The author thanks the National Institutes of Health for the past funding and thanks Michael R. Green for synthesizing oligonucleotides.

NOTE: Figure 1 provides an overview of phosphorothioate mutagenesis and summarizes key steps in the process.
1. Generation of a Library of Mutants &#8212; Doping DNA Template with Non-wild Type Nucleotides
<ol>
	<li>Synthesize T7 Primer (5&#8217;-GTAATACGACTCACTATAG-3&#8217;) by chemical synthesis on a DNA synthesizer.</li>
	<li>Synthesize a doped oligonucleotide (complementary strand) by chemical synthesis on a DNA synthesizer corresponding to the protein binding site. Use an appropriate mixture of phosphoramidites during chemical synthesis for each site of doping (X below) with a ratio of 90% A as the wild-type nucleotide and 10% T as the non-wild type nucleotide (see Representative Results for more detail on this ratio). 
	Note: Here, the sequence of the doped oligonucleotide is 5&#8217;-GTTCACTACACTXGAXAXAXAXCAXCXAXAXGXTGCCCTATAGTGAGTCGTATTAC-3&#8217;. The underlined sequence is the reverse complement of SXL protein binding site, plus additional nucleotides outside the binding site that provide useful controls confirming that not every change in the RNA affects binding, as well as being loading controls for comparison and normalization of nucleotides within the binding site. The SXL-binding site sequence UUUUUGUUGUUUUUUUU, which is present in transformer pre-mRNA14,15,16,17, was used to devise the proposed methodology. The sequence in italics is complementary to the T7 primer sequence and is the T7 promoter for in vitro transcription.</li>
</ol>
2. Synthesis of RNA
<ol>
	<li>Synthesize RNA in a 20 &#181;L transcription reaction, as previously described18.
	<ol>
		<li>Mix T7 transcription buffer and 1 &#181;M T7 oligonucleotide, 1 &#181;M doped oligonucleotide, 10 mM dithiothreitol (DTT), 2 mM GTP, 1 mM each ATP, CTP, and UTP (guanosine, adenosine, cytidine, and uridine triphosphate), and 2 U/&#181;L T7 RNA polymerase.</li>
		<li>Add, in two separate microcentrifuge tubes, 0.167 mM &#945;-thio ATP or 0.05 mM &#945;-thio UTP to incorporate phosphorothioates (see schematics in Figure 2) into RNAs. 
		NOTE: For alternative protocols, add 0.2 mM &#945;-thio CTP or 0.2 mM &#945;-thio GTP to an appropriate transcription reaction containing a doped oligonucleotide to test the two remaining nucleotide substitutions.</li>
		<li>Incubate the RNA synthesis reaction mixture for 2 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Add 2.5 &#181;L heat-labile alkaline phosphatase and 2.5 &#181;L 10xphosphatase buffer to the RNA sample. Incubate this 25 &#181;L reaction for 10&#8211;30 min at 37 &#176;C to remove 5&#8217; phosphates.</li>
	<li>Inactivate the alkaline phosphatase enzyme by heating at 80 &#176;C for 2&#8211;5 min.</li>
	<li>Radiolabel the 5&#8217; end of dephosphorylated RNA (5 pmole) using 1 &#181;L T4 polynucleotide kinase and 1 &#181;L &#947;-32P ATP in a 10 &#181;L reaction volume. Incubate the reaction mix for 30&#8211;60 min at 37 &#176;C.</li>
	<li>Inactivate the T4 polynucleotide kinase enzyme by heating at 65 &#176;C for 20&#8211;30 min.</li>
	<li>Gel purify the RNA by electrophoresis in a 10% denaturing polyacrylamide gel. Locate RNA on the gel by autoradiography. Excise the gel slice containing radiolabeled RNA, crush in a microcentrifuge tube by pressing against the walls with a pipette tip, and soak in proteinase K (PK) buffer (100 mM Tris, pH 7.5, 12.5 mM EDTA, 150 mM NaCl, 1% Sodium dodecyl sulfate). Rotate the tube at room temperature from 2 h to overnight.</li>
	<li>Centrifuge the gel slurry, discard the gel and collect the buffer solution.</li>
	<li>Extract the solution twice with equal volume of phenol-chloroform and once with chloroform and collect the aqueous phase.</li>
	<li>Add to the aqueous phase 0.1 volume of 3M Sodium Acetate, pH 5.2, carrier tRNA or glycogen, and 2.5 volume of ethanol. Keep the sample at -80 &#176;C for 1 h.</li>
	<li>Centrifuge the sample for 5&#8211;10 min in a high speed microcentrifuge at 16,873 x g. Remove the buffer/ethanol solution carefully without disturbing the RNA pellet.</li>
	<li>Wash the pellet with 70% ethanol and centrifuge for 2&#8211;5 min. Remove ethanol carefully. Dry the pellet in air.</li>
	<li>Resuspend the pellet in 20&#8211;50 &#181;L diethyl pyrocarbonate (DEPC)-treated water. Store at -20 &#176;C until use. 
