Measuring gene expression via RNA

Northern blot RNase protection Primer extension RT-PCR Q-RT-PCR Microarray RNAseq Measuring gene expression via RNA

Northern blotting Denaturing gel for true MW (urea, formamide) Alternative polyadenylation sites  2 dhfr mRNAs http://www.gene-quantification.de/mrna.html#northern

RNase protection (RPA) dhfr mRNA Wild type Mutant-exon3 1-2-3-4-5-6 1-2-4-5-6

Primer extension: map the 5’ end of an mRNA 1 3 minor start major start

Cap trapping to isolate cDNAs that go to the 5’ end of the mRNA First biotinylate the ribose residues that carry adjacent ring hydroxyls (diols):

Next: Use an XhoI-tailed adapter-primer to copy the RNA into cDNA Use RNaseI to digest SS RNA. Biotinylated 3’ end cleaved. 5’ incomplete cDNAs lose their cap-biotin. Isolate the surviving capped DS molecules with avidin beads. Get rid of the RNA with RNase A. dG tail. Make second strand with SacI-tailed oligo dC Cut with SacI and XhoI and clone. Full length Truncated Magnetic avidin beads

“Nanostrings” to quantify mRNA levels by single molecule counting Geiss et al. Nat. Biotech. 26:317, 2008 900 nt m13 segments labeled with one of 4fluorescent dyes. Make a unique color-code, ligate to 30-50- nt mRNA-specific seq and to a 5’ universal repeat. Can make up to 800 of these. Strrech out via electrophoresis and then anchor far end. Ligate a universal 3’ repeat to the 3’ end of an mRNA-specific sequence (35-50 nt) Avidin coated surface.B=biotinylated Fluorescent RNA: T7 promoted transcription of m13 segment PCR product using amino-allyl-UTP; then conjugate to dye. 4 colors, 7 positions, 37=2100 [diff. neighbors]

λ=average no. of occurrences f= probability of k occurrences For k=0, f0=e-λ Observe f, calculateλ Digital droplet PCR, or digital PCT, dPCR QX100™ Droplet Digital™(Bio-Rad) Poisson distribution: Aqueous microspheres in water-in-oil emulsion PCR in droplets Read + or – in instrument Data Positive (green, here) microspheres had >= 1 templates. All positives have same intensity, as PCR  plateau.

Bioanalyzer 2100 (Agilent): gel electrophoresis in microfluidic channels Area under curves  quantitative results Virtual gel image (for Luddites)

Protein-protein interactions Yeast 2-hybrid system Yeast 3-hybrid and 1 hybrid systems Co-immunoprecipitation Pull-downs Far western blots Biacore (surface plasmon resonance, SPR) Fragment complementation

Yeast 2-hybrid system: To discover proteins that interact with each other, or To test for interaction based on a hypothesis for a specific protein. Positive control: (bait) ? Y = e.g., a candidate protein being tested for possible interaction with X Or: Y = e.g., a cDNA library used to discover a protein that interacts with X ? (prey) BD =(DNA) binding domain AD =activation domain UAS =upstream activating sequence http://www.mblab.gla.ac.uk/~maria/Y2H/Y2H.html

No interaction between X and Y: no reporter expression Yes, interaction between X and Y: reporter protein is expressed Y = e.g., a cDNA fusion protein library used to discover a protein that interacts with X Recover the Y sequence from reporter+ colonies by PCR to idenify protein Y

Fusion library Bait protein is the known target proteinfor whom partners are sought =“prey” Two different assays help, as there are often many false positives. BD= DNA binding domain; TA = transactiavting domain http://www.mblab.gla.ac.uk/~maria/Y2H/Y2H.html

3-HYBRID: select for proteins domains that bind a particular RNA sequence Use a known tight protein-RNA interaction (e.g., from RNA phage MS2) Prey RNA binding site in question Bait Prey could be proteins from a cDNA library

Yeast one-hybrid: Insert a DNA sequence upstream of the selectable or reporter Transform with candidate DNA-binding proteins (e.g., cDNA library) fused to an activator domain. Each T = one copy of a DNA target sequence

