SELEX to Identify Protein-Binding Sites on RNA

Abstract

SELEX (systematic evolution of ligands by exponential enrichment) is a very powerful method for determining the binding site of a protein on RNA. It relies on the ability of an RNA-binding protein to select high-affinity RNA ligands from a randomized pool of RNAs. SELEX experiments are carried out over several “rounds,” with each round resulting in increased enrichment of RNAs capable of binding to the protein. There are many variations of SELEX strategies, but they all rely on the ability to separate bound RNA from unbound RNA. The method presented here uses tagged proteins; however, it is also possible to use other separation techniques (e.g., filter binding or antibodies).

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Reagents

• ATP, CTP, GTP, UTP (100 mM each)

• BamHI (200 μL)

• DNase I (RNase-free; 2 units/μL)

• dNTP mix (10 mM each of dATP, dCTP, dGTP, dTTP)

• Ethanol (70%, 100%)

• Klenow buffer (5X) <R>

• Klenow enzyme

• NT2 buffer <R>

• Phenol:chloroform:isoamyl alcohol (PCA) (25:24:1)

• Primers:

  • ○ “Forward” primer sequence: 5′-CGC GAA TTC TAA TAC GAC TCA CTA TAG GGG CCA CCA ACG ACA TT-3′

    The forward primer must contain a T7 promoter sequence and a restriction enzyme site for cloning. Here, the EcoRI restriction site is nucleotides 4–9, and the T7 promoter is nucleotides 10–29.

  • ○ “Reverse” primer sequence: 5′-CCC GAC ACC GCG GGA TCC ATG GGC ACT ATT TAT ATC AA-3′

    The reverse primer must contain a restriction enzyme site for cloning and hybridization sequence for reverse transcription. Here, the BamHI restriction site is nucleotides 13–18.

  • ○ “Library” primer sequence: 5′-TTACAGCAACCACCG GG GAT CCA TGG GCA CTA TTT ATA TCA AC (N)₂₅ AAT GTC GTT GGT GGC CC-3′

    The library primer must contain a region of randomized sequence as well as sequences that anneal to the forward and reverse primers.

• Proteinase K (10 mg/mL)

• Proteinase K buffer for SELEX (2X) <R>

• Protein of interest fused to a tag for purification (such as GST or histidine)

• Reverse transcriptase (M-MuLV 200 units/μL)

• Reverse transcriptase buffer for SELEX (10X) <R>

• RNase inhibitor (40 units/μL)

• SDS extraction buffer <R>

• Sepharose or agarose beads for purification of complexes

• Sodium acetate (3 m, pH 5.2)

• T7 RNA polymerase

• T7 transcription buffer (10X) <R>

• Taq DNA polymerase (1 unit/μL) and buffer

• Polyacrylamide gel (15%; 19:1 acrylamide:bis-acrylamide) containing 8 m urea, prepared in 1X TBE

• Polyacrylamide gel (15%; 19:1 acrylamide:bis-acrylamide), prepared in 1X TBE TE buffer (pH 8.0)

• Vector for cloning and bacteria for transformation (see Step 46)

• Yeast tRNA (10 mg/mL)

Equipment

• Beaker with 50 mL of H₂O at 75°C

• Conical centrifuge tube (15 mL)

• Dry ice

• Ice

• Microcentrifuge

• Microcentrifuge tubes (1.5 mL)

• PCR tubes

• Polypropylene tubes (12 mL)

• Shaker

• Spectrophotometer

• Thermal cycler

• Tube rocker

• Ultraviolet (UV) light source (254 nm, handheld)

• Vortex mixer

• Water baths (37°C, 42°C, 55°C, and 75°C)

• X-ray intensifying screen for UV shadowing

METHOD

Before beginning this protocol, it is necessary to understand the experimental flow (see Fig. 1).

Figure 1.

Schematic representation of a “round” of SELEX. Each “round” of SELEX consists of the same sequence of steps. First, it is necessary to construct DNA templates for synthesizing the randomized RNA pool. The pool is then synthesized, labeled, and allowed to bind to the protein of interest. Bound RNAs are then converted back into DNA templates by reverse-transcriptase–polymerase chain reaction (RT-PCR). This process is reiterated until the bulk (≥90%) of the pool is capable of binding to the protein. At this point, the “final” pool is converted back to double-stranded DNA, cloned, and sequenced.

