Cell culture. A431, Bend3, and HeLa cells were obtained from the American Tissue Culture Collection (ATTC, Manassas, VA) and cultured in Dulbecco's modified Eagle medium (DMEM) (ATCC) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). LnCAP cells were also purchased from ATCC and were cultured in RPMI-1640 (Invitrogen) supplemented with 10% FBS.
Min6B1 cells were a kind gift of Dr Jeffrey Pessin at the Diabetes Center at the Albert Einstein College of Medicine, New York, and were cultured in DMEM supplemented with 10% FBS. HBEC, A549, and primary NHBEC cells were a kind gift of Dr Simon Spivack at the Albert Einstein College of Medicine, New York and were cultured in F12 Kaighn's medium supplemented with 10% FBS. HeLa-PSMAs cells were generated in our lab using a PSMA expression vector in which the open reading frame (positions −128 to 2,253) was inserted between the Kpn1 and Xba1 of the EF1 promoter driven vector pEF6-V5HisA (Invitrogen).
All cells were grown at 37 °C with 5% CO2 and 99% humidity.
Aptamer libraries, aptamers, and oligonucleotides. All primers used for PCR as well the biotinylated oligonucleotides used for flow cytometry were purchased from IDT (Coralville, IA). All pools and minimized aptamers were synthesized in our lab on an Expedite 8909 DNA synthesizer (Applied Biosystems, Carlsbad, CA) using standard synthesis procedures. Unless noted otherwise, all reagents were purchased from Glen Research (Sterling, VA).
Libraries were synthesized such that N regions contained an equal probability of containing A, T, G, or C, as previously described.34
Following deprotection, libraries were gel purified by denaturing (7 mol/l urea) gel electrophoresis on an 8% polyacrylamide gel. The single-stranded DNA library was amplified by PCR to generate a double-stranded DNA bearing a T7 promoter and transcribed in vitro
using the Y639F mutant of T7 RNA polymerase35,36
or Durascribe kits and 2′-fluoro-pyrimidines. The RNA was purified on a denaturing (7 mol/l urea) 8% polyacrylamide gel.
Biotinylated, minimized aptamer variants were chemically synthesized in our lab using 2′-fluoro-dC and 2′-fluoro-dU phosphoramidites (Metkinen, Kuusisto, Finland) bearing an inverted dT residue for added stability. Thiolated, minimized aptamer variants were synthesized using a thiol-modifier C6 S-S phosphoramidite. All aptamers were synthesized with 4,4′-dimethoxytrityl on. Following deprotection, aptamers were purified by reversed phase high-performance liquid chromatography (HPLC) on a 10 × 50 mm Xbridge C18 column (Waters, Milford, MA) using a linear gradient of acetonitrile in 0.1 mol/l triethylammonium acetate at pH 7.0.
Thiolated aptamers were used to generate the AlexaFluor488 (A488)-labeled aptamer used for in vivo microscopy studies. Labeling was performed using AF488-C5-malemide as follows: 10 nmol of thiolated aptamer was reduced using 10 mmol/l tricarboxyethylphosphine in 100 µl of 0.1 mol/l triethylammonium acetate. Samples were heated at 70 °C for 3 minutes followed by incubation at room temperature for 1 hour. The reduced aptamers were desalted using on a Biospin 6 column (Bio-Rad, Hercules, CA) into PBS supplemented with 50 mmol/l phosphate, pH 7.5. To this, 10 µl of dimethyl sulfoxide (DMSO) was added containing 50 nmol AF488-C5-malemide. Following an overnight reaction at 4 °C, the oligonucleotide was recovered by ethanol precipitation and desalted an additional time using a Biospin 6 column. Dye to aptamer ratios were determined at 260 and 488 nm and were typically ~1. The absence of free dye in the final product was confirmed by reversed phase HPLC.
Real-time PCR analysis of aptamer binding and internalization.
