1 Introduction of Aptamer, SELEX, and Different SELEX Variants
5
NECEEM partitioning. Aptamers were again collected into h-Ras in the vial to establish the second aptamer-enriched equilibrium mixture. The procedure was repeated
one more time to collect the third aptamer-enriched fraction. Three rounds of NonSELEX improved the affinity of a DNA library from 104 to 0.3 μM, and the selected
aptamers had Kd values of 0.2 μM. The whole procedure took only 1 h, significantly
shortening the selection time.
Besides, Qu’s team has also attempted to improve the CE-SELEX efficiency in
recent years. They developed LpH-CE-SELEX mode [14] for the selection of Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c). In this mode,
the injection time and sample volume can be increased to collect a relatively large
amount of protein-ssDNA complex at the cathode end of the capillary without the
contamination of unbound ssDNA library. To further simply the operation step, ssCESELEX mode was developed [15]. In this mode, the mixing, reaction, separation,
detection and complex collection can be accomplished in a single step online process
and take only about 10 min.
1.3.2 Furnishing More Targets Applicability
1.3.2.1 Magnetic Beads-Based SELEX
Magnetic beads-based SELEX is one kind of target immobilization-based SELEX,
which has been widely used to select aptamers against small molecules, polypeptides, proteins, and so on. In magnetic beads-based SELEX, magnetic nanoparticles (also be called magnetic beads) are used as immobilization matrix instead
of microwell plates, agarose beads, SPR chips, etc. Target molecules are fixed
to the surface of the magnetic nanoparticles through chemical modification and
covalent attachment. Following the incubation between the oligonucleotide library
and the magnetic nanoparticle-target conjugates, the magnetic nanoparticle-targetoligonucleotide complex can be separated from the unbound free oligonucleotides in
the reaction solution by magnetic separation. The weakly bound oligonucleotides can
be removed by washing the magnetic nanoparticle-target-oligonucleotide complex
with buffer for some times. Then, the affinity sequences can be dissociated from the
complex by heating or alkali treatment, followed by PCR amplification, single strand
preparation, purification, etc.
Bruno [16] first applied the magnetic bead (MB)-based affinity separation to
in vitro selection of DNA aptamers against carcinogenic pollutants, including
chloroaromatics, 4-chloroaniline, 2, 4, 6-trichloroaniline and pentachlorophenol
(Fig. 1.3). Magnetic beads with a linker arm were utilized to immobilize these target
molecules. Incubating them with an oligonucleotide library resulted in target-aptamer
complexes that were separated from unbound oligonucleotides by a magnetic separator. Use of MBs was advantageous in obviating elution and precipitation of DNA
because conjugated MBs with surface-captured template DNA could be directly
added to PCR mixture. Then, a simplified PCR scheme requiring only one type of
5
NECEEM partitioning. Aptamers were again collected into h-Ras in the vial to establish the second aptamer-enriched equilibrium mixture. The procedure was repeated
one more time to collect the third aptamer-enriched fraction. Three rounds of NonSELEX improved the affinity of a DNA library from 104 to 0.3 μM, and the selected
aptamers had Kd values of 0.2 μM. The whole procedure took only 1 h, significantly
shortening the selection time.
Besides, Qu’s team has also attempted to improve the CE-SELEX efficiency in
recent years. They developed LpH-CE-SELEX mode [14] for the selection of Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c). In this mode,
the injection time and sample volume can be increased to collect a relatively large
amount of protein-ssDNA complex at the cathode end of the capillary without the
contamination of unbound ssDNA library. To further simply the operation step, ssCESELEX mode was developed [15]. In this mode, the mixing, reaction, separation,
detection and complex collection can be accomplished in a single step online process
and take only about 10 min.
1.3.2 Furnishing More Targets Applicability
1.3.2.1 Magnetic Beads-Based SELEX
Magnetic beads-based SELEX is one kind of target immobilization-based SELEX,
which has been widely used to select aptamers against small molecules, polypeptides, proteins, and so on. In magnetic beads-based SELEX, magnetic nanoparticles (also be called magnetic beads) are used as immobilization matrix instead
of microwell plates, agarose beads, SPR chips, etc. Target molecules are fixed
to the surface of the magnetic nanoparticles through chemical modification and
covalent attachment. Following the incubation between the oligonucleotide library
and the magnetic nanoparticle-target conjugates, the magnetic nanoparticle-targetoligonucleotide complex can be separated from the unbound free oligonucleotides in
the reaction solution by magnetic separation. The weakly bound oligonucleotides can
be removed by washing the magnetic nanoparticle-target-oligonucleotide complex
with buffer for some times. Then, the affinity sequences can be dissociated from the
complex by heating or alkali treatment, followed by PCR amplification, single strand
preparation, purification, etc.
Bruno [16] first applied the magnetic bead (MB)-based affinity separation to
in vitro selection of DNA aptamers against carcinogenic pollutants, including
chloroaromatics, 4-chloroaniline, 2, 4, 6-trichloroaniline and pentachlorophenol
(Fig. 1.3). Magnetic beads with a linker arm were utilized to immobilize these target
molecules. Incubating them with an oligonucleotide library resulted in target-aptamer
complexes that were separated from unbound oligonucleotides by a magnetic separator. Use of MBs was advantageous in obviating elution and precipitation of DNA
because conjugated MBs with surface-captured template DNA could be directly
added to PCR mixture. Then, a simplified PCR scheme requiring only one type of