1. Functional RNA - Introduction Part 2
Biochemistry 4000
Dr. Ute Kothe

2. in vitro selection of RNAs
SELEX = Systematic evolution of ligands by exponential enrichment
Generates Aptamers
= oligonucleotides (RNA or ssDNA) which bind to their target with high selectivity and sensitivity because of their 3-dimensional shape
Targets:
single molecules to whole organisms
Chiral molecules
Recognition of distinct epitopes
Applications:
pharmaceutical research
drug development
proteomics
molecular biology

3. SELEX Library: 1013 – 1015 sequences In vitro selection
Binding to target
Partitioning from unbound oligos
Elution of selected oligos
Amplification
PCR for DNA or RT-PCR for RNA
Conditioning: transformation of dsDNA into new pool of ssDNA or RNA for seletion
Iterative process

4. Random oligonucleotide library
Chemically synthesized
DNA oligonucleotides:
Randomized sequence flanked by 2 fixed sequences used as primer binding sites

5. Selection of catalytic RNA
more complex RNA
– often random pool is further enlarged by mutagenic PCR
reaction must result in self-modification
such that active molecules can be selected
Example: Selection of an RNA ligase
???

6. In vitro evolution of proteins
Principle:
selection based on protein properties, genes must be selected simultaneously
Physical linkage between genotype & phenotype
Methods:
Cell-surface display
Phage display
mRNA display
Ribosome display
In vitro compartmentalization

7. Selection of proteins: mRNA Display
random mRNA is translated in vitro
mRNA is linked to DNA oligo
with puromycin
puromycin covalently attaches
mRNA to produced protein
Puromycin: analog of Tyr-tRNA
can not be hydrolyzed

8. Selection of proteins: mRNA Display
By binding to
target of interest
specific for
Each problem

9. In vitro evolution of proteins
Ribosome Display
In vitro compartmentalization
mRNA linked to microbeads emulsified with substrate-biotin conjugate
product-biotin binds to beads via streptavidin
detection of product by fluorescent-labeled anti-product antibody, sorting by FACS
In vitro translation of mRNA without stop codon
mRNA is linked to protein in ternary complex with ribosome

10. Enzyme/ribozyme kinetics
Kinetics = study of chemical reaction rates
Why Kinetics?
Understanding of enzyme function: affinity, maximum catalytic rate
Identification of intermediates
Insight into catalytic mechanism
Investigation of inhibitors, activators
k k k k4
E + S ES ES* EP E + P
k k k-3 k-4

11. Michaelis-Menten Kinetics
k k2
E + S ES EP E + P
k-1
Assumed Mechanism:
Assumption of steady-state,
i.e. [ES] = constant, then:
k-1 + k2
KM = k1
kcat [E0] [S]
v =
KM + [S]
vmax = kcat [E0]
Follow reaction under multiple-turnover conditions to obtain kcat & KM
Problem: KM ╪ KD and kcat ╪ k2 (kchem) if not Michaelis-Menten mechanism
no information on intermediate steps and their rate constants

12. Pre-steady state Kinetics
Solution: Follow reaction
in real-time, i.e. pre-steady state by rapidly mixing substrates and enzymes and detection in ms to s range
under single-turnover conditions ([E] >> [S])
Quench-Flow: observation of chemcial reactions (S P)
Stopped-Flow: observation of conformational changes by
absorbance or fluorescence
k k k k4
E + S ES ES* EP E + P
k k k-3 k-4

13. Rate constants v = d[P] / dt = - d[S] / dt = k [S]
First order reaction:
v = d[P] / dt = - d[S] / dt = k [S]
S P
ln[S] = ln [S0] –kt
[S] = [S0] exp (-kt)
Second order reaction:
v = d[P] / dt = - d[S1] / dt
= - d[S2] / dt = k [S1] [S2]
S1 + S P
[S1] = ???
measure at pseudo-first order conditions: [S1] >> [S2]
[S1] = constant
v = - d[S2] / dt = k’ [S2] with k’ = k [S1]
[S2] = [S20] exp (-k’t)
measure apparent rate constant k’ at various [S1]
to determine rate constant k

14. Quench-Flow rapidly mix samples
stop reaction after desired time (ms) with quencher (strong acid, base etc.)
analyze (radioactive) reaction product by HPLC, thin-layer chromatography etc.
One time point at a time, several mixing events required to obtain time curve

15. Quench-Flow data EPSP synthase: PEP + S3P I EPSP + Pi
shikimate 3-phosphate (S3P), 5-enolpyruvoylshikimate 3-phosphate (EPSP)

16. Stopped-flow Rapidly mix samples,
stop the flow of mixed solutions such that it stays in cuvette
Detect change in fluorescence/absorbance in real time
One mixing event generates data of whole time curve

17. Stopped-Flow data Analyze data by exponential fitting:
F = Amp * exp (-kapp*t)
Generates apparent rate constant kapp
(e.g. for particular concentrations)
Titrate different substrate concentrations to determine real rate constant k from kapp