	NOTE: Perform all steps with radiolabeled RNA using appropriate precautions and a plexiglass shield to protect from radioactivity. Presently, spin columns are more commonly used and more convenient to remove unincorporated radioactivity, as an alternative to gel purification of RNA.</li>
</ol>
3. Protein Binding Reaction and Separation of Bound RNA
<ol>
	<li>Express the recombinant protein in and purify from E. coli.</li>
	<li>Estimate recombinant protein concentration by spectrophotometry or by separating in an SDS-polyacrylamide gel next to a known protein standard, bovine serum albumin (BSA). Visualize the protein of interest and the BSA standard by staining the gel with Coomassie Brilliant Blue R-250. Quantitate the protein in comparison to known quantities of BSA dilutions on the same gel. 
	NOTE: Store recombinant protein at -80 &#176;C until use and dilute in 20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 8.0, 1 mM dithiothreitol (DTT), 0.2 mM Ethylenediaminetetraacetic acid (EDTA), 0.05% NP-40, 20% glycerol. Use of 0.5&#8211;1.0 mM protease inhibitor phenylmethane sulfonyl fluoride (PMSF) is optional.</li>
	<li>Perform RNA-binding reaction (20&#8211;100 &#181;L) in 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 50 mM KCl, 0.5 units/&#181;L RNase inhibitor, 0.09 &#181;g/&#181;L acetylated bovine serum albumin, 1 mM EDTA, 0.15 &#181;g/&#181;L tRNA, 5&#8217;-end radiolabeled RNA, and 6 &#181;L of appropriate concentration (a concentration at which ~50% of RNA binds to protein) of the protein. 
	NOTE: This binding buffer works for three RNA&#8211;binding proteins (SXL, U2AF65, and PTB), but must be standardized for a protein of interest. Estimate protein concentration empirically, using various dilutions for a given protein preparation, that is required to obtain approximately 50% RNA binding. This condition or Kd represents the most sensitive part of the RNA-binding curve.</li>
	<li>Incubate the protein binding reaction for 20&#8211;30 min at 25 &#176;C (or on ice).</li>
	<li>Separate the protein-bound RNA fraction from the unbound fraction using one of two approaches:
	<ol>
		<li>Nitrocellulose filter binding
		<ol>
			<li>Apply the binding reaction (20&#8211;100 &#181;L) onto a nitrocellulose filter connected to a vacuum manifold at room temperature. 
