Transcript Advantages of aptamers

Methods developed for SELEX1
Presented by: chang chu
Guided by: Prof. Liu
Prof. Jiang
1. Subash Chandra Bose Gopinath, Anal Bioanal Chem (2007) 387:171–182
1. Introduction
Basic processes
Features
2. Methods
3. Aptamer base
1. SELEX (systematic evolution of ligands by exponential
enrichment)
is a process that involves the progressive purification from a
combinatorial library of nucleic acid ligands with a high affinity for a
particular target by repeated rounds of partitioning and amplification.
Three Processes
Selection of ligand sequences that bind to a target
partitioning of aptamers from
non-aptamers via affinity methods
amplification of bound aptamers
Antibody VS aptamer in therapeutic use1
Advantages of antibodies
• Pharmacokinetic
other systemic properties of antibodies are often
Advantages of and
aptamers
sufficient
to support
product chemically
development
• Aptamers
are produced
in a readily scalable process
• Comparatively
long circulating
half-lives
• Chemical production
process
is not prone to viral or bacterial
• Not
susceptible to nuclease degradation
contamination
• Antibody
technologies are widely distributed
• Non-immunogenic
• Smaller size allows more efficient entry into biological compartments
Limitations of antibodies
• Antibodies
are produced
biologically in a process
• Limitations
of aptamers
• Viral
or bacterial contamination
• Pharmacokinetic
and other systemic properties are variable and often
• Large
limits
hard size
to predict
• Shorter half-life
• Unmodified aptamers are highly susceptible to serum degradation
1. Anthony D. Keefe, Supriya Pai and Andrew Ellington(2010). Aptamers as therapeutics. Nature
2. Methods
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Nitrocellulose membrane filtration
Using affinity surfaces
Using affinity tags
Using column matrices or ligands
Cross-linking
Antibody-based
Using gel electrophoresis
Surface plasmon resonance
Flow cytometry
Capillary electrophoresis
Automated selection
• Initial rounds of selection: long incubation times & less stringent conditions
Later cycles: stringent conditions, such as changing the buffer conditions, reaction volume
and time of incubation.
• Monovalent & divalent cations
• Pre-negative selection
Using affinity surfaces
• Affinity surfaces: allow proteins and small
molecules to bind with them & have
affinities with RNA or DNA.
• Magnetic beads, affinity titer plates
• RNA Aptamer against Panama influenza
virus subtype A2
2. Kumar PKR, Gopinath SCB, Misono T, Kawasaki K (2004) Japanese patent JP2004-293679
STEP2
STEP4
Step
Step
31STEP5
Negative
selection
Amplification
Counterselection
selection
•Coating the whole virus onto
•The •Incubating
unbound molecules
the was
pool were again
beads
•The
unbound RNA
collected
incubated
with beads
RNA and
with
collected
withthe
theBSAhelp of
coated
with beads
the target
A/Panama virus
coated
in binding
•Reverse
transcription
magnet
and
applied
onto
•BSA Blocking
buffer
for with
10 min.
beads
coated
the same
•After
thisA/Aichi
incubation,
thethe
beads were
subtype
virus as
•PCR
•Washing
thetimes
coated
beads.
washed
three
300 μl of
counter-selection
towith
remove
binding
buffer.
molecules
specific to A/Aichi.
•In vitro transcription
•Denaturing
the
•Incubating
for
10pool
min.RNA(90 °C
for 2 min
andwere
allowed
to coolwith
at a
•Bound
RNAs
recovered
room
hot
7M temperature
urea solution.for 10 min)
•Bound molecules were precipitated
by ethanol.
Flow cytometry
• Detecting the fluorescence
Leu3a-FITC
RNA aptamerFITC
Using gel electrophoresis
0.7% native agarose gel
Using gel electrophoresis
Electrophoresis through
4 %, polyacrylamide, TBE,
0.05 % SDS, and then
recovered from the gel by
the crush-and-soak
method.
Smith D, Kirschenheuter GP, Charlton J, Guidot DM, Repine JE(1995) Chem Biol 2:741–750
Using capillary electrophoresis
• The nucleic acid sequences that bind the target undergo a mobility
shift, migrating at a different rate, allowing them to be separated
from the inactive sequences. 4
Thus, there is no need to wash the active sequences off a column
as in conventional SELEX, eliminating any kinetic bias.
• Higher speed, better resolution capacity,
minimal sample dilution, fewer cycles.
4.Mendonsa SD, Bowser MT (2004) Anal Chem 76:5387–5392
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a poly (vinyl alcohol)-coated capillary 40.2 cm long and with an inner
diameter of 50 μm
The sample was applied to the capillary at 5 psi for 5 s and monitored under
UV detection at 254 nm
After the nonspecific species had migrated out, the CE fractions containing
specific DNA sequences were collected
PCR
Anti-IgE aptamers with dissociation constants as low as 27 nM were
obtained in only two rounds of selection.
5. Jose Cruz-Toledo1,*,y, Maureen McKeague2,*,y, Xueru Zhang2, Amanda Giamberardino2, Erin McConnell2, Tariq Francis2,
Maria C. DeRosa2,3,* and Michel Dumontier1,3,4,* Database, Vol. 2012, Article ID bas006, doi:10.1093/database/bas006