Convert Library of Oligonucleotides into Double-stranded DNA Transcription Templates

1. Anneal the “forward” and “library” primers by mixing the following in a 1.5-mL tube:

   Klenow buffer (5×)	200 μL
   100 μm “forward” primer	85 μL
   100 μm “library” primer	85 μL
   H2O	590 μL

2. Heat the mixture for 15 min to 75°C and then transfer it to a beaker containing 50 mL of H₂O at 75°C. Allow the tube to cool slowly to room temperature. Centrifuge briefly for 5 sec at room temperature.

3. Extend the annealed primers by adding 20 μL of dNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP) and 20 μL of Klenow enzyme. Incubate for 1 h at 37°C.

4. Add 100 μL of 3 m sodium acetate (pH 5.2).

5. Divide the sample into four 275-μL aliquots and add 600 μL of 100% ethanol to each.

6. Freeze on dry ice for 10 min and then centrifuge at ≥12,000g for 15 min at room temperature.

7. Decant the supernatant and wash the pellet with 70% ethanol. Centrifuge at ≥12,000g for 5 min at room temperature. Decant the supernatant and air-dry the pellet.

8. Resuspend the double-stranded DNA library from Step 7 and gel-purify using a 15% nondenaturing TBE polyacrylamide (19:1 acrylamide:bis-acrylamide) gel. Electrophorese until the xylene cyanol dye is close to the bottom of a 15-cm-long gel.

9. Visualize the nucleic acids by UV shadowing.

10. Excise the band containing the full-length double-stranded library. Crush the excised gel band and elute the DNA using 1–2 mL of 0.5 m sodium acetate overnight with shaking.

11. Collect supernatant, precipitate the DNA with ethanol, and wash the precipitate with 70% ethanol. Repeat the elution once to increase recovery if desired.

12. Dissolve the DNA in ~100 μL of TE buffer (pH 8.0).

13. Determine the OD₂₆₀ and convert the value to molar concentration.

    For example, for 50 μg/mL per OD₂₆₀, the 95-nucleotide product has a molecular weight of 62,440 g/mol.

14. Dilute the DNA to 1.7 nmol/100 μL and divide it into 100-μL aliquots.

Transcribe the Double-stranded DNA Template for First Round

• 15
  Set up the transcription reaction in a 15-mL conical centrifuge tube as follows:

  1.7-nmol double-stranded DNA library (from Step 14)	100 μL
  10× T7 transcription buffer	1 mL
  100 mm ATP	100 μL
  100 mm CTP	100 μL
  100 mm GTP	100 μL
  100 mm UTP	100 μL
  T7 RNA polymerase	1 mL
  H2O	7.5 mL

• 16
  Mix the reagents gently by inverting and then incubate for 2 h at 37°C.

  The solution should become cloudy.

• 17Add 100 μL of RNase-free DNase I to the transcription reaction and incubate for a further 15 min at 37°C.
• 18Split into 2.5-mL aliquots in 12-mL polypropylene tubes and precipitate the RNA with ethanol.
• 19Purify the full-length RNA transcripts on an 8 m urea, 15% polyacrylamide gel.
• 20Visualize the nucleic acids by UV shadowing.
• 21Excise the band containing full-length RNA transcripts. Elute RNA overnight as in Step 10. Collect the supernatant, precipitate with ethanol, and wash the precipitate with 70% ethanol.
• 22Dissolve the RNA in ~100 μL of TE buffer (pH 8.0) or in SDS extraction buffer.
• 23
  Determine the OD₂₆₀ and convert the value to molar concentration.

  For example, for 40 μg/mL per OD₂₆₀, the 69-nucleotide, single-stranded RNA has a molecular weight of 23,630 g/mol.

• 24
  Dilute the RNA to 17 nmol/100 μL and store in 100-μL aliquots at –80°C.

  Each aliquot on average contains 10 RNA copies of each of the 1 × 10¹⁵ DNA templates.

