Imaging Genetic Information - Nanotechnology for Biomedical Imaging and Diagnostics - page 371

to DNA sequence from normal tissue, as the cost for doing this has been rapidly
declining with the advent of Nexgen sequencing technologies [62-64]. In cancers
that arise from retroviruses, such as HPV, a unique mRNA may be present that can
be detected. Another target RNA would be one that is overexpressed and could be
again detected for a specific cancer or a biopsy by DNA chip analysis in comparison
to a sample of normal tissue. In some conditions, such as inflammation, there may be
highly upregulated mRNAs. The RNA could also be an miRNA [60, 65], or other
ncRNA, or an RNA, such as rRNA from a pathogen.

13.3.2 Identifying antisense accessible sites on rna targets

Once candidate RNAs are identified, high-affinity antisense binding sites must be iden-
tified. Though RNA, unlike DNA, is single stranded, all parts of the sequence are not
equally accessible to an antisense agent because RNA molecules will spontaneously
fold into complicated structures, which may also bind proteins [37]. Thus, it would
appear to be important to be able to identify sequences along the RNA that would be
able to bind to an antisense agent with high affinity. In humans, to be unique, a sequence
generally has to be about 18 nucleotides long, but 1 may not necessarily require that
all 18 nucleotides of the sequence be accessible. It may be sufficient for an antisense
agent to get a toehold on a single-strand section of the RNA, which can then displace
an adjacent base-paired section of RNA by a strand displacement process that becomes
favored by the base-pairing properties of the antisense agent used [66]. even if the
antisense agent is able to bind to the RNA before folding, thermodynamically favor-
able RNA folding could result in the displacement of the probe. Therefore, the challenge
for selection of a suitable antisense agent is to find antisense accessible sites on the
RNA inside a cell. generally, candidate antisense agents have been selected through
the use of RNA folding calculations and screening the candidates for activity [67].
There are a number of experimental methods for mapping antisense accessible
sites on in vitro transcribed RNA involving hybridization to random oligonucleotide
libraries [68, 69] or for initiating reverse transcriptase PcR [70]. This latter approach
has been extended to determine the accessible sites on mRNAs produced by in vitro
transcription [69] or isolated directly from cells (Fig. 13.4) [71]. This mapping
method uses a library of random 10-mer sequences fused to a PcR tag to prime
reverse transcriptase from sites accessible to the primer. The resulting cDNA is then
PcR amplified using an mRNA-specific primer and the PcR tag to produce bands
that can be aligned with the known mRNA sequence to map the antisense accessible
sites. Antisense ODNs are then synthesized to these sites, and their affinity for the
mRNA is determined by a binding assay. The affinity of chemical analogs of the high-
est-affinity antisense ODNs can then be determined directly or through a competition
assay. These techniques have been successfully applied for the identification of high-
affinity sites for the unr mRNA that is overexpressed in mcF-7 cells [69] and iNOS
mRNA that is overexpressed in the mouse macrophage cell line in an inflammatory
response to gIFN and LPS [71]. The problem with using in vitro transcribed RNA is
that it lacks the 5′-capping sequence and may not fold in the same way as in vivo and
would certainly lack any bound proteins that may alter the conformation of the mRNA