			NOTE: Only the RNA-protein complex is retained on the filter and unbound RNA flows through the filter. The filter binding approach allows higher recovery of the bound RNA and is faster and simpler, compared to the gel mobility shift assay. By placing a DEAE membrane underneath the nitrocellulose filter it is also possible to collect the unbound RNA fraction or free RNA for comparison with the bound fraction.</li>
			<li>Cut the portion of the nitrocellulose filter containing the retained radioactive RNA into smaller pieces to fit into a microcentrifuge tube, soak in sufficient PK buffer (300&#8211;500 &#181;L containing 10&#8211;20 &#181;g PK) to immerse the filter pieces, and elute RNA from the filter for 2&#8211;3 h or overnight.</li>
			<li>Extract with phenol-chloroform, chloroform, and collect aqueous phase. Add sodium acetate and ethanol and, after incubation in freezer, centrifuge, wash, dry and resuspend RNA in DEPC-treated water. Follow these steps as detailed in steps 2.10 to 2.14 above.</li>
		</ol>
		</li>
		<li>Gel mobility shift
		<ol>
			<li>Prepare and polymerize a 5% native polyacrylamide gel (60:1 Acrylamide:bis-acrylamide) in 0.5x TBE (standard Tris-Borate-EDTA buffer) before starting the binding reaction, as an alternative to the filter binding method.</li>
			<li>Pre-run the gel for 15 min at 250 V in a cold room (4 &#176;C).</li>
			<li>Load each binding reaction (above) into separate wells of the above pre-run gel. 
			NOTE: The protein storage buffer provides sufficient glycerol for sample loading into wells.</li>
			<li>Separate the protein-bound RNA by gel electrophoresis in a cold room at 250 V for 1 to 2 h, depending on the RNA size and specific protein.</li>
			<li>Locate the position of the RNA-protein complex on the gel by autoradiography. Excise the gel slice containing the RNA-protein complex. Elute the RNA from the gel slice by crushing the gel slice and soaking in the PK buffer.</li>
			<li>Extract with phenol-chloroform, chloroform, and collect aqueous phase. add Sodium acetate and ethanol and, after incubation in freezer, centrifuge, wash, air dry, and resuspend RNA in DEPC-treated water. Follow these steps as detailed in steps 2.10 to 2.14 above.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Analysis of Iodine-cleaved Phosphorothioate Products for Detection of Mutant Nucleotide Positions
<ol>
	<li>Add 1 mM iodine in a 20 &#181;L DEPC-treated water containing up to 10 &#181;g carrier tRNA to cleave RNAs (bound and total RNAs) at the sites of phosphorothioate incorporation. Incubate at room temperature for 5 min. 
	NOTE: Further details on iodine cleavage with slightly different conditions &#8212; 7% (v/v) iodoethanol, heating at 95 &#176;C for 3 min &#8212; can be found in Gish &#38; Eckstein 198819.</li>
	<li>Precipitate cleaved RNA by adding sodium acetate/ethanol, as described above. Resuspend in a loading dye for denaturing gels. Heat and load the sample to separate RNA fragments by electrophoresis in a 15&#8211;20% denaturing polyacrylamide gel.</li>
	<li>Expose the polyacrylamide gel to an X-ray film.</li>
	<li>Detect bands in the bound fraction versus total pool using autoradiography. 
	NOTE: Perform all steps with iodine in an exhaust hood. For RNA separation shown here, a wedge-shaped gel is employed, which is achieved by doubling the thickness of the both spacers at the bottom of the gel plates by inserting an extra inch-long spacer at the bottom of the gel. This shape allows more uniform or closer spacing between shorter RNA fragments. Alternatively, a phosphorimager can be used rather than X-ray films for detection and quantitation of radioactivity.</li>
</ol>

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Drosophila as a paradigm.</a> Bioessays. 14 (8), 513-518 (1992).</li><li>Salz, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex,+stem+cells+and+tumors+in+the+Drosophila+ovary.">Sex, stem cells and tumors in the Drosophila ovary.</a> Fly (Austin). 7 (1), 3-7 (2013).</li><li>Gawande, B., Robida, M. D., Rahn, A., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Sex-lethal+protein+mediates+polyadenylation+switching+in+the+female+germline.">Drosophila Sex-lethal protein mediates polyadenylation switching in the female germline.</a> Embo J. 25 (6), 1263-1272 (2006).</li><li>Robida, M. D., Rahn, A., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+alternatively+spliced+mRNA+targets+of+specific+RNA-binding+proteins.">Genome-wide identification of alternatively spliced mRNA targets of specific RNA-binding proteins.</a> PLoS One. 2 (6), e520 (2007).</li><li>Samuels, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+by+Sxl+proteins+in+vitro+and+in+vivo.">RNA binding by Sxl proteins in vitro and in vivo.</a> Mol. Cell. Biol. 14 (7), 4975-4990 (1994).</li><li>Fujii, S., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+expressed+in+the+Drosophila+head+reveal+a+role+for+fat+cells+in+sex-specific+physiology.">Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology.</a> EMBO J. 21 (20), 5353-5363 (2002).</li><li>Kelley, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+msl-2+causes+assembly+of+dosage+compensation+regulators+on+the+X+chromosomes+and+female+lethality+in+Drosophila.">Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila.</a> Cell. 81 (6), 867-877 (1995).</li><li>Hager, J., Cline, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+female+Sex-lethal+RNA+splicing+in+male+germ+cells:+implications+for+Drosophila+germline+sex+determination.">Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination.</a> Development. 124 (24), 5033-5048 (1997).</li><li>Vied, C., Halachmi, N., Salzberg, A., Horabin, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antizyme+is+a+target+of+sex-lethal+in+the+Drosophila+germline+and+appears+to+act+downstream+of+hedgehog+to+regulate+sex-lethal+and+cyclin+B.">Antizyme is a target of sex-lethal in the Drosophila germline and appears to act downstream of hedgehog to regulate sex-lethal and cyclin B.</a> Dev Biol. 253 (2), 214-229 (2003).</li><li>Chau, J., Kulnane, L. S., Salz, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-lethal+facilitates+the+transition+from+germline+stem+cell+to+committed+daughter+cell+in+the+Drosophila+ovary.">Sex-lethal facilitates the transition from germline stem cell to committed daughter cell in the Drosophila ovary.</a> Genetics. 182 (1), 121-132 (2009).</li><li>Chau, J., Kulnane, L. S., Salz, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-lethal+enables+germline+stem+cell+differentiation+by+down-regulating+Nanos+protein+levels+during+Drosophila+oogenesis.">Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.</a> Proc Natl Acad Sci U S A. 109 (24), 9465-9470 (2012).</li><li>Singh, R., Valcarcel, J., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+binding+specificities+and+functions+of+higher+eukaryotic+polypyrimidine+tract-binding+proteins.">Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.</a> Science. 268 (5214), 1173-1176 (1995).</li><li>Singh, R., Banerjee, H., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+recognition+of+the+polypyrimidine-tract+by+the+general+splicing+factor+U2AF65+and+the+splicing+repressor+sex-lethal.">Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal.</a> RNA. 6 (6), 901-911 (2000).</li><li>Sakashita, E., Sakamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RNA+binding+specificity+of+the+Drosophila+sex-+lethal+protein+by+in+vitro+ligand+selection.">Characterization of RNA binding specificity of the Drosophila sex- lethal protein by in vitro ligand selection.</a> Nucleic Acids Res. 22 (20), 4082-4086 (1994).</li><li>Kanaar, R., Lee, A. L., Rudner, D. Z., Wemmer, D. E., Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+sex-lethal+RNA+binding+domains+with+RNA.">Interaction of the sex-lethal RNA binding domains with RNA.</a> EMBO J. 14 (18), 4530-4539 (1995).</li><li>Milligan, J. F., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+small+RNAs+using+T7+RNA+polymerase.">Synthesis of small RNAs using T7 RNA polymerase.</a> Methods Enzymol. 180, 51-62 (1989).</li><li>Gish, G., Eckstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA+sequence+determination+based+on+phosphorothioate+chemistry.">DNA and RNA sequence determination based on phosphorothioate chemistry.</a> Science. 240 (4858), 1520-1522 (1988).</li></ol>

The author declares no competing financial interests.

Mutagenesis has long been used to characterize protein binding sites. First, a series of mutants can be constructed and individually tested in binding assays to analyze their effects on binding affinity. While a standard mutagenesis approach offers a way to analyze several sequences, multiple steps involved in the standard approach, such as constructing mutants and performing a series of binding reactions for each mutant, is laborious and time consuming and may not allow saturation mutagenesis, especially for longer sequences. Second, a sequence can be randomized and the pool used for multiple cycles of binding and polymerase chain reaction (PCR) amplification. These sequences that bind protein will have to be cloned and sequenced to identify and validate a binding site from the consensus sequence. Nonetheless, this iterative binding and amplification option involves multiple steps and is laborious in identifying residues that are important within the binding site. Moreover, repeated sequence amplifications inherent to PCR may introduce sequence bias. Third, sequences selected from the random pool can be sequenced using a faster option of high throughput sequencing, although it is relatively expensive. Therefore, to overcome these limitations of multiple steps, laborious and/or expensive methods, we devised a faster, inexpensive, and most importantly a single-step method, described here, to accomplish saturation mutagenesis of a binding site (PTM).