Protein–RNA Binding and Selection

• 25
  Select RNA from Step 24 using a matrix bound to the protein of interest (e.g., glutathione– Sepharose beads with a GST-tagged protein or Ni-NTA agarose with a histidine-tagged protein).

  Immunoprecipitation, filter binding, or a mobility shift assay can also be used to separate protein–RNA complexes from free RNA.

• 26Prepare sufficient beads for 2 mg of each protein of interest and an equal volume of beads to preclear the RNA library. Wash the beads in NT2 buffer.
• 27Bind 1–10 μg of protein to Sepharose or agarose beads for at least 2 h at 4°C with rocking.
• 28Pellet the beads by centrifugation at 8000g for 10 sec at room temperature. Then, wash the beads three times with 1 mL of NT2 buffer each time.
• 29Preclear a 17-nmol aliquot of the RNA library from Step 24 by binding it to beads for 30 min at 4°C. Pellet the beads by centrifugation at 8000g for 10 sec at room temperature. Keep the supernatant, which contains the precleared RNA library.
• 30Bind the precleared RNA library from Step 29 to the proteins from Step 28 by incubating for at least 2 h at 4°C with rocking.
• 31Wash the beads five times with 1 mL of NT2 buffer each time. After the last wash, leave 100 μL of NT2 buffer on the beads.
• 32Add 100 μL of H₂O, 200 μL of 2× proteinase K buffer for SELEX, and 5 μL of proteinase K. Incubate for 15 min at 37°C.
• 33Add 400 μL of PCA (25:24:1), vortex for 30 sec, and then centrifuge at 12,000g for 1 min at room temperature.
• 34Transfer aqueous layer, which contains the RNA, to a clean tube. Add 2 μL of 10 mg/mL of yeast tRNA, 40 μL of 3 m sodium acetate (pH 5.2), and 1 mL of 100% ethanol. Mix thoroughly, freeze on dry ice for 10 min, and then centrifuge at ≥12,000g for 15 min at room temperature.
• 35Decant the supernatant, wash the RNA pellet with 70% ethanol, and centrifuge at ≥12,000g for 5 min at room temperature. Decant the supernatant and air-dry the pellet.

Reverse Transcription PCR

• 36
  Resuspend the RNA pellet from Step 35 in 13 μL of H₂O. Add the following:

  “Reverse” primer	3 μL
  Reverse transcriptase buffer for SELEX(10X)	2 μL
  dNTP mix	2 μL
  Reverse transcriptase	0.5 μL
  Incubate the reaction for 5 min at 55°C and then for 1 h at 42°C.

  Incubate the reaction for 5 min at 55°C and then for 1 h at 42°C.

• 37
  Transfer 6 μL from the reverse transcription reaction into a PCR tube and add the following:

  H2O	75.5 μL
  Taq buffer (10×)	10 μL
  dNTP mix	2 μL
  “Forward” primer	3 μL
  “Reverse” primer	3 μL
  Taq polymerase	0.5 μL

• 38Denature for 5 min at 94°C. Then, amplify for 25 cycles of 30 sec at 94°C, 30 sec at 50°C, and 30 sec at 72°C followed by a final extension of 10 min at 72°C.
• 39Add 1 μL of BamHI to the reaction and incubate at 37°C to regenerate the 3′ ends of original library.
• 40Extract with an equal volume of PCA. Precipitate the double-stranded DNA template with ethanol, air-dry the pellet, and resuspend it in 15 μL of H₂O.

Transcribe Double-stranded DNA for Subsequent Rounds

• 41
  Combine the following:

  T7 transcription buffer (10×)	2 μL
  NTP mix (2.5 mm each ATP, CTP, UTP, GTP)	8 μL
  Double-stranded DNA template (from RT-PCR of previous round [Step 40])	3 μL
  RNase inhibitor (40 units/μL)	1 μL
  H2O	4 μL
  T7 RNA polymerase	2 μL
  Incubate for 1 h at 37°C.