The anti-PSMA aptamer, A9,7
was used to proof internalization into cells. Approximately 50,000 PSMA-positive LnCAP cells were plated per well in a 24-well plate and allowed to grow overnight such that cells would be at ~105
cells/well at assay time. The cells were washed in DPBS (Invitrogen) and treated with one of four conditions: 50 nmol/l anti-PSMA aptamer was added directly to the media on cells (RNA); cells were first treated for 10 minutes with 10 mmol/l Az and 50 mmol/l dG to prevent endocytosis, then RNA was added to the media after washing (Az-dG); RNA was added to cells, and then cells were nuclease-treated with 0.02 U/µl Riboshredder (Epicentre, Madison, WI) to digest species bound to the cell surface (Rb); or cells were treated with Az-dG, washed and treated with RNA and nuclease-treated (Az-dG/Rb). Each treatment was performed in triplicate. Total cell RNA from each of the treatments was extracted with Trizol (Invitrogen) following manufacturer's protocols. For reverse transcription, 500 ng total cell RNA was incubated with 5 pmol aptamer-specific reverse primer (5′-TCGGGCGAGTCGTCTG-3′), 5 pmol pool-specific primer (5′-AAGCTTCGTCAAGTCTGCAGTGAA-3′), and 5 pmol glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reverse primer (5′-GAAGGTGAAGGTCGGAGT-3′) in First Strand Buffer. The solution was heated to 70 °C for 3 minutes and then cooled on ice. A solution of 50 nmol dithiothreitol, 5 pmol each deoxynucleotide triphosphate and 50 U Superscript III reverse transcriptase (Invitrogen) was added to the primer mix for a final total volume of 10 µl and incubated at 50 °C for 1 hour followed by an inactivation step at 70 °C for 15 minutes.
Real-time PCR was performed using a 7300 Real Time PCR System (Applied Biosystems). PCR reactions consisted of 1 µl of reverse transcription reaction, 12.5 µl SYBR Green Mastermix (Applied Biosystems), 10 pmol target-specific reverse primer, and 10 pmol target-specific forward primer in a total volume of 25 µl. Separate reactions were performed to amplify the aptamer (forward primer: 5′-TTCTAATACGACTCACTATAGGGAGGACGATGCGG-3′), or GAPDH (forward primer: 5′-TCGACCGTGTGCGCTCGCTG-3′). Cycling conditions started with an initial denaturation step at 95 °C for 10 minutes followed by 40 cycles of 95 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 1 minute. A final cycle of 95 °C for 15 seconds, 60 °C for 30 seconds, and 95 °C for 15 seconds was used to generate dissociation curves to check the integrity of the products. The levels of each species were calculated by the ΔΔCt method by taking the difference in Ct between each sample and cells control and then normalizing to GAPDH levels for each sample.
Doped-A9 selection. The sequence of the doped-A9 library used for the selection was, 5′-GGGAGGACGATGCGGACCGAAAAAGACCTGACT TCTATACTAAGUCUACGTTCCCAGACGACTCGCCCGATTGAA TTAAATGCCCGCCATCACCAG-3′, where the underlined residues were doped to a level of 30%. Library preparation was performed as described above.
