Delivery of Single siRNA Molecules - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 99

them is the diverse class of double-stranded RNA binding proteins
(DRBP) that contain certain domains able to bind double-stranded
RNA. These double-stranded RNA binding domains (DRBDs) are
highly abundant and can be found in many different species. The
DRBPs have different functions but are similar in that they contain
varying copies of DRBDs.
A consensus sequence of about 65-68 amino acid residues
specific for binding dsRNA was identified in 1992 (St Johnston
et al., 1992). It was later shown that a minimum number of 16

base pairs of siRNAs are required for binding, however, for longer
RNAs, only 11 base pairs are required (Bevilacqua and Cech,
1996). Moreover, DRBDs are known to bind the compressed A-
form of the siRNA sequence independently and almost without
changing the structure. The DRBD binds the 2

′

-hydroxyl groups
on the sugar phosphate backbone, and every 11 mer is involved in
the interaction. Consequently, DRBDs cannot bind dsDNA or even
RNA/DNA hybrids, since dsDNA is predominantly found in the
more open B-form (Bevilacqua and Cech, 1996). Three regions are
responsible for binding the dsRNA, where two out of three bind
the minor groove, whereas the third binds the major groove. One of
the most commonly used PTDs belongs to the HIV-1 and is derived
from the TAT protein (Vivès 1997). By designing a fusion

protein of three TAT peptides and a DRBD (PTD-DRBD), the previous
obstacles in siRNA delivery can be overcome (Eguchi

et al.,

et al., 2009,

Palm-Apergi et al., 2011).

4.2.4 Delivery of Single siRNA Molecules by PTD-DRBD

Initially, while developing this new delivery strategy, several PTDs
were assessed for their ability to deliver siRNA. The goal was
to conjugate a single PTD to a single siRNA molecule. However,
the positive charges of the PTD and the negative charges on the
siRNA lead to precipitation, aggregation, and charge neutralization
during each conjugation attempt, resulting in an inactivated
PTD. Those observations lead to the conclusion that the anionic
phosphodiester backbone of the siRNA needs to be masked, before
any PTD conjugation can occur without any precipitation. Since
several TAT fusion proteins had been developed to study cellular
mechanisms (Nagahara 1998), it seemed natural to design a

fusion protein with a PTD delivery domain and a DRBD, able to bind
and neutralize the anionic charges on the siRNA. Since the DRBD is

et al.,