• 42Add 1.5 μL of RNase-free DNase I to the reaction and incubate for an additional 10 min at 37°C.
• 43Add 78.5 μL of H₂O and 10 μL of 3 m sodium acetate (pH 5.2). Extract the reaction with an equal volume of PCA, precipitate with ethanol, air-dry the RNA pellet, and then resuspend RNA in 20 μL of H₂O.
• 44Use one-quarter (5 μL) of the RNA from Step 43 to repeat the cycle for the binding to protein–RNA-binding step (Steps 25–35). Repeat the RT-PCR (Steps 36– 40). Usually 4–10 cycles of enrichment are required.
• 45Check for protein–RNA binding by electrophoretic mobility shift assay using radiolabeled RNA (either by incorporation of radioactive nucleotide during transcription or by end-labeling RNA transcripts) and the purified protein of interest.

Determine the Sequence Recognized by the Protein

• 46After sufficient enrichment has been achieved (≥90% of the transcribed RNA is bound by the protein), clone the double-stranded DNA templates into a suitable vector and transform bacteria.
• 47Pick a large number (50–100) of transformants and determine the nucleotide sequence of the inserts.

DISCUSSION

SELEX (Kenan and Keene 1999; Bouvet 2001) is extremely useful for determining preferred RNA-binding sites for specific RNA-binding proteins. Consensus sequences for binding sites should emerge from the analysis. It is often the case that suboptimal (but functional) sequence elements can be determined if the cloning and sequencing are carried out at earlier “rounds” of selection. In general, the number of iterative selection rounds required is determined by the size of the starting pool (e.g., RNA containing seven randomized positions contains only ~16,000 individual molecules, whereas an RNA randomized at 10 positions has a pool size of ~10⁶ individual molecules) and the stringency of selection. To ensure adequate coverage, it is important that ~10 times the theoretical pool size be synthesized for each reaction. It is essential that the investigator know and keep in mind the number of distinct molecules required for adequate representation of the pool and take steps to ensure that each molecule of distinct sequence is present in the starting pool.

RECIPES

Klenow buffer (5X)

Reagent	Quantity (for 1 mL)	Final concentration
NaCl (5 m)	50 μL	250 mm
Tris-HCl (1 m, pH 7.9)	50 μL	50 mm
DTT (dithiothreitol; 1 m)	5 μL	5 mm
H2O	895 μL

Store for 6 mo at –20°C.

NT2 buffer

Reagent	Quantity (for 100 mL)	Final concentration
Tris-HCl (1 m, pH 7.4)	5 mL	50 mm
NaCl (5 m)	3 mL	150 mm
MgCl2 (1 m)	0.1 mL	1 mm
Nonidet P-40 (NP-40; 10%)	0.5 mL	0.05%
H2O	91.4 mL

Store for 6 mo at 4°C.

Proteinase K buffer for SELEX (2X)

Reagent	Quantity (for 50 mL)	Final concentration
SDS (10%)	2.5 mL	0.5%
EDTA (0.5 m, pH 8.0)	0.4 mL	4 mm
H2O	47.1 mL

Store indefinitely at room temperature.

Reverse transcriptase buffer for SELEX (10X)

Reagent	Quantity (for 1 mL)	Final concentration
Tris-HCl (1 m, pH 8.3)	500 μL	500 mm
KCl (2 m)	200 μL	400 mm
MgCl2 (1 m)	60 μL	60 mm
DTT (dithiothreitol; 1 m)	10 μL	10 mm
H2O	230 μL

Store for 6 mo at –20°C.

SDS extraction buffer

Reagent	Quantity (for 500 mL)	Final concentration
Tris-HCl (1 m, pH 7.5)	10 mL	20 mm
EDTA (0.5 m, pH 8.0)	1 mL	1 mm
SDS (10% w/v)	25 mL	0.5% (w/v)
H2O	464 mL	-

Store indefinitely at room temperature.

T7 transcription buffer (10X)

Reagent	Quantity (for 1 mL)	Final concentration
Tris-HCl (1 m, pH 8.0)	400 μL	400 mm
MgCl2 (1 m)	200 μL	200 mm
DTT (dithiothreitol; 1 m)	50 μL	50 mm
H2O	350 μL

Store for 6 mo at –20°C.