The key steps in this approach include identification or tagging of the non-wild-type nucleotide(s). We used phosphorothioate nucleotides, in which one of the oxygens in the phosphate backbone is substituted with sulfur, as described (Figure 2). The advantage is that iodine can be used to chemically cleave the backbone of the phosphorothioate nucleotide. Thus, iodine cleavage of the RNA substrate containing a phosphorothioate backbone generates a sequencing-type ladder, where the site of cleavage is a proxy or tag for the presence of one of the four bases at that position. A comparison of the unbound and total fractions identifies residues that are preferentially excluded from the bound fraction and thus are important for protein binding. In contrast, residues that are not important for binding remain equally distributed between the two fractions. This approach can be used to define binding sites for any RNA-binding protein.
In summary, this one-step saturation mutagenesis approach, combining doping, phosphorothioates, and iodine cleavage, offers a powerful means for the characterization of any binding site in RNA. However, this approach requires that the binding site is already known prior to mutagenesis. Furthermore, protein&#8211;binding conditions need to optimized for each protein. The following precautions need to be emphasized. Precautions for RNA handling must be exercised, such as wearing gloves, preparation of solutions and buffers in DEPC-treated water, autoclaving pipette tips and tubes, and dedicating equipment for RNA use only to avoid RNAses. Similarly, care involving radioactive shields, radioactive monitoring while using radioactive material is essential, and use of exhaust hood while using hazardous chemicals are necessary.

Gene regulation plays an important role in biology. Genes can be regulated at the level of transcription, pre-mRNA splicing, 3&#39; end formation, RNA export, translation, mRNA localization, decay, post-translational modification/stability, etc. Both DNA- and RNA-binding proteins play key roles in gene regulation. While molecular genetic analyses have identified numerous regulatory proteins, only a small subset of them have been characterized fully for their cellular functions or binding sites in vivo. Phylogenetic sequence analysis and mutagenesis offer complementary approaches to characterize DNA- or RNA-protein interactions.
RNA-binding proteins are important in developmental processes, including sexual differentiation. The Drosophila protein Sex-lethal (SXL) or the master sex-switch protein is absent in males, but present in females. It recognizes uridine-rich sequences or pyrimidine-tracts adjacent to specific splice sites in downstream pre-mRNA targets (transformer, Sex-lethal, and male-specific lethal2) in somatic cells1,2,3,4. In addition, it regulates polyadenylation site switching by binding to uridine-rich polyadenylation enhancer sequences in the enhancer of rudimentary (e(r)) transcript5,6. SXL likely regulates additional targets in the female germline that remain to be identified1,7,8,9,10,11,12,13.
Typically, characterization of a binding site involves mutagenesis, for example, by deletion or substitution of single or multiple nucleotides. Each mutant binding site, relative to the wild-type RNA sequence, is then analyzed using a series of protein concentrations to determine its binding affinity (Kd or equilibrium dissociation constant) for the protein of interest; Kd is the protein concentration required to obtain 50% RNA binding. This labor-intensive process of detailed mutagenesis involves generation and analysis of numerous mutants &#8212; three non-wild type nucleotides for each position in the binding site. Thus, there is a need for an alternative approach for faster, simpler, and inexpensive saturation mutagenesis of protein binding sites in RNA.
Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

<table>
	<tbody>
		<tr>
			<td>Uridine 5&#8217; a-thio triphosphate</td>
			<td>NEN (Boston, Massachusetts)</td>
			<td>NLP-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217; a-thio triphosphate</td>
			<td>NEN (Boston, Massachusetts)</td>
			<td>NLP-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Fisher Scientific</td>
			<td>XX1002500</td>
			<td>Millipore&#160;25&#160;mm Glass Microanalysis Vacuum Filter</td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Millipore</td>
			<td>XX2702552</td>
			<td>1225 Sampling Vacuum Manifold</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Millipore</td>
			<td>HAWP</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Schleicher &#38; Schuell</td>
			<td>PROTRAN</td>
			<td></td>
		</tr>
		<tr>
			<td>Dephosphorlyation Kit</td>
			<td>NEB</td>
			<td>M0508</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>NEB</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td></td>
			<td>RNase inhibitor</td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Electrophoresis equipment</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray films</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide gel solutions</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel saturation mutagenesis approach: single step characterization of regulatory protein binding sites in rna using phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Principle of saturation mutagenesis using doping:
For an appropriate molar ratio of wild-type and other nucleotides, use an equal mixture of all four nucleotides if only one position is to be analyzed. However, if multiple positions are analyzed simultaneously, the ratio of non-wild type to wild- type nucleotides must be adjusted, i.e., reduced. Otherwise, in addition to single substitutions, which is desired, there will also be templates with multiple non-wild type nucleotides in a molecule, precluding analysis of the effect of single-nucleotide substitutions. Thus, as a rule of thumb, use a ratio of non-wild type to wild-type nucleotides of 1/n, where n is the number of positions to be analyzed. Here, we analyzed 10 positions simultaneously and used a mixture containing 90% of the wild-type nucleotide and 10% of the non-wild type nucleotide for doping the DNA template. Separate transcription reactions are done, in which each transcription reaction is performed with one of the four phosphorothioates. Thus, each reaction monitors a specific nucleotide at the doped positions.
Principle of partitioning due to interference:
In the mutagenesis approach presented here, RNAs have on an average fewer than one phosphorothioate residue per molecule. During the process of binding, RNA molecules partition between the protein-bound fraction and the unbound fraction. Since a particular nucleotide is linked to a phosphorothioate linkage, cleavage of the phosphorothioate backbone linkage is a readout of the presence of a specific nucleotide at that position. It is expected that at any given position, when a nucleotide is changed it may have either no effect on binding or it may inhibit binding, partially or completely. If presence of a specific nucleotide has no effect on binding it will partition equally between the protein-bound and unbound fractions. However, if a specific nucleotide at a given position interferes with protein binding it will be preferentially excluded from the protein-bound fraction. The degree of interference can be quantitatively monitored for each of the positions in the same reaction following gel electrophoresis. This concept is illustrated in a schematic (Figure 3). In total RNA fraction (lane T), band intensity is approximately equal for all doped positions (bands 1, 3&#8211;7). In protein-bound RNA fraction (lane B), at positions 1, 4, and 7, the nucleotide has no effect on binding. However, at positions 3 and 6, it interferes with binding and thus is excluded from the bound fraction. At position 5, interference is partial. Thus, comparisons of four paired lanes, T and B for each nucleotide, allow for analysis of all four nucleotides. It should be noted that wild-type nucleotide for a given position reflects the effect, if any, of sulfur in the RNA backbone on protein binding.
The accompanying autoradiograph (Figure 4) shows two pairs (&#945;-thio A and &#945;-thio U) of lanes from a denaturing gel (T for Total pool and B for Bound fraction). Several observations can be made by comparing the intensities of bands at each position between each pair of lanes (T and B). First, the majority of the signal is at the top of the gel, i.e., uncleaved product, for both &#945;-thio A lane and &#945;-thio U lane. Second, for &#945;-thio A pair lanes, several bands (iodine cleavage products) are identical between the bound and the total RNA fractions for example, bands above 1; relevant bands within the binding site for the &#945;-thio A lane are numbered for reference. Third, bands at the bottom of the gel are more closely spaced (e.g., bands below 6) than is usual for a sequencing gel. Fourth, several bands are present in the total RNA fraction but absent or significantly reduced in the bound RNA fraction (e.g., bands 1, 2, 3, and 5). Fifth, for the &#945;-thio U lane pair, while most bands are comparable between the total and bound lanes, some bands are relatively less intense in the bound fraction (e.g., bands 7 and 8).
