsDNA bands which contained the desired aptamer sequences were confirmed by size comparison with a low range DNA ladder. The identified bands of interest were excised from the gel using a scalpel blade and stored at -20 °C until required.
2.4.7 Extraction of dsDNA molecules
The desired aptamer sequences were extracted from the excised gels using a Qiagen mini elute dsDNA extraction kit. The protocol used for extraction was specified in the manufacturer’s guidelines with following modifications: after dissolving the excised gels in QG buffer and addition of isopropanol, the resulting solution was vortexed for 10 sec before being centrifuged at 6,000 x g for 1 minute using mini elute columns. To maximise the retention of aptamers on the column matrix, each aliquot of the flow-through solutions was passed through 5 times before being discarded. This was followed by washing the column with 500 µL buffer QG and then centrifuged at 6,000 x g for 1 min. Thereafter, all column washing and centrifugation steps were carried out as stated in manufacturer’s guidelines. The dsDNA preparations were eluted from each column membrane, using a 1:50 dilution of EB buffer. The first elution step was carried out using 50 μL of EB followed by a second elution step using 30 μL of EB. To stabilise the dsDNA products and to enable an improved strand separation yield, 80 μL of 2X BWB was added to the eluate. The eluted dsDNA preparations were then stored at -20 °C until future use
2.4.8 Strand separation
Alkaline denaturation of dsDNA preparations was the method of choice for generating ssDNA aptamers for either the sequential round of SELEX or an intended-dot blot assay. 2.4.8.1 Strand separation to generate ssDNA aptamers for selection
To carry out the next round of SELEX, dsDNA preparations were subjected to a strand separation process, since only forward strands are utilised during SELEX. In brief, a 30 μL aliquot of streptavidin-coated magnetic beads (SMBs) was pipetted into a micro- centrifuge tube before being recovered using a magnetic pipette (PickPen) with silicone tips and transferred to a microcentrifuge tube containing 50 μL of 1X BWB. The SMBs were stirred using pipette tip for a thorough washing and then transferred to a new tube. This washing step was repeated three times, and the washed SMBs were incubated with 160 µL of each biotin-labelled dsDNA preparation. The resulting solution was incubated at RT for 20 mins with periodic mixing to allow for the formation of SMB-biotin-dsDNA complexes. Following incubation, the complexes were washed with 50 µL of 1X BWB three times, and then mixed with 30 μL of 0.3 M NaOH. The mixture was then incubated for 3 mins at RT, followed by removing SMB complexes (i.e. the SMB-biotin-reverse strands) using the PickPen.The leftover solution containing only the forward strands was neutralised immediately by adding 8.5 µL of 1 M HCl. The resulting solution containing ssDNA aptamers was used as the aptamer library for the subsequent round of selection. 2.4.8.2 Strand separation to generate ssDNA aptamers for dot blot assay
Preparation of biotin-labelled ssDNA aptamers for dot blot assay was similar to that for selection (see Section 2.4.8.1) with minor modifications. In brief, the biotin-labelled dsDNA preparations (each 160 µL) were incubated with the washed SMBs (30 µL for each preparation). The protocol for SMB washing and incubation with the biotin-labelled dsDNA molecules was performed as described previously (see Section 2.4.8.1). After formation of SMB-biotin-dsDNA complexes, strand separation was performed by adding 30 μL of 0.3 M NaOH, which produced the SMB bound forward strands (retained on the beads) and the unbound reverse strands (in solution).
The forward strand-biotin-SMB complexes were then transferred to a new tube and treated with 30 µL of 0.3 M NaOH for a further 1 min to ensure complete dissociation of the reverse strands from the complexes. Thereafter, the forward strand-SMB complexes were washed with 50 μL dH2O for three times. Following washing steps, the forward
strands were dissociated from the SMB-biotin forward strand complexes by heating the complexes in an 85 °C water bath for 2 mins to break biotin-streptavidin bonds. The SMBs were removed using the PickPen and the leftover solution containing the biotin-labelled forward strands was then used for the subsequent dot blot assay.