Functional RNA- Introduction Part 2

Biochemistry 4000

Dr. Ute Kothe

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

Selection of proteins: mRNA Display

By binding to target of interest- specific forEach problem

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

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