Functional RNA
- Introduction Part 2
Biochemistry 4000
Dr. Ute Kothe
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 3dimensional shape
Targets:
• single molecules to whole organisms
• Chiral molecules
• Recognition of distinct epitopes
Applications:
• pharmaceutical research
• drug development
• proteomics
• molecular biology
SELEX
Library: 1013 – 1015 sequences
1. In vitro selection
•
Binding to target
•
Partitioning from unbound
oligos
•
Elution of selected oligos
2. Amplification
•
PCR for DNA or RT-PCR for
RNA
•
Conditioning:
transformation of dsDNA
into new pool of ssDNA or
RNA for seletion
 Iterative process
Random oligonucleotide library
Chemically synthesized
DNA oligonucleotides:
Randomized sequence
flanked by 2 fixed
sequences used as
primer binding sites
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
???
In vitro evolution of proteins
Principle:
selection based on protein
properties, genes must be
selected simultaneously
 Physical linkage between
genotype & phenotype
Methods:
a.Cell-surface display
b.Phage display
c.mRNA display
d.Ribosome display
e.In vitro compartmentalization
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
Selection of proteins: mRNA Display
By binding to
target of interest
- specific for
Each problem
In vitro evolution of proteins
Ribosome Display
In vitro translation of mRNA without
stop codon
 mRNA is linked to protein in
ternary complex with ribosome
In vitro compartmentalization
• mRNA linked to microbeads emulsified
with substrate-biotin conjugate
• product-biotin binds to beads via
streptavidin
• detection of product by fluorescentlabeled anti-product antibody, sorting by
FACS
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
k1
E+S
k2
ES
k-1
k3
ES*
k-2
k4
EP
k-3
E+P
k-4
Michaelis-Menten Kinetics
Assumed
Mechanism: E + S
k1
k2
ES
EP
E+P
k-1
Assumption of steady-state,
i.e. [ES] = constant, then:
v=
kcat [E0] [S]
KM =
KM + [S]
k-1 + k2
k1
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
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])
1.Quench-Flow: observation of chemcial reactions (S
P)
2.Stopped-Flow: observation of conformational changes by
absorbance or fluorescence
k1
E+S
k2
ES
k-1
k3
ES*
k-2
k4
EP
k-3
E+P
k-4
Rate constants
First order reaction:
S
P
v=
d[P] / dt
= - d[S] / dt = k [S]
 ln[S] = ln [S0] –kt
 [S] = [S0] exp (-kt)
Second order reaction:
S1 + S2
P
v=
d[P] / dt
= - d[S1] / dt
= - d[S2] / dt = k [S1] [S2]
 [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
Quench-Flow
1. rapidly mix samples
2. stop reaction after
desired time (ms) with
quencher (strong acid,
base etc.)
3. analyze (radioactive)
reaction product by
HPLC, thin-layer
chromatography etc.
 One time point at a
time, several mixing
events required to
obtain time curve
Quench-Flow data
EPSP synthase:
PEP + S3P
I
EPSP + Pi
shikimate 3-phosphate (S3P), 5-enolpyruvoylshikimate 3-phosphate (EPSP)
Stopped-flow
1. Rapidly mix samples,
2. stop the flow of mixed solutions such that it stays in cuvette
3. Detect change in fluorescence/absorbance in real time
 One mixing event generates data of whole time curve
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