functional rna - introduction part 2 biochemistry 4000 dr. ute kothe
Post on 20-Dec-2015
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in vitro selection of RNAsSELEX = 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
SELEXLibrary: 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 synthesizedDNA 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 displayb.Phage displayc.mRNA displayd.Ribosome displaye.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
In vitro evolution of proteinsRibosome Display In vitro compartmentalization
In vitro translation of mRNA without stop codon
mRNA is linked to protein in ternary complex with ribosome
• 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
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 k2 k3 k4
E + S ES ES* EP E + P
k-1 k-2 k-3 k-4
Michaelis-Menten Kinetics
kcat [E0] [S]v = KM + [S]
vmax = kcat [E0]
k-1 + k2KM = k1
k1 k2
E + S ES EP E + P
k-1
Assumed Mechanism:
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
Assumption of steady-state, i.e. [ES] = constant, then:
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 k2 k3 k4
E + S ES ES* EP E + P
k-1 k-2 k-3 k-4
Rate constants
v = d[P] / dt = - d[S] / dt = k [S]S P
First order reaction:
v = d[P] / dt = - d[S1] / dt
= - d[S2] / dt = k [S1] [S2]S1 + S2 P
Second order reaction:
ln[S] = ln [S0] –kt
[S] = [S0] exp (-kt)
[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-Flow1. 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