• Reproducibility. The methodology must be reproducible with no significant result
variations.
In this context, the design of the aptamer-based probes begins with the correct
aptamer selection.
First, systematic evolution of ligands by exponential (SELEX) enrichment is the
most established methodology of aptamer obtention and initially restricts the affinity
and specificity parameters [1, 2]. At this moment, it is important to decide if DNA or
RNA will be used [12]. Both have no trivial differences in stability and chemical
composition. While the use of unnatural nucleic acids [13, 14] has emerged as an
interesting tool, DNA and RNA native libraries are used in most SELEX procedures.
Since RNA plays an important role in gene regulation, it represents the majority of
the libraries used in genomic SELEX [15]. Additionally, some authors suggest
RNAs have more conformational motifs, which result in high affinity binders
[16]. However, DNA has clear advantages, since it is reportedly more chemically
and biologically stable [17]. In fact, most of the commercial-developed aptamers are
from DNA [18].
Typically, the SELEX library includes a randomized region between two constant
primer binding sequences on the ends. Consequently, the binding depends on the
constant and random regions, as well as their length and GC percentage. The library
design comprises variations [19], and the introduction of artificial nucleotides,
polymers, and functional groups into the library results in a challenge to find a
good starting point [12].
The molecular diversity can be improved using an extend alphabet of SELEX
starting libraries. This expanded alphabet includes unnatural hydrophobic bases and
functional groups that mimic amino acid side chains [20]. The probability to select
more affine aptamers is higher due to the increased number of possible interactions
between aptamers and target.
Additionally, stabilization of aptamer structures with a 2’OH group of RNA [21]
or locked nucleic acids (LNA) may improve their target binding affinities [22]. Nonetheless, the development of modified SELEX libraries requires the adaptation of
compatible steps of SELEX, PCR amplification, and cloning [23].
Second, target presentation could be determinant. Correct antigenic fragments
can be exposed to the library in a soluble resin, in target fragments, and by tissues or
whole cells [24]. The assays’ settings must be approximate to the real conditions. At
this point, the correct folding of the oligonucleotides is fundamental in the presentation of the target. Ionic force, salts, pH, and temperature are some parameters to be
considered in order to properly reproduce the binding conditions. In order to control
the media conditions, the buffer is usually enriched with magnesium. Addition of
magnesium chloride to the aptamer enhances G-quadruplex folding [25].
Successful SELEX also requires efficient separation of bound and unbound
sequences, allowing increase of target bound/unbound ratio and progress in the
enrichment. There are diverse variants of the methodology, which showed to
improve the success rate [26, 27].
Aptamers in Diagnostic and Molecular Imaging Applications
145
variations.
In this context, the design of the aptamer-based probes begins with the correct
aptamer selection.
First, systematic evolution of ligands by exponential (SELEX) enrichment is the
most established methodology of aptamer obtention and initially restricts the affinity
and specificity parameters [1, 2]. At this moment, it is important to decide if DNA or
RNA will be used [12]. Both have no trivial differences in stability and chemical
composition. While the use of unnatural nucleic acids [13, 14] has emerged as an
interesting tool, DNA and RNA native libraries are used in most SELEX procedures.
Since RNA plays an important role in gene regulation, it represents the majority of
the libraries used in genomic SELEX [15]. Additionally, some authors suggest
RNAs have more conformational motifs, which result in high affinity binders
[16]. However, DNA has clear advantages, since it is reportedly more chemically
and biologically stable [17]. In fact, most of the commercial-developed aptamers are
from DNA [18].
Typically, the SELEX library includes a randomized region between two constant
primer binding sequences on the ends. Consequently, the binding depends on the
constant and random regions, as well as their length and GC percentage. The library
design comprises variations [19], and the introduction of artificial nucleotides,
polymers, and functional groups into the library results in a challenge to find a
good starting point [12].
The molecular diversity can be improved using an extend alphabet of SELEX
starting libraries. This expanded alphabet includes unnatural hydrophobic bases and
functional groups that mimic amino acid side chains [20]. The probability to select
more affine aptamers is higher due to the increased number of possible interactions
between aptamers and target.
Additionally, stabilization of aptamer structures with a 2’OH group of RNA [21]
or locked nucleic acids (LNA) may improve their target binding affinities [22]. Nonetheless, the development of modified SELEX libraries requires the adaptation of
compatible steps of SELEX, PCR amplification, and cloning [23].
Second, target presentation could be determinant. Correct antigenic fragments
can be exposed to the library in a soluble resin, in target fragments, and by tissues or
whole cells [24]. The assays’ settings must be approximate to the real conditions. At
this point, the correct folding of the oligonucleotides is fundamental in the presentation of the target. Ionic force, salts, pH, and temperature are some parameters to be
considered in order to properly reproduce the binding conditions. In order to control
the media conditions, the buffer is usually enriched with magnesium. Addition of
magnesium chloride to the aptamer enhances G-quadruplex folding [25].
Successful SELEX also requires efficient separation of bound and unbound
sequences, allowing increase of target bound/unbound ratio and progress in the
enrichment. There are diverse variants of the methodology, which showed to
improve the success rate [26, 27].
Aptamers in Diagnostic and Molecular Imaging Applications
145