Indirect selection using a yeast 3-hybrid system:toward a more efficient glycosynthase enzyme Directed Evolution of a Glycosynthase via Chemical Complementation Hening Lin,† Haiyan Tao, and Virginia W. Cornish J. AM. CHEM. SOC. 2004, 126, 15051-15059 Turning a glycosidase into a glyco-synthase Glycosidase: Glucose-Glucose (e.g., maltose) + H2O  2 Glucose

Indirect selection using a yeast 3-hybrid system(one of the hybrid molecules here is a small molecule) e.g., from a mutated library of enzyme glycosynthase genes glucose Leu2 gene Leu2 gene Transform a yeast leucine auxotroph. Provide synthetic chimeric substrate molecules. Select in leucine-free medium. DHFR = dihydrofolate reductase GR = glucocorticoid receptor (trancription factor ) MTX = methotrexate (enzyme inhibitor of DHFR) DEX = dexamethasone, a glucocorticoid agonist, binds to GR AD = activation domain, DBD = DNA binding domain

Selection of improved cellulases via the yeast 2-hybrid system Survivors are enriched for cellulase genes that will cleave cellulose with greater efficiency (kcat / Km) Yeast cell Cellobiose (disaccharide) URA-3 (toxic) cellulase Directed Evolution of Cellulases via Chemical Complementation. P. Peralta-Yahya, B. T. Carter, H. Lin, H. Tao. V.W. Cornish. x x x x Library of cellulase mutant genes (one per cell)

Detail

URA-3 = gene for orotidine phosphate (OMP) decarboxylase Pathway to pyrimidine nucleotides: How does the URA-3 “suicide” system work? analog 5-fluoroorotic acid 5-fluoro-OMP URA-3 decarboxylation (pyr-4) 5-fluoro-UMP uridine kinase exogenousuridine thymidylate synthetase inhibition RNA death

Measuring protein-protein interactions in vitro X=one protein Y= another protein Pull-downs: Binding between defined purified proteins, at least one being purified. Tag each protein differently by making the appropriate cDNA clone. Examples: His6-X + HA-Y; Bind to nickel ion column via X, elute (his), Western with HA Ab for Y GST-X + HA-Y; Bind to glutathione ion column, elute (glutathione), Western with HA Ab His6-X + 35S-Y (made in vitro); Bind Ni column, elute (his),  gel + autoradiography. No antibody needed. GST = glutathione-S-transferase(HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.

Co-immunoprecipitation Protein A (bacterial) binds most IgG’s Ig-hc protein 1 protein 2 Ig-lc Boil, SDS PAGE molecularsciences.org

Without a specific antibody for your target protein, express it as a fusion protein carrying a standard epitope tag such as FLAG, myc, hemagglutinin (HA), etc. Or, do a pulldown, without antibodies using pairs of strongly interacting molecules: His6 + chelated nickel or cobalt on beads glutathione-s-tranferase (a small enzyme, GST) + Glutathione (tetrapeptide) on beads maltose binding protein (MBP) + amylose (poly-alpha-glucose) on beads

Pulldowns: GST or poly-His tags bait prey Elute with glutathione or imidazole GST = glutathione-S-transferase =GST or His6 =glutathione or cobalt chelate

Example of a result of a pull-down experiment Total protein: no antibody/Western (stained with Coomassie Blue or silver stain) Also identify by MW (or mass spec)

WB = western blot FLAG and Myc are epitopes for which there are good antibodies available. GST = glutathione-S-transferase PABP2 = PolyA binding protein 2 RRMs = PABP2 RNA recognition motif PABP2-FL full length protein PABP2-N N-terminal fragment

Western blotting * To detect the antibody use a secondary antibody against the primary antibody (e.g, goat anti-rabbit IgG). The secondary antibody is a commercial fusion protein with an enzyme activity (e.g., alkaline phosphatase). The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound. * http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

Detection of antibody binding in western blots Antibody to protein on membrane Alkaline phosphatase fusion Non-luminescent substrate-PO4 = Y Y Luminescent product + PO4= (chemiluminescence) Secondary antibody Detect by exposing to film (minutes or hours) Protein band on membrane

