SELEX
(systematic evolution of ligands by exponential enrichment)
• is a method that was originally developed to
discover short RNA sequences (aptamers) that
bind to particular molecules.
• In the first step: these DNAs are transcribed in vitro, using
the phage T7 RNA polymerase, which recognizes the T7
promoter in the upstream constant region of every DNA in
the pool.
• In the next step: the aptamers are selected by affinity
chromatography, using a resin with the target molecule
immobilized.
The selected RNAs bind to the resin and then can be
released with a solution containing the target molecule.
These selected RNAs are then reverse-transcribed to yield
double-stranded DNA, which is then subjected to PCR, using
primers specific for the DNAs’ constant ends.
Aptamer
• Aptamers (from the Latin aptus - fit, and
Greek meros - part) are oligonucleic
acid or peptide molecules that bind to a specific
target molecule. Aptamers are usually created by
selecting them from a large
random sequence pool, but natural aptamers also
exist in riboswitches.
• Aptamers can be used for both basic research
and clinical purposes as macromolecular drugs.
• More specifically, aptamers can be classified as:
• DNA or RNA or XNA aptamers.
They consist of (usually short) strands of
oligonucleotides.
• Peptide aptamers.
They consist of a short variable peptide domain,
attached at both ends to a protein scaffold.
Post-Segregational Killing (PSK)
or
Toxin-antitoxin system
• A toxin-antitoxin system is a set of two or more closely
linked genes that together encode both a protein 'poison' and
a corresponding 'antidote'.
• When these systems are contained on plasmids – transferable
genetic elements – they ensure that only the daughter cells
that inherit the plasmid survive after cell division.
• If the plasmid is absent in a daughter cell, the unstable antitoxin is degraded and the stable toxic protein kills the new
cell; this is known as 'post-segregational killing'(PSK).
Type I
• Type I toxin-antitoxin systems rely on the basepairing of complementary antitoxin RNA with
the toxin's mRNA.
• Translation of the mRNA is then inhibited either
by degradation via RNase III or by occluding
the Shine-Dalgarno sequence or ribosome
binding site.
Example systems
Toxin
Antitoxin Notes
Hok
Sok
The original and best-understood type I toxin-antitoxin system
(pictured), which stabilises plasmids in a number of gramnegative bacteria
fst
RNAII
The first type I system to be identified in gram-positive bacteria
TisB
IstR
Responds to DNA damage
LdrD
RdlD
A chromosomal system in Enterobacteriaceae
FlmA
FlmB
A hok/sok homologue, which also stabilises the F plasmid
Discovered in E. coli intergenic regions, the antitoxin was
originally named QUAD RNA
TxpA/Brn
Ensures the inheritance of the skin element
RatA
T
during sporulation in Bacillus subtilis
SymE
SymR
A chromosomal system induced as an SOS response
A system identified in Xanthomonas campestris with erratic
XCV2162 ptaRNA1
phylogenetic distribution.
Ibs
Sib
Hok sok system
Type II
• In this system a labile protein antitoxin tightly
binds and inhibits the activity of a stable
toxin.
Type III
• Type III toxin-antitoxin systems rely on direct
interaction between a toxic protein and an
RNA antitoxin.
• The toxic effects of the protein are neutralised
by the RNA gene.
Biotechnological applications
•
•
•
•
In fermentation
As a future target for antibiotics
In DNA cloning
Control of the Genetically modified organisms
Genome editing
Genome editing
• Genome editing, or genome
editing with engineered
nucleases (GEEN) is a type
of genetic engineering in
which DNA is inserted,
replaced, or removed from
a genome using artificially
engineered nucleases, or
"molecular scissors."
Mechanism
• The nucleases create specific double-stranded
break (DSBs) at desired locations in the genome,
and harness the cell’s endogenous mechanisms
to repair the induced break by natural processes
of :
– homologous recombination (HR)
– nonhomologous end-joining (NHEJ).
There are currently four families of engineered nucleases
being used:
– Zinc finger nucleases (ZFNs)
– Transcription Activator-Like Effector Nucleases (TALENs)
– Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)
– Engineered meganuclease
• It is commonly practiced in genetic analysis that in order to
understand the function of a gene or a protein function one
interferes with it in a sequence-specific way and monitors its effects
on the organism.
