Zhen Huang

03/20/98

General Introduction (GTP aptamer selectionas an example)

GTP aptamers were isolated by iterative rounds of selection (6 rounds)from mutants of the ATP aptamer. The selection was done by affinity chromatographyon GTP-agarose column followed by reverse transcription, PCR amplificationand transcription. The initial pool of DNA containing a central regionof 15%-doped ATP aptamer (40 nt.) flanked by constant primer regions waschemically synthesized and its complexity was 6x10¹⁵.

1X Binding Buffer

300 mM NaCl, 5 mM MgCl₂, 20 mM Tris-HCl,pH 7.6

Selection Procedure

The initial pool of RNA was transcribed from the DNA pool in the presenceof [a-³²P]-GTPor [a-³²P]-UTP.RNA was purified by polyacrylamide gel electrophoresis and was loaded onGTP-agarose (linked at 2'- or 3'-position to the matrix via adipticacid linker). The column (Bio-rad) of GTP-agarose bead (0.5-1.5 mL, 2.2mM GTP) was pre-equilibrated with binding buffer (10-30 mL). The RNA (100mL, 0.1-2 mM) was heated at 65°C in a waterbath for 1 min, allowed to stand at room temperature for 2 min, 10x bindingbuffer was then added, and the solution allowed to stand at at room temperaturefor 15 min. After the RNA was loaded on the GTP-agarose column and incubatedat at room temperature for 1 hr, the column was eluted with binding buffer(6 column-volumes) and fractions were collected. This elution precedurecan remove upto 99.9% of unbounded RNA. The remaining RNA was specificallyeluted with 3 column-volumes of GTP solution (4 mM GTP in 1X binding buffer)and fractions were collected. The fractions were counted by a Scintillationcounter. The fractions from GTP elution were precipitated by ethanol precipitation,and the residual GTP was removed by G-25 NAP column. The RNA was used asa template for reverse transcription, then degraded using NaOH, and thecDNA was amplified by nested PCR. The DNA was transcribed into RNA forthe subsequent cycle of selection.

Reverse Transcription.

The reactions were carried out using conditions suggested by the producer.Superscript II RNase H- (GibcoBRL) reaction mixturesconsisted of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂,10 mM DTT, 1 mM each dNTP, 0.5 mM RNA, 2 mM3'-primer and 10U/mL reverse transcriptase.RNA template and primer were heated to 80°C with all buffer componentsexcept MgCl₂ and enzyme, and allowed thetemperature to reach room temperature over 10 min., then the MgCl₂was added. After equilibration at 42°C, the enzyme was added to initiatethe reaction. After 1 hr incubation, NaOH was added to 100 mM and the reactionmixture was heated at 90°C for 10 min. to degrade the template. Thesolution was neutralized with diluted HCl finally.

Nested PCR.

The reactions were carried out using conventional conditions. PCR reactionmixtures consisted of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100,2.5 mM MgCl₂, 0.2 mM dNTPs, 0.5-1 mMprimers, 0.001-1 nM template and 0.05 U/mL Taqpolymerase. Thermal cycling conditions were 94°C 45s, 55°C 90s,and 72°C 120s. PCR reactions were controlled by 2.5% agarose gel electrophoresis.

Transcription.

The reactions were carried out using conventional conditions. Transcriptionreaction mixtures consisted of 40 mM Tris-HCl (pH 8.0), 2.5 mM Spermidine,26 mM MgCl₂, 0.01% Triton X-100, 10 mMDTT, 9 mM GTP, 5 mM other NTPs, 0.2-1 mM DNAtemplate, 0.08 U/mL RNase inhibitor and 4 U/mLT7 RNA polymerase. Reaction mixtures were incubated at 37°C overnight,and RNA was purified by polyacrylamide gel electrophorosis.