12 Aptamers for the Diagnosis and Therapy …
363
Diagnostic Strategies Targeting PrP
In 2003, Rhie et al. first selected and characterized RNA aptamers that bind preferentially to PrP
Sc . In order to enhance stability, 2
-fluoro-RNA aptamers was synthesized,
and one of these aptamers, called SAF-93, had more than tenfold higher affinity for
PrP
Sc than PrP
C because of the existence of two specific heparin-binding sites within
the PrP
Sc molecule [94]. However, because these aptamers are too long and complex,
they are not ideal tools for the clinical diagnose of TSEs. Subsequently, in 2004,
Sayer et al. identified the minimal portions of aptamer and determined its secondary
structures by a combination of modeling and solution probing. Consequently, they
identified an internal site for biotinylation of a minimized and synthesized aptamer
SAF-93 (1-34,35bioU,36-60) to detect the PrP
Sc in vitro [95]. These aptamers targeting
PrP
Sc could be potentially used in detecting PrP in blood, cerebrospinal fluid, brain
homogenate, or other biological materials.
Because TSEs are often occurring in various animals, the aptamers against PrP
should also be species-specific. The first aptamer for PrP was selected by Weiss
et al. in 1997 directly against recombinant hamster full-length prion protein. They
found the isolated RNA aptamers form a G-quartet structure that could interact
with the N-terminal amino acids 23–52 of PrP [96]. In order to enhance the
RNA aptamer resistance to nucleases, the same group selected and synthesized
2
-amino-2
-deoxypyrimidine-modified RNA aptamer (DP7). Interestingly, it was
highly specific to human PrP
90−129 , a region involved in PrP pathological conversion, and its binding was sustained even for full-length PrP from different species,
including humans, mice, and hamsters [97]. In order to recognize mouse PrP, aptamer
60-3 was found by Satoshi et al. with a high affinity (K d in the range of 4–7 nM).
They also determined the binding region of mPrP-aptamer and the results showed
that the binding sites of aptamer located between amino acids 23–108 [98]. To identify sheep PrP, RM312, an RNA aptamer, was selected by Mercey groups with a
high affinity (with a K d of 20 nM) in 2006. They identified the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites
[99]. Additionally, an RNA aptamer targeting bovine PrP, called R12, was selected
by Tsukasa Mashima et al. in 2009. The GGAGGAGGAGGA sequence from R12
aptamer forms an intramolecular parallel G-quadruplex structure to recognize bovine
PrP with high affinity [100].
Therapeutic Strategies Targeting PrP
The conversion of PrP
C into its abnormal isoform PrP
Sc is associated with the pathogenesis of TSEs, so blocking the generation of PrP
Sc from PrP
C would be a potential
approach to treat TSEs. SAF-93, an RNA aptamer against PrP
Sc , could inhibit the
conversion of PrP
C into PrP
Sc in cell-free conversion assay [94]. DP7, another RNA
aptamer against PrP
Sc , could reduce PrP
Sc accumulation in prion-infected cells [97].
These two aptamers could be used as tools to treat TSEs.
In summary, isoform- and species-specific aptamers against PrP have been used
as tools to discriminate between PrP
C and PrP
Sc , as well as inhibit the transformation
from PrP
C into PrP
Sc (Table 12.3), which suggests aptamers could be employed as
tools to diagnose and treat TSEs.
363
Diagnostic Strategies Targeting PrP
In 2003, Rhie et al. first selected and characterized RNA aptamers that bind preferentially to PrP
Sc . In order to enhance stability, 2
-fluoro-RNA aptamers was synthesized,
and one of these aptamers, called SAF-93, had more than tenfold higher affinity for
PrP
Sc than PrP
C because of the existence of two specific heparin-binding sites within
the PrP
Sc molecule [94]. However, because these aptamers are too long and complex,
they are not ideal tools for the clinical diagnose of TSEs. Subsequently, in 2004,
Sayer et al. identified the minimal portions of aptamer and determined its secondary
structures by a combination of modeling and solution probing. Consequently, they
identified an internal site for biotinylation of a minimized and synthesized aptamer
SAF-93 (1-34,35bioU,36-60) to detect the PrP
Sc in vitro [95]. These aptamers targeting
PrP
Sc could be potentially used in detecting PrP in blood, cerebrospinal fluid, brain
homogenate, or other biological materials.
Because TSEs are often occurring in various animals, the aptamers against PrP
should also be species-specific. The first aptamer for PrP was selected by Weiss
et al. in 1997 directly against recombinant hamster full-length prion protein. They
found the isolated RNA aptamers form a G-quartet structure that could interact
with the N-terminal amino acids 23–52 of PrP [96]. In order to enhance the
RNA aptamer resistance to nucleases, the same group selected and synthesized
2
-amino-2
-deoxypyrimidine-modified RNA aptamer (DP7). Interestingly, it was
highly specific to human PrP
90−129 , a region involved in PrP pathological conversion, and its binding was sustained even for full-length PrP from different species,
including humans, mice, and hamsters [97]. In order to recognize mouse PrP, aptamer
60-3 was found by Satoshi et al. with a high affinity (K d in the range of 4–7 nM).
They also determined the binding region of mPrP-aptamer and the results showed
that the binding sites of aptamer located between amino acids 23–108 [98]. To identify sheep PrP, RM312, an RNA aptamer, was selected by Mercey groups with a
high affinity (with a K d of 20 nM) in 2006. They identified the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites
[99]. Additionally, an RNA aptamer targeting bovine PrP, called R12, was selected
by Tsukasa Mashima et al. in 2009. The GGAGGAGGAGGA sequence from R12
aptamer forms an intramolecular parallel G-quadruplex structure to recognize bovine
PrP with high affinity [100].
Therapeutic Strategies Targeting PrP
The conversion of PrP
C into its abnormal isoform PrP
Sc is associated with the pathogenesis of TSEs, so blocking the generation of PrP
Sc from PrP
C would be a potential
approach to treat TSEs. SAF-93, an RNA aptamer against PrP
Sc , could inhibit the
conversion of PrP
C into PrP
Sc in cell-free conversion assay [94]. DP7, another RNA
aptamer against PrP
Sc , could reduce PrP
Sc accumulation in prion-infected cells [97].
These two aptamers could be used as tools to treat TSEs.
In summary, isoform- and species-specific aptamers against PrP have been used
as tools to discriminate between PrP
C and PrP
Sc , as well as inhibit the transformation
from PrP
C into PrP
Sc (Table 12.3), which suggests aptamers could be employed as
tools to diagnose and treat TSEs.