• However, in some organisms it is difficult or impossible to perform
site-specific mutagenesis, and therefore more indirect methods have
to be used, such as silencing the gene of interest by short RNA
interference (siRNA) .
•
Yet gene disruption by siRNA can be variable and incomplete.
• Genome editing with nucleases such as ZFN is different from siRNA
in that the engineered nuclease is able to modify DNA-binding
specificity and therefore can in principle cut any targeted position in
the genome, and introduce modification of the endogenous
sequences for genes that are impossible to specifically target by
conventional RNAi.
Double stranded breaks and their repair
• non-homologous end joining (NHEJ) is a error
prone process.
• Thus if one is able to create a DSB at a desired
gene in multiple samples, it is very likely that
mutations will be generated at that site in
some of the treatments because of errors
created by the NHEJ infidelity.
Double stranded breaks and their repair
• On the other hand, the dependency of HDR on
a homologous sequence to repair DSBs can be
exploited by inserting a desired sequence
within a sequence that is homologous to the
flanking sequences of a DSB which, when used
as a template by HDR system, would lead to
the creation of the desired change within the
genomic region of interest.
• So based on these principles if one is able to
create a DSB at a specific location within the
genome, then the cell’s own repair systems
will help in creating the desired mutations.
Site-specific double stranded breaks
• if genomic DNA is treated with a particular
restriction endonuclease many DSBs will be created.
• To overcome this challenge and create site-specific
DSB, three distinct classes of nucleases have been
discovered and bioengineered to date:
– Zinc finger nucleases (ZFNs),
– Transcription-Activator like Effector Nucleases (TALENs)
– Meganucleases
Meganucleases
• Meganucleases, found commonly in microbial
species, have the unique property of having
very long recognition sequences (>14bp) thus
making them naturally very specific.
• But the challenge is that:
not enough meganucleases are known, or may
ever be known, to cover all possible target
sequences.
To overcome this challenge:
• Mutagenesis and high throughput screening
methods have been used to create
meganuclease variants that recognize unique
sequences.
Meganuclease
advantages and disadvantages.
• less toxicity (compared to ZFNs) because of
more stringent DNA sequence recognition.
• very specific
• the construction of sequence specific enzymes
for all possible sequences is costly and time
consuming.
• As opposed to meganucleases, the concept
behind ZFNs and TALENs is more based on a
non-specific DNA cutting enzyme which would
then be linked to specific DNA sequence
recognizing peptides such as:
– zinc fingers and
– transcription activator-like effectors (TALEs)
• The key to this was to find an endonuclease
whose DNA recognition site and cleaving site
were separate from each other, a situation that
is not common among restriction enzymes.
A restriction enzyme with such properties is
FokI.
FokI enzyme
• The enzyme FokI, naturally found
in Flavobacterium okeanokoites, is a
bacterial type IISrestriction
endonuclease consisting of:
– an N-terminal DNA-binding domain and
– a non-specific DNA cleavage domain at
the C-terminal.
FokI dimerization
• FokI has the advantage of requiring dimerization
to have nuclease activity and this means the
specificity increases dramatically as each
nuclease partner would recognize a unique DNA
sequence.
Applications
•
•
•
•
•
•
Targeted gene mutation
Creating chromosome rearrangement
Study gene function with stem cells
Transgenic animals
Endogenous gene labeling
Targeted transgene addition
TAL effector
• TAL (transcription activator-like) effectors are proteins secreted
by Xanthomonas bacteria via their type III secretion
system when they infect various plant species.
•
These proteins can bind promoter sequences in the host plant
and activate the expression of plant genes that aid bacterial
infection.
Transcription activator-like effector nucleases
(TALENs)
Asparagine
Asn
N
Glycine
Gly
G
Histidine
His
H
Aspartic acid
Asp
D
Isoleucine
Ile
I
Transcription activator-like effector
nucleases (TALENs)
• Transcription activator-like effector nucleases
(TALENs) are artificial restriction enzymes generated
by fusing a TAL effector DNA binding domain to a
DNA cleavage domain.
