Measuring gene expression via RNA
• Northern blot
• RNase protection
•
Primer extension
• RT-PCR
• Q-RT-PCR
• Microarray
• RNAseq
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Northern blotting
Denaturing gel for true MW (urea, formamide)
Alternative polyadenylation sites  2 dhfr mRNAs
2 http://www.gene-quantification.de/mrna.html#northern

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

Primer extension: map the 5’ end of an mRNA
4
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):
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Next:
Magnetic avidin beads
Full length
Truncated
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 capbiotin.
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.
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“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 4 fluorescent dyes.
Make a unique colorcode, ligate to 30-50- nt mRNA-specific seq and to a 5’ universal repeat.
Can make up to 800 of these.
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Strrech out via electrophoresis and then anchor far end.
Avidin coated surface.
B=biotinylated
Ligate a universal 3’ repeat to the 3’ end of an mRNA-specific sequence (35-50 nt)
Fluorescent RNA: T7 promoted transcription of m13 segment PCR product using amino-allyl-UTP; then conjugate to dye.
4 colors, 7 positions, 3 7 =2100 [diff. neighbors]

Digital droplet PCR, or digital PCT, dPCR
QX100™ Droplet Digital™ (Bio-Rad)
λ=average no. of occurrences f= probability of k occurrences
For k=0, f
0
=e -
λ
Observe f, calculate
λ
Poisson distribution:
PCR in droplets
Aqueous microspheres in water-in-oil emulsion
Read + or – in instrument
Positive (green, here) microspheres had >= 1 templates.
All positives have same intensity, as PCR  plateau.
Data
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Bioanalyzer 2100 (Agilent): gel electrophoresis in microfluidic channels
9
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
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Positive control:
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 .
(bait)
?
11
Y = e.g., a candidate protein being tested for possible interaction with X
?
(prey)
Or: Y = e.g., a cDNA library used to discover a protein that interacts with X
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
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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

Bait protein is the known target protein for whom partners are sought
Fusion library
=“prey”
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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
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Use a known tight protein-RNA interaction (e.g., from
RNA phage MS2)
Bait
Prey
RNA binding site in question
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.
15
Each T = one copy of a DNA target sequence

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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) + H
2
O  2 Glucose

Indirect selection using a yeast 3-hybrid system
(one of the hybrid molecules here is a small molecule) glucose e.g., from a mutated library of enzyme glycosynthase genes
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Leu2 gene
Transform a yeast leucine auxotroph.
Provide synthetic chimeric substrate molecules.
Select in leucine-free medium.
Leu2 gene
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

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

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

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:
His
6
-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
His
6
-X + 35 S-Y (made in vitro); Bind Ni column, elute (his),  gel + autoradiography.
No antibody needed.
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GST = glutathione-S-transferase
(HA = flu hemagglutinin) glutathione = gamma-glutamyl-cysteinyl-glycine.

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

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.
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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

bait
Pulldowns: GST or poly-His tags 24 prey
GST = glutathione-S-transferase
=glutathione or cobalt chelate
Elute with glutathione or imidazole
=GST or His6

Example of a result of a pull-down experiment
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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
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Western blotting
* http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
*
To detect the antibody use a secondary antibody against the primary antibody (e.g, goat antirabbit 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.
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Antibody to protein on membrane
Alkaline phosphatase fusion
Non-luminescent substrate-PO
4
=
Luminescent product + PO
4
=
(chemiluminescence)
Secondary antibody
Detect by exposing to film
(minutes or hours)
Protein band on membrane
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protein protein
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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.
OR:
Use a radioactively labeled protein of interest and detect by autoradiography http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

in vitro
in vitro
T7 RNA polymerase binding site (17-21 nt)
VECTOR
….ACC
ATG G…..
cDNA
Radioactively labeled protein
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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 ( 35 S-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”

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
31 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

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

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

Dihydrofolate reductase (DHFR): role in metabolism
Folic acid
DHFR
(FH
2
)
DHFR
(FH
4
)
35 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
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DHFR fragments
Rapamycin promotes the association of the 2 protein domains fMTX
Cell growth assay: CHO
DHFR
mutant cells
Fluorescein – MTX binding assay
IN PURINE-FREE MEDIUM
DHFR = dihydrofolate reductase
DHF=dihydrofolate = FH
2
THF=tetrahydrofolate = FH
4 fMTX=fluorescent methotrexate
FK506 = immunosuppressant drug
FKBP = FK506 binding protein
FRAP = FKBP
FRB= FKBP
–rapamycin binding protein
–rapamycin binding domain of FRAP

FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase a phosphatase
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Claim detection of
0.05 nM rapamycin
??
[rapamycin]
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Fluorescent methotrexate
(fMTX) assay:
Wash in, wash out
Background association of
FKBP and FRB without rapamycin
(compare mixed input)
CHO cells
(permanent transfection)
39 cos cells
(transient transfection)
Leucine zipper protein fragments instead of rapamycin binding proteins (positive contro)

Log of fluorescence intensity
Fuorescence-activated flow cytometer
(FACS is this, plus more)
Allows quantitation of fluorescence per cell
40
8-fold increase in fluorescence per cell
Measure affinity for a drug in vivo
[rapamycin]

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

FACS =
Fluorescence-activated cell sorter
Impart a charge on the recognized cell
Can be used purely anaytically without the sorting capability. Then called “flow cytometry”, or also called FACS anyway.
Less than one cell or particle per droplet. Thus the most that most droplets contain is one particle.
Charged plates attract droplets containing a particle of the opposite charge
Cells remain viable if treated with care.
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No fluorescence (background autofluorescence)
Red stained
Usually a log scale
Having this much fluorescence

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Analysis on 2 colors
One cell
Amount of red fluorescence (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.

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