Far western blotting to detect specific protein-protein interactions. Use a specific purified protein as a probe instead of the primary antibody To detect the protein probe use an antibody against it. Then a secondary antibody against the first antibody, a fusion protein with an enzyme activity. The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound. protein protein OR:Use a radioactively labeledprotein of interest and detect by autoradiography http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

How to make a radioactively labeled protein:Expression via in vitro transcription followed by in vitro translation T7 RNA polymerase binding site (17-21 nt) VECTOR cDNA ….ACCATGG….. Radioactively labeled protein 1. Transcription to mRNA via the T7 promoter + T7 polymerase 2. Add a translation system: rabbit reticulocyte lysate or wheat germ lysate Or: E. coli lysate (combined transcription + translation, TnT) All commercially available as kits Add ATP, GTP, tRNAs, amino acids, label(35S-met), May need to add RNase (Ca++-dependent, stop with EGTA) to remove endogenous mRNA In lysate NOTE: Protein is NOT at all pure (1000s of lysate proteins present), just ~“radio-pure”

Surface plasmon resonance (SPR) Popular instrument is a Biacore The binding events are monitored in real-time and it is not necessary to label the interacting biomolecules. In a flow cell glass plate Reflection angle changes depending on the mass of the material on the surface. Binding increases this mass. Follow as a function of concentration  Kd’s Or time : Measure on-time, off time; Kd = off-time/on-time http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm

A Biacore result Ligand added Ligand removed

Back to protein-protein interactions: Reporterenzyme Enzyme fragments themselves do not associate well enough to reconstitute an active enzyme F = reporter protein fragment SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif

Dihydrofolate reductase (DHFR): role in metabolism Folic acid DHFR (FH2) DHFR (FH4) http://www.nature.com/onc/journal/v22/n47/images/1206946f1.gif

Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays, I. Remy and S.W. Michnick PNAS 96, 394–5399, 1999 DHFR fragments Rapamycin promotes the association of the 2 protein domains fMTX Cell growth assay: CHO DHFR- mutantcells Fluorescein – MTXbinding assay IN PURINE-FREE MEDIUM DHFR = dihydrofolate reductase DHF=dihydrofolate = FH2 THF=tetrahydrofolate = FH4 fMTX=fluorescent methotrexate FK506 = immunosuppressant drug FKBP = FK506 binding protein FRAP = FKBP–rapamycin binding proteinFRB= FKBP–rapamycin binding domain of FRAP

FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase a phosphatase

Claim detection of 0.05 nM rapamycin ?? No. of CHO colonies [rapamycin]

Fluorescent methotrexate (fMTX) assay: Wash in, wash out CHO cells (permanent transfection) cos cells (transient transfection) Leucine zipper protein fragments instead of rapamycin binding proteins (positive contro) Background association of FKBP and FRB without rapamycin (compare mixed input)

Fuorescence-activated flow cytometer (FACS is this, plus more) Allows quantitation of fluorescence per cell No. of cells 8-fold increase in fluorescence per cell Log of fluorescence intensity Fluorescence intensity Measure affinity for a drug in vivo [rapamycin] Competition with a molecule that binds only one

Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic EPO EPO bp2 EPO bp1 Erytropoietin (EPO) receptor In vivo assay of drug effectiveness (EMP1) (inexpensive substitute for erythropoietin?) EMP1 = Erythropoietin mimetic peptide 1 Erythropoietin

FACS = Fluorescence-activated cell sorter Impart a charge on the recognized cell Less than one cell or particle per droplet. Thus the most that most droplets contain is one particle. Can be used purely anaytically without the sorting capability. Then called “flow cytometry”, or also called FACS anyway. Charged plates attract droplets containing a particle of the opposite charge Cells remain viable if treated with care.

Histogram-type display No fluorescence (background autofluorescence) No. of cells Red stained Usually a log scale Having this much fluorescence

Scatter plot display Analysis on 2 colors One cell Amount of greenfluorescence (log) You decide on the positions of of demarcations Say, want high reds but low greens: Instruct the FACS to deflect cells in this quadrant only. Collect and grow or analyze further. Amount of red fluorescence (log)

Measuring gene expression via RNA