Transcription activator-like effector
nucleases (TALENs)
Genetargeting,ontheotherhand,reliesonHRbetweentheendogenoustargetlocusandan
exogenously introduced homologous DNA fragment, also referred to as the "donor
DNA".In the absenceofaDSBat the targetlocus, typically fewer than1in105
of targetedcellswillcontain the desired genetic modification, a frequency too low to be
useful for gene therapy[14]. However, proof‐of‐principle experiments involving the
meganuclease I‐SceI, which binds to an 18‐bp recognition site, demonstrated that the
insertion of a DSB in the target locus stimulates
recombination
withanexogenousdonorDNAbyseveralordersofmagnitude[15,16].Aconcernhas
been thatNHEJwillcompetewithHR to seal the
brokenchromosome.Recentexperimentsin two
differenthumancelllinesindicatedthatHR‐mediatedgenetargetingaccountedfor60%to70
%ofall
DSBrepaireventsatanendogenouslocus[17],suggestingthatinthepresenceoflargeamoun
tsofa
donorDNAHRisthepreferredpathwaytorepairDSBs.
Zinc finger nuclease
• Zinc-finger nucleases (ZFNs) are
artificial restriction enzymes generated by
fusing a zinc finger DNA-binding domain to
a DNA-cleavage domain.
• A pair of two ZFNs with three zinc fingers each are shown
introducing a double stranded break. Subsequently, the
double strand break is being repaired through either
homologous recombination or non-homologous end joining.
Zinc finger nuclease
• DNA-binding domain
– The DNA-binding domains of individual ZFNs typically
contain between three and six individual zinc
finger repeats and can each recognize between 9 and
18 basepairs.
• cleavage domain
– The non-specific cleavage domain from the type
IIs restriction endonuclease FokI is typically used as
the cleavage domain in ZFNs.
Zinc Finger Nucleases for Genome Editing
Types of genome editing made possible using
zinc finger nucleases
Gateway Technology
 The Gateway cloning System, invented and
commercialized by Invitrogen since the late 1990s,
 is a molecular biology method that enables
researchers to efficiently transfer DNA-fragments
between plasmids using a proprietary set of
recombination sequences, the "Gateway att" sites,
and two proprietary enzyme mixes, called "LR
Clonase", and "BP Clonase".
Gateway cloning steps
• Making Gateway Entry clone
• Transfer of target gene from Entry clone into
any Gateway Destination vector
Entry clones are often made in two steps:
• 1) “Gateway attB1, and attB2” sequences are added to the 5’,
and 3’ end of a gene fragment, respectively, using gene specific
PCR primers and PCR-amplification;
• 2) the PCR amplification products are then mixed with
special plasmids called Gateway “Donor vectors” (containing
attP1 & attP2 )and the proprietary “BP Clonase” enzyme mix.
recombination sequences are referred to as the Gateway “att L”
type.
Destination vector
• Invitrogen nomenclature for any Gateway
plasmid that contains Gateway “att R”
recombination sequences and elements such
as promoters and epitope tags, but not ORFs
Transfer into Destination vector
• The gene cassette in the Gateway Entry clone
can then be simply and efficiently transferred
into any Gateway Destination vector using the
proprietary enzyme mix, “LR Clonase”.
To summarize the different steps involved in
Gateway cloning:
• Gateway BP reaction:
PCR-product with flanking att B sites (e.g., amplified from cDNA
library) + Donor vector containing att P sites + BP clonase =>
Gateway Entry clone, containing att L sites, flanking gene of
interest
– this step can be replaced by other cloning methods
• Gateway LR reaction:
Entry clone containing att L sites + Destination vector containing
att R sites, and promoters and tags + LR clonase => Expression
clone containing att B sites, flanking gene of interest, ready for
gene expression.
Advantages
• Fast—1-hour room temperature cloning reactions
• Accurate—cloning efficiency >99% delivers the clone you need
• Easy—no need for restriction enzymes or ligation to maintain
orientations and reading frames for expression-ready clones
• Convenient—no resequencing required; use the same clone
from target identification to validation for consistency
• Flexible—shuttle DNA inserts from one expression vector to
the next
• Versatile—select from E. coli, yeast, insect, or mammalian cell
Destination vectors
Building an
Entry Clone
• The three possible methods that lead to the Entry clone are depicted below: A.
BP cloning, B. TOPO® cloning and C. restriction enzyme and ligase cloning.