Biomedical Engineering Reference
In-Depth Information
have a complexity of
y
N
. However, for practical purposes, and as mentioned
above, the maximum number of unique sequences that can be screened in a
library is limited to 10
13
-10
18
. It is interesting to note, however, that further diver-
sity may be introduced during the SELEX process itself (see Section 20.3.2), in
particular due to the infidelity of the DNA polymerase used in the polymerase
chain reaction (PCR) step, which may not be 100% accurate.
The nucleotide chemistry
: This is important because, firstly, it defines the range of
possible three-dimensional structures into which the aptamer can fold, and sec-
ondly, the nucleotide chemistry plays an extremely important role in relation to
the aptamer's stability to degradation. Indeed, the susceptibility of single-
stranded oligonucleotides to enzymatic or chemical cleavage is a severe practi-
cal handicap to the use of aptamers that needs to be overcome for the molecules
to be stable in biological fluids. Three different approaches have been adopted to
overcome this problem:
●
Modification of nucleotide bases, which has been the most commonly used
method. The modification of pyrimidines at the 5
●
position with I, Br, Cl, NH
3
,
position with NH
2
, F, and OCH, for example, has been
described by Pieken et al. (58). These modifications can increase the RNA
oligonucleotide half-life up to 15 h (54). It should be noted, however, that any
modified nucleotides should still be compatible with the enzymes used in the
SELEX protocol (e.g., DNA polymerase). Interestingly, some problems associ-
ated with modified nucleotides have been overcome by a modification of the
SELEX protocol known as “Transcription Free SELEX”, in which random RNA
fragments bind random DNA templates, after which the fragments are ligated
either enzymatically or using a standard chemical condensation reaction. The
RNA aptamer can then be recovered by melting the duplex.
●
Modification of the phosphodiester backbone, for example through the use of
and N
3
and at the 2
-thio substituted deoxynucleotide triphosphates, although this technique has
been more successful with DNA as opposed to RNA aptamers.
The use of enantiomeric aptamers, known as spiegelmers (from the German
word meaning “mirror”). This technique consists in creating a mirror image of
the target and selecting an aptamer for this mirror image. A stereo isomer of the
selected aptamer is then created (i.e., the spiegelmer), which will be specific for
the target but will not be susceptible to normal enzymatic degradation due to the
substitution of the natural
D
-ribose with
L
-ribose (54).
●
The constant region primer design
: The random aptamer sequence has to be flanked
by 5
●
constant sequences, usually 20-25 base pairs in length, which provide
hybridization sites during a number of steps of the SELEX process (51). The 3
and 3
flanking sequence generally acts as an attachment site for the reverse-transcriptase
primer; and the 5
flanking sequence acts as the attachment site for the PCR primers
during the amplification step of the SELEX protocol. The design of the constant
region for the SELEX protocol is even more important than for normal PCR given
that a complete SELEX protocol may include up to 200 cycles of PCR. Any artifacts
would thus be drastically amplified in the final aptamer population.
20.3.2
SELEX
After a suitable aptamer library has been prepared, it can undergo the SELEX protocol, as
shown in Figure 20.6. This technique essentially consists of the repeated binding, selection,
and amplification of aptamers from the initial library until one (or more) aptamer(s) dis-
playing the desired characteristic(s) has (have) been isolated (53).
