Imaging Genetic Information - Nanotechnology for Biomedical Imaging and Diagnostics - page 374

binding affinity for RNA, such as the LNAs and PNAs. Analogs based on the phospho-
diester backbone can be conveniently synthesized by standard automated DNA
synthesis, whereas PNAs can be synthesized by standard automated peptide synthesis.
Various types of conjugation strategies have been developed for attaching reporter
groups to the nucleotide analogs. generally, these involve postsynthetic modification of
the antisense agent by amide bond formation between an NHS ester of the reporter and
amino group linked to the antisense agent or a maleimide of the reporter and a thiol on
the antisense agent [77]. In the case of PNAs that are synthesized by solid-phase pep-
tide methods, coupling can be done directly on column by amide bond-forming chem-
istry. more recently, click chemistry has been used to form bioconjugates [77, 78].

13.4 reporter systems for rna ImagIng

There are two general classes of reporter systems that can be used. Always ON and
turn-on reporter systems. Always ON reporters are those reporters that always give
a signal, whether or not bound to the target mRNA, such as a radioactive PeT imaging
agent, a fluorescent probe, or a relaxation agent. The problem with the always ON
probes is that an RNA-specific signal will only arise if unbound probe can efficiently
exit cells and the tissue environment, which is problematic for antisense agents due
to their extremely low membrane permeability (Fig. 13.6).
This problem is greatly diminished with fluorescent turn -on probes, which only
become significantly activated upon binding to the target mRNA through a variety of
mechanisms (Fig. 13.6) [79-88]. A large number of turn -on probes are based on FReT,
which is a distance-dependent singlet excitation energy transfer mechanism. The FReT
mechanism has been utilized for the construction of molecular beacons, quenched
strand displacement probes, and binary FReT imaging agents. In the molecular beacon
design, the antisense sequence is made to form a hairpin with a short stem, one end of
which is attached to a fluorescent reporter group and the other end is attached to a
quencher (Fig. 13.7a) [89-91]. In the absence of the mRNA target, the fluorescence of
the reporter is quenched. In the presence of the target, the hairpin opens and the quencher
is then held at quite some distance from the reporter and fluorescence is restored.
The quenched strand displacement probe operates in a similar fashion to molec-
ular beacons, only that the probe consists of a bimolecular hybrid between the anti-
sense agent bearing the reporter and a shorter strand bearing the quencher (Fig. 13.7b)
[92, 93]. In the presence of the target, the shorter quencher strand is displaced by the
longer RNA target and fluorescence is restored. The potential problem with these
two types of FReT-based agents is that the reporter and quenchers are susceptible to
separation by other mechanisms than by target RNA binding, such as protein binding
and degradation of the linkers and/or nucleic acid strands. Binary FReT probes do
not suffer from this problem, as the signal only arises when the antisense strand
bearing the donor fluorophore is bound to the mRNA next to the antisense strand
bearing the acceptor fluorophore (Fig. 13.7c) [81]. The downside of the binary FReT
system is that it requires a larger accessible site on the mRNA and requires separate
delivery of the donor and acceptor probes to avoid FReT arising from close proximity