These observations provide evidence for successful development of this new method and lead to the following conclusions: First, for both &#945;-thio A pair lanes and &#945;-thio U pair lanes, because most of the RNA is uncleaved, it provides evidence that only a tiny fraction of the RNA contains modified or phosphorothioate residues. This is important because it ascertains that RNA molecules contain no more than one phosphorothioate residue. Second, identical or similar intensities for several bands outside of the binding site between the two lanes, for both &#945;-thio A and &#945;-thio U, demonstrate that loading is comparable for both lanes, allowing easy comparison between lanes. Third, more closely spaced bands are achieved, for &#945;-thio A and &#945;-thio U, by the wedge-shape of the gel (thinner at the top and thicker at the bottom), thus allowing analysis of longer sequence reads and offering higher resolution. Typically, shorter fragments are more widely spaced in a gel with uniform thickness. Fourth, disappearance or reduced intensity of certain bands in the bound fraction indicates that the non-wild-type nucleotide is preferentially excluded from the bound fraction (&#945;-thio A). Relative intensities of different bands within the bound lane also offer quantitative information about the extent of interference from non-wild type residues at different locations. In other words, the mutant or non-wild type nucleotides at specific positions interfere with protein binding. Finally, testing the effect of backbone sulfur substitution (phosphorothioate) at one of the non-bridging oxygens (&#945;-thio U), shows that three positions show minor effects from backbone sulfurs (e.g., 6, 7 and the one below 7). Our combined results show that several positions within the binding site show preferential disappearance or reduced intensities of specific bands in the bound fraction. This is due to base rather than backbone substitution, indicating protein interactions with specific bases in the binding site (&#945;-thio A). Meanwhile, incorporation of &#945;-thio C shows an interference pattern comparable to &#945;-thio A. However, only 3&#8211;4 &#945;-thio G substitutions show small but detectable interference centered around, for example, positions numbered 2 and 3 (data not shown). This method has been used to reveal differences in how the splicing repressor SXL and the general splicing factor U2 snRNP Auxiliary Factor (U2AF65) bind to an identical pre-mRNA splicing signal sequence (polypyrimidine-tract) in distinct manner15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57816/57816fig1.jpg" /> 
Figure 1: Flow chart of key steps in phosphorothioate mutagenesis (PTM). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57816/57816fig2.jpg" /> 
Figure 2: Schematic of a phosphorothioate linkage between two nucleotides, which can be chemically cleaved by iodine. Sulfur replaces one of the non-bridging phosphate oxygens. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57816/57816fig3.jpg" /> 
Figure 3: Principle of partitioning due to interference. Hypothetical RNA contains representative &#945;-thio nucleotides at six positions, including a protein-binding site. Following protein binding, RNA is cleaved at sites of phosphorothioate incorporation by iodine. Positions 1&#8211;7 within and around the binding site are arbitrarily numbered for reference in the text. Position 2 or missing band represents no phosphorothioate incorporation or non-doped nucleotide. Lane T is total RNA and lane B is protein-bound RNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57816/57816fig4.jpg" /> 
Figure 4: Phosphorothioate mutagenesis (PTM) approach allows saturation mutagenesis of a polypyrimidine-tract/3&#39; splice site present in the transformer pre-mRNA. Lane T is total RNA and lane B is protein-bound RNA. &#945;-thio A represents RNA synthesized in the presence of &#945;-thio ATP and identifies doped positions with &#945;-thio adenosines in the sequence. Three strong bands correspond to adenosines in the sequence at those positions. &#945;-thio U represents RNA synthesized in the presence of &#945;-thio UTP and identifies all uridines, including doped positions, in the sequence. Vertical line on the left marks the SXL binding site. Positions 1&#8211;8 within the binding site are numbered for reference purposes and for ease of description in the results section. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates at JoVE.com

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

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Nanyang Technological University

Research

JoVE Journal

Genetics

6.5K Views.  University of Colorado at Boulder. Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Video: A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...