Cell-Specific Aptamer-Functionalized RNAi: A New Prospect for Targeted siRNA Delivery - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 110

(A) SELEX & Aptamer (B) RNAi & siRNA

Initial ssDNA library sequence

Double-stranded RNA

Fixed Random (20-60 nt) Fixed

PCR
T7 transcription or strand separation TRBP

Dicer

PACT

siRNA

RNA or ssDNA pool

RISC
AGO2

or Target

Guide strand

m 7 G

mRNA

AAAA

Purified cell membrane proteins

Whole cells

mRNA cleavage

2-18 Selection rounds

Clone and sequence

Cell specific aptamers

(C) Aptamer-functionalized siRNAs (D) Aptamer-functionalized therapeutic

nanocarriers

Linker

siRNA
nanoparticles

Cell-specific aptamer

Figure 5.1 Through the combination of SELEX (aptamers) and RNAi (siRNA)

techniques, cell-specific aptamer-functionalized RNAi can be
achieved. (A) SELEX procedure for aptamer isolation. In order
to generate a cell-specific aptamer, the selection procedure can
vary from traditional purified protein-based SELEX to whole
cell-based SELEX. Generally, in a typical SELEX procedure, the
initial single-stranded DNA pool contains a 20-60 nt random
sequence, which guarantees the presence of active structures
with high binding affinity to the target protein. By repeating
selection rounds, aptamers against any given targets can be
routinely isolated from an initial combinatorial oligonucleotide
library. (B) Mechanisms of RNA interference in mammalian cells.
Cytoplasmic double-stranded RNAs (dsRNAs) are processed by
a complex consisting of Dicer, TAR RNA-binding protein (TRBP)
and protein activator of protein kinase PKR (PACT) into siRNAs,
which are loaded into Argonaute 2 (AGO2) and the RNA-induced
silencing complex (RISC). The siRNA guide strand recognizes
target sites to direct mRNA cleavage, which is carried out by the
catalytic domain of AGO2. siRNAs complementary to promoter
regions direct transcriptional gene silencing in the nucleus
through chromatin changes involving histone methylation (m7G,
7-methylguanylate). (C