Imaging Genetic Information - Nanotechnology for Biomedical Imaging and Diagnostics - page 372

X

3′-XN 8 5′

Isolate
total
mRNA

5′

X

5′

RT, dNTPs

Cell
culture

5′ A n -3′

A n -3′

5′

T n

5′* 3′

3′- 5′

1) PAGE
2) Phosphor
-image

5′* 3′

5′* XN 8

PCR

X=A, C, G or T 5′* XN 8

X=A C G T

Align with
mRNA sequence

5′----GGCTACN 8 ----3′

Antisense
accessible site

fIgure 13.4 general method for determining antisense accessible sites on native mRNA.
mRNA is first extracted from whole cells using a complementary oligoT bead. It is then incu-
bated with a random 8 mer oligonucleotide terminating in a known base with a PcR tag (black)
and extended with reverse transcriptase. It is then PcR amplified with the reverse primer
(black) and forward gene-specific primer (gray) that is 5'- 32 P-endlabeled (denoted with *) and
run on a denaturing gel. From the length and pattern of bands, an antisense accessible site on
the mRNA can be identified.

or block otherwise accessible sites. While mRNA directly isolated from cells would
have the native sequence, it too would lack the native bound proteins unless methods
were used to maintain their presence. more recently, methods have been reported
for mapping mRNA structure in vivo through structure-specific chemical probes in
conjunction with next-generation high-throughput sequencing technologies that
might be able to identify antisense accessible sites [72].

13.3.3 stable antisense odn analogs for Imaging

The problem with DNA as an antisense agent is that the DNA•RNA hybrid that forms
is a substrate for RNAseH that will cleave the RNA strand of the DNA•RNA hybrid and
thereby destroy the binding affinity of the DNA for the target mRNA. In addition, DNA
is susceptible to degradation in the serum and in cells. Likewise, RNA is even more
susceptible to enzymatic degradation and can likewise induce mRNA strand cleavage
through the RIcS complex (RNAi). To circumvent these problems, nucleic acid analogs