In vivo SELEX

All oligonucleotides, including DNA libraries and primers, were synthesised by Ella Biotech GmbH (Munich, Germany). Two separate 80-nt single-stranded DNA libraries, D3 and D3P, were used consisting in a 43-nt random region. D3P library contained an 11 kDa polyethylene glycol (PEG) moiety on the 5′-end. Both libraries were amplified by PCR using the following primers: forward primer (Fw) 5′-GCTGTGTGACTCCTGCAA-3′, with 5′–11kDa PEG moiety in the case of D3P library, and reverse primer (Rv-Pho) 5′-Phosphate-GGAGACAAGATACAGCTGC-3′). PCR reaction was performed by using GoTaq® G2 Flexi DNA Polymerase (Promega) and 1 µM of both Fw and Rv-Pho primers with the following cycling program (2 min 95 °C; 30 sec. 95 °C, 30 sec. 64 °C, 45 sec. 72 °C; hold 10 °C) in a Veriti 96 well thermal cycler (Applied Biosystems).

In vivo selection was done using tumour-bearing animals. Mice were either treated intravenously with one of two aptamer libraries (D3 or D3P) or left untreated. Five nmol of libraries (consisting of one copy of each sequence) were injected in cycle 1 and the amount was reduced to 2, 1 and 0.5 nmol for cycles 2, 3 and 4 respectively. From cycle 5 to 10, 0.1 nmol of the libraries were injected. Prior injection, libraries were prepared in 110 µL of Dulbecco’s phosphate-buffered saline (DPBS) containing Ca⁺⁺ and Mg⁺⁺ (Gibco), denatured at 80 °C for 3 min and slowly cooled down to RT for proper folding. After 20 min, animals were perfused with DPBS, killed by cervical dislocation and tumours and kidney were collected. Tumours and kidney were snap-frozen in liquid nitrogen and stored at −80 °C before recovering the nucleic acids from the tissues. Weight of the tumours and kidneys used during in vivo SELEX are summarised in Supp. Table ^S1. Extraction of the nucleic acids from 3 tumours and 3 kidneys from injected mice was performed by first homogenising the organs with a 7 mL dounce tissue grinder with large and small clearance pistils (Landgraf Laborsysteme HLL GmbH). To lyse the cells, TE-SDS Lysis buffer (0.1 M Tris-HCl pH 8, 1 mM EDTA pH 8, 0.5% SDS) supplemented with 0.5 µg/µL of Proteinase K (Roth) was used for D3 injected tumours or kidney, followed by 10 min incubation at 95 °C. For D3P library, buffers A1, A2 and A3 from NucleoSpin® Plasmid kit (Macherey-Nagel) were used for lysis of the cells followed by 10 min centrifugation at 4000 rcf to remove cellular debris. Purification of extracted oligonucleotides was performed by means of phenol/chloroform extraction and ethanol precipitation for D3 library, and by silica columns (DNA Clean & Concentrator^TM −500 (Zymo Research)) in the case of D3P library. A negative control was always included with tumours extracted from control mice (injected with DPBS) following the same procedure described above. Purified oligonucleotides were then re-dissolved in milliQ water and a first PCR amplification was performed. RNA digestion was performed before PCR amplification by using a 1:1 mixture of RNase T1 (Roche) and RNase A (Macharey-Nagel). Agarose gel (4%) purification was then performed in order to separate the library band from genomic DNA and primers with the NucleoSpin® Clean-Up kit. Further PCR amplification was then performed to reach the required amount of library the next cycle. All tissue homogenisations and PCR preparations were performed in two different PCR workstations (Peqlab) in order to avoid contaminations. Single strand displacement of the purified PCR product was carried out by λ-exonuclease digestion in 1 × λ-exonuclease reaction buffer and 5000 U/mL of λ-exonuclease (Thermo Scientific). After 30 min incubation at 37 °C, λ-exonuclease was inactivated at 80 °C for 10 min. Subsequently the samples were purified with the NucleoSpin® Clean-Up kit using the NTC buffer and resulting DNA libraries were freeze dried and frozen prior usage for next selection cycle. Detailed selection conditions are summarised in Supp. Table ^S2.