The internalization selection was performed using HeLa-PSMA cells. For each round, ~105 adherent cells were incubated in 300 µl of DMEM supplemented with 10% FBS and 1 mg/ml transfer RNA (tRNA) and ssDNA (Sigma, St Louis, MO) as blocking agents. The first round of selection used 30 µg of the A9 doped pool encompassing ~1013 unique variants. Subsequent rounds were performed using 3 µg of RNA. For each round of selection, the pool was combined with a 1.5-fold molar excess of reverse primer bearing a 5′ fluorescein dye. The mixture was denatured at 70 °C for 3 minutes and allowed to refold at room temperature for at least 15 minutes before addition to cells. RNA was added to the blocked cells and incubated for 1 hour at 37 °C. Following incubation, the cells were washed three times with 1 ml HBSS (Hank's buffered saline solution; Invitrogen) containing 0.1% Az, once with 1 ml cold 200 mmol/l glycine and 150 mmol/l NaCl at pH 4, and three more times with HBSS. Cells were lifted with 500 µl trypsin-EDTA (Mediatech, Manassas, VA) containing 0.1% Az, removed from the plate, washed with 1 ml HBSS and resuspended in 100 µl HBSS containing 5 µl Riboshredder RNAse cocktail (Epicentre). Following a 15 minute incubation at room temperature, the cells were washed an additional three times with 1 ml of HBSS. Cells were lysed, and RNA was recovered using Trizol extraction according to manufacturer's protocol. Recovered RNA was reverse-transcribed and amplified by PCR. Double-stranded DNA was ethanol precipitated and transcribed back into RNA. The progress of the selection was monitored by flow cytometry as described below.
Following the third round of selection, the pool was cloned using the TOPO TA kit (Invitrogen) and 19 clones were sequenced, transcribed, and assayed by flow cytometry for their ability to bind to wild-type HeLa cells or HeLa-PSMA cells.
HeLa cell internalization selection. The sequence of the N62 library used for selection against HeLa cells was: 5′-GGCGCTCCGACCTTAGTCTCTG-N62-GAACCGTGTAGCACAGCAGA-3′. Library preparation was performed as described above. Each round of selection employed 1 nmol RNA, which corresponds to a starting library size of ~1014. Thermally equilibrated RNA was added to 5 ml of media in a T-75 flask containing ~6 × 106 HeLa cells and incubated for 30 minutes at 37 °C with 5% CO2. Cells were then washed with 10 ml DPBS three times and lifted with trypsin. The cells were suspended in 5 ml of DMEM supplemented with 10% FBS and recovered by centrifugation at 200g for 5 minutes. The cell pellets were each resuspended in 100 µl DPBS, combined, pelleted, and washed three more times in 1 ml DPBS. Total cell RNA was recovered using the mirVana Kit (Invitrogen) according to manufacturer's protocol, and eluted in 40 µl of elution buffer; 20 µl of the isolated RNA was used in a 40 µl reverse transcription reaction using SuperScript III reverse transcriptase (Invitrogen) and the N62 reverse primer according to manufacturer's protocol. Following PCR amplification, the double-stranded DNA was ethanol precipitated, transcribed back into RNA and purified as described above. After Round 2, the cells were washed four times as described above, then resuspended in 100 µl DPBS containing 5 mmol/l MgCl2 and 10 µl Riboshredder RNase. The reaction was allowed to proceed for 15 minutes at room temperature after which the cells were washed five times in 1 ml DPBS and resuspended in 100 µl DPBS supplemented with 10 µl of RNase Out (Invitrogen). The mixture was left at room temperature for 2 minutes after which the cells were pelleted, and the RNA was isolated as described above.
Min6B1 cell internalization selection. The sequence of the N50 library used for selection against Min6B1 cells was: 5′-GGGAGGTGAATGGTTCTACGAT-N50-TTACATGCGAGATGACCACGTAATTGAATTAAATGCCCGCCATGACCAG-3′. Library preparation was performed as described above. For the first round of selection, Min6B1 cells were grown as an adherent monolayer and cultured in a 6-well plate to about 90% confluence (~106 cells). Cells were blocked with 0.1 µg/µl tRNA and ssDNA in 1 ml DMEM supplemented with 10% FBS for 30 minutes at 37 °C before the addition of 1.2 nmol of thermally equilibrated RNA library (~1014 sequences). Following a 1 hour incubation at 37 °C, the cells were washed three times with 1 ml HBSS to remove unbound RNA, and then cells were lifted by the addition of trypsin in the presence of 10 mmol/l EDTA for 20 minutes at 37 °C. Cells were recovered by centrifugation and washed three times with 1 ml HBSS. Total cell RNA was Trizol extracted, according to the manufacturer's protocol. Recovered RNA was reverse-transcribed using MMLV reverse transcriptase (Invitrogen) and the N50 reverse primer according to the manufacturer's protocol. Following PCR amplification, the double-stranded DNA was ethanol precipitated and transcribed back into RNA and purified as described above. For Rounds 7–10, an RNase treatment step followed trypsinization. Cells were incubated in 500 µl of HBSS containing 0.1% azide and 5 µl of RNAse RiboShredder (Epicentre) at room temperature for 20 minutes. Cells were then washed three times with 1 ml HBSS containing 0.1% azide, and total RNA was recovered by Trizol extraction. Rounds 11 and 12 were performed exactly as previously described, except that freshly isolated islets were used as the target cells.
Preparation of adult mouse islets. Pancreatic islets were isolated from 5- to 8-week old male BALB/c mice. The pancreas was first distended by intraductal injection of collagenase solution (1 mg/ml in HBSS). Collagenase digestion was performed at 37 °C for 13 minutes. Subsequently, the pancreas was excised, and the digestion was quenched by addition of cold HBSS. The islets suspension was washed three times with cold HBSS, and islets were separated by centrifugation on a Histopaque discontinuous gradient for 20 minutes at 1700g at 4 °C. Islets were picked and counted under an inverted microscope. The islets were cultured in 100 mm dishes at 37 °C in humidified 5% CO2 in RPMI-1640 medium supplemented with 10% FBS and 1× penicillin/streptomycin solution (Invitrogen) for 5 days.
Binding assay by flow cytometric analysis. Aptamer binding and uptake was assessed by flow cytometry. Rounds from each selection or isolated clones were first hybridized to a biotinylated oligonucleotide (B-CTGGTCATGGCGGGCATTTAATTC) which was complementary to the 3′ end of the library. The population or individual aptamer was incubated at 1 µmol/l with 1.5 µmol/l biotinylated oligonucleotide in DPBS heated to 70 °C for 3 minutes and then allowed to cool on the bench top for 15 minutes. Following hybridization, the aptamers were complexed with 1.5 µmol/l streptavidin-R-phycoerythrin (Prozyme, Hayward, CA) for an additional 15 minutes. For assays performed with biotinylated minimized aptamers, following thermal equilibration, 1 µmol/l aptamer was incubated with 1.5 µmol/l streptavidin-R-phycoerythrin and incubated for 15 minutes before addition to cells.
For the assay, cells were first blocked with 0.1 µg/µl tRNA and ssDNA in DMEM supplemented with 10% FBS for 30 minutes at 37 °C. Aptamer complexes were added to cells at a final concentration of 50–100 nmol/l (as indicated) for 30–60 minutes (as indicated) at 37 °C and 5% CO2. Cells were then washed three times with DPBS, trypsinized and washed with fluorescence-activated cell sorting (FACS) buffer (HBSS, 1% bovine serum albumin, 0.1% Az). Samples designated to test internalization were treated with Riboshredder cocktail as described above for the selection. After incubation, cells were washed twice with FACS buffer and resuspended in FACS buffer with 7-aminoactinomycin D stain (7AAD) to exclude dead cells in the analysis. Flow cytometry was performed on a FACScan or FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Microscopy. For microscopic analysis of Min6B1, Hela, and Bend3 cells, cells were grown as adherent monolayers in Labtek 8-chamber glass slides systems (Thermo Scientific, Rochester, NY). Thirty minutes before aptamer treatment, cells were blocked with 0.1 µg/µl tRNA and ssDNA in DMEM supplemented with 10% FBS at 37 °C. The blocked cells were then incubated with 100 nmol/l 5′-biotinylated c1 or 5′-biotinylated control aptamer conjugated to streptavidin-R-phycoerythrin (Prozyme) at 37 °C for 1 hour. Cells were then washed three times with PBS and fixed with 4% formaldehyde solution for 15 minutes at room temperature. The fixed cells were washed three times with PBS and mounted in mounting media (0.1 mol/l propylgalate, 50% glycerol in PBS) containing 1 µg/ml 4′-6-diamidino-2-phenylindole (DAPI). Cells were also assayed without fixation and visualized on a Zeiss Axio Observer fluorescence microscope (Thornwood, NY).
