The affinity column for
selection was prepared as described.
In brief, guanine-rich RNA was synthesized by run-off
transcription with T7 RNA polymerase from the DNA template to which the T7 promotor was annealed and purified by PAGE (polyacrylamide gel electrophoresis) and gel elution of the crush and soak method.
The gel-purified guanine-rich RNA was oxidized at the 3'-terminal sugar with NaIO
and then coupled to Sepharose-adipic acid hydrazide resin (Amersham Pharmacia Biotech). Selection was performed with the guanine-rich RNA-attached column. In order to minimize the enrichment of undesirable RNA species binding to the Sepharose resin itself, we pre-selected the RNA pool on the uncoupled Sepharose-adipic acid hydrazide resin. After being passed through the pre-column of Sepharose-adipic acid hydrazide resin, RNA library was passed through the affinity column and then RNA species bound to the column were eluted by reducing the ionic strength and chelating the Mg
with EDTA. In other words, we loaded the RNA pool onto the guanine-rich RNA-attached affinity column. We washed the column with binding buffer and then eluted the bound RNA with three column volumes of the elution buffer (25 mM Na-EDTA, pH 8.0). We recovered the selected RNAs by ethanol precipitation and reverse-transcribed it with an M-MLV reverse transcriptase (RT) (bioneer, Korea) (or AMV reverse transcriptase, promega) using a cDNA primer (5'-AAGCTTGCATGCGGATCC-3'). Then, the cDNAs were amplified by PCR with the cDNA primer and T7 primer (5'-TAATACGACTCACTATAGGTG-3'). A new pool of RNA, enriched in the guanine-rich RNA-binding motifs, was prepared by transcription from the PCR-amplified DNA and used for the next round of selection and amplification. The stringency of the selection was given to lead to a more cohesive sets of RNA isolates by reducing the concentration of the guanine-rich RNA to make the affinity column as the number of selection cycle increased. After the 11th round of selection, the amplified cDNAs were cloned into the pGEMT Easy vector (Promega), and their sequences were determined.
Enzymatic Protection Experiment.
RNA aptamer 11-30-24 was synthesized by run-off
transcription with T7 RNA polymerase from the DNA template to which the T7 promotor was annealed and purified by PAGE and gel elution of the crush and soak method. The purified RNA was treated with CIP (calf intestinal alkaline phosphatase) to remove 5' terminal phosphate and then labeled at 5'-end with [𝛾-32P]ATP (GE Healthcare) and T4 polynucleotide kinase (New England Biolab). The 5' end radiolabeled RNA was purified by PAGE and renatured. The labeled RNA alone or mixed with the non-labeled cognate RNA was used for nuclease-cleavage reactions. The reaction volume was adjusted to 100 μL with binding buffer (30 mM Tris-acetate, pH 7.5, 60 mM magnesium acetate, 120 mM potassium acetate, and 120 mM ammonium acetate) and incubated for 20 min at room temperature. Then, 1 U of RNase T1 (Industrial Research Limited) or 0.1 U of RNase V1 (Pierce Molecular Biology) was added to the above mixture. The reaction mixture was incubated for another 20 min at room temperature. The cleavage products were recovered by ethanol precipitation and electrophoresed on 15% polyacrylamide- 7 M urea gel.
We thank Prof. Younghoon Lee (Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology) for his helpful support.
Huppert J. L.
Nat. Chem. Biol.
Wallace S. T.
Ahn J. K.
Bull. Korean Chem. Soc.
Fritsch E. F.
Molecular Cloning: A Laboratory Manual
Cold Spring Harbor Laboratory Press
Cold Spring Harbor, NY