1 Introduction of Aptamer, SELEX, and Different SELEX Variants
23
1.3.6.3 In Silico SELEX
Different from conventional SELEX modes based on experiments, in silico selection
generated computational aptamer via molecular simulation and tuning structural
complexities by algorithm filtering. The advantages of in silico selection include
avoiding the blindness of the SELEX experiment and the possible failure caused
by experiment operation such as nucleic acid pollution as well as increasing the
structural diversity of the starting oligonucleotide pool to enhance the possibility of
finding novel aptamers with improved affinity thereof.
Probably, the first in silico SELEX was reported by Chushak and Stone [75]
when they proposed a computational method to develop RNA aptamers for codeine,
gentamicin, theophylline, etc. The process of in silico SELEX was described as
follows. First, RNA sequences were selected from the randomly generated RNA
pools to obtain the ones with stable low-energy structures. The Rosetta package
was used for the prediction of tertiary structures of these selected RNA sequences,
the structures were minimized using the AMBER force field and generalized Born
implicit solvent, and then ensemble docking with a modified DOVIS package having
AutoDock4 software as the docking engine and run in parallel on Linux clusters for a
library of RNA molecules with lowest energy structures. RNA aptamers of codeine,
gentamicin and theophylline were selected via in silico SELEX and verified by
experiments in vitro.
When the targets are not compounds or irons and so on that are with known
structures, in silico SELEX can show the specific advantages of generating special
structure DNA/RNA sequences. Wondergem et al. [76] performed in silico SELEX
against nucleosome and obtained overwound DNA rings that showed high affinity
to nucleosome by using Dubbed Mutation Monte Carlo (MMC) method (Fig. 1.16).
Similarly, with the purpose of obtaining shorter sequences of aptamer, Ahirwar [77]
developed a selection method of human estrogen receptor ERα-aptamer selection
based on in silico SELEX. RNA analogs of human estrogen response elements
(EREs) were used to obtain aptamer-like sequences. AutoDockVina, HADDOCK,
and PatchDock docking were applied to examine the likelihood of near-native RNA
analogs of selected single-stranded EREs emerge as ERα aptamer.
1.3.6.4 Genomic SELEX
Genomic SELEX is based on the generation of RNA species that are derived from a
library of an organism’s entire genomic DNA in vitro. The targets that possess bioactive molecules such as proteins, polysaccharides and so on are suitable for genomic
SELEX. The generated RNA pool will undergo successive rounds of association
with a given RNA-binding protein, partitioning, and reamplification. This method
does not require isolation of non-coding RNAs (ncRNAs) from an organism or cell.
Genomic SELEX is an efficient tool to reveal the potential binding sites of targets.
Valentin-Hansen et al. applied genomic SELEX to identify new Hfq-binding RNAs
from Escherichia coli. A representative library of the E. coli genome is constructed
23
1.3.6.3 In Silico SELEX
Different from conventional SELEX modes based on experiments, in silico selection
generated computational aptamer via molecular simulation and tuning structural
complexities by algorithm filtering. The advantages of in silico selection include
avoiding the blindness of the SELEX experiment and the possible failure caused
by experiment operation such as nucleic acid pollution as well as increasing the
structural diversity of the starting oligonucleotide pool to enhance the possibility of
finding novel aptamers with improved affinity thereof.
Probably, the first in silico SELEX was reported by Chushak and Stone [75]
when they proposed a computational method to develop RNA aptamers for codeine,
gentamicin, theophylline, etc. The process of in silico SELEX was described as
follows. First, RNA sequences were selected from the randomly generated RNA
pools to obtain the ones with stable low-energy structures. The Rosetta package
was used for the prediction of tertiary structures of these selected RNA sequences,
the structures were minimized using the AMBER force field and generalized Born
implicit solvent, and then ensemble docking with a modified DOVIS package having
AutoDock4 software as the docking engine and run in parallel on Linux clusters for a
library of RNA molecules with lowest energy structures. RNA aptamers of codeine,
gentamicin and theophylline were selected via in silico SELEX and verified by
experiments in vitro.
When the targets are not compounds or irons and so on that are with known
structures, in silico SELEX can show the specific advantages of generating special
structure DNA/RNA sequences. Wondergem et al. [76] performed in silico SELEX
against nucleosome and obtained overwound DNA rings that showed high affinity
to nucleosome by using Dubbed Mutation Monte Carlo (MMC) method (Fig. 1.16).
Similarly, with the purpose of obtaining shorter sequences of aptamer, Ahirwar [77]
developed a selection method of human estrogen receptor ERα-aptamer selection
based on in silico SELEX. RNA analogs of human estrogen response elements
(EREs) were used to obtain aptamer-like sequences. AutoDockVina, HADDOCK,
and PatchDock docking were applied to examine the likelihood of near-native RNA
analogs of selected single-stranded EREs emerge as ERα aptamer.
1.3.6.4 Genomic SELEX
Genomic SELEX is based on the generation of RNA species that are derived from a
library of an organism’s entire genomic DNA in vitro. The targets that possess bioactive molecules such as proteins, polysaccharides and so on are suitable for genomic
SELEX. The generated RNA pool will undergo successive rounds of association
with a given RNA-binding protein, partitioning, and reamplification. This method
does not require isolation of non-coding RNAs (ncRNAs) from an organism or cell.
Genomic SELEX is an efficient tool to reveal the potential binding sites of targets.
Valentin-Hansen et al. applied genomic SELEX to identify new Hfq-binding RNAs
from Escherichia coli. A representative library of the E. coli genome is constructed