Competition binding assay by flow cytometry analysis. Approximately 5 × 104 Jurkat cells were blocked with 0.1 µg/µl tRNA and ssDNA in DMEM supplemented with 10% FBS for 30 minutes at 37 °C. Cells were then incubated with 400 nmol/l 5′-biotinylated Otter aptamer conjugated to AlexaFluor-647-labeled streptavidin (Invitrogen) and increasing concentrations (0, 0.2, 0.4, 0.8, 2, and 4 µmol/l) of either 5′-biotinylated c1 or 5′-biotinylated Otter conjugated to FITC-labeled streptavidin (Invitrogen) at 37 °C for 1 hour. Cells were washed with FACS buffer and analyzed by flow cytometry; 7AAD was used to exclude the presence of dead cells.
Endocytosis inhibition. Jurkat, HEK EBNA 293, or NHBE cells were incubated with either 10 µmol/l phenylarsine oxide or 100 µmol/l indomethacin in RPMI-1640 media at 37 °C during 30 minutes. Cells were then blocked with RPMI-1640 medium supplemented with 10% FBS and 0.1 µg/µl tRNA and ssDNA for 30 minutes at 37 °C; 100 nmol/l 5′-biotinylated c1 or 5′-biotinylated control aptamer conjugated to streptavidin-R-phycoerythrin (Prozyme) was added to the media and incubated at 37 °C for 1 hour. RNase treated samples were further incubated with 10 µl of RNase Riboshredder (Epicentre) in RPMI-1640 supplemented with 10% FBS. Cells were washed with HBSS and analyzed by flow cytometry.
Vaginal uptake experiments. C57BL/6 mice (Taconic, Hudson, NY) were subcutaneously injected with 2 mg medroxyprogesterone acetate (Greenstone, Peapack, NJ) to maintain them in diestrus. One week later, 150 pmol c1 aptamer or control c36 aptamer labeled with AlexaFluor A488 were administered intravaginally in 15 µl of DPBS. Twenty-four hours later, vaginal tissue was dissected, placed in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA) and snap-frozen in liquid nitrogen; 10 µm sections were fixed in acetone and mounted. Images were acquired by fluorescence microscopy (Leica SP5) using Leica LAS-AF software (Leica Microsystems, Buffalo Grove, IL) and analyzed by ImageJ (NIH, Bethesda, MD). All animal studies have been approved by the Albert Einstein College of Medicine institutional review board.
Representative real-time PCR data from analysis of A9 uptake by LnCAP cells.
Cytometric analysis of clones identified from a doped- A9 internalization selection on HeLa-PSMA cells.
Cytometric analysis of clones identified from a doped-A9 internalization selection.
Sequences of functional clones identified from the A9 doped selection.
Sequence analysis of Round 6 of N62 internalization selection on HeLa cells.
Riboshredder is effective at destroying 2′-fluoro–modified RNA at the concentration and conditions used in the internalization selection.
Sequence analysis of Round 12 of the N50 internalization selection on Min6B1 cells.
Early fitness of the N62 pool internalization selection.
Retention of fluorescent signal of selected aptamers in cells after ribonuclease treatment.
Analysis of c1 binding to different mammalian cell lines.
HEK-EBNA 293 endocytosis inhibition.
Sequences tested for the minimization of c1.
Clones from separate selections compete for binding on cells and co-localize in A431 cells.
c1 is internalized into primary cells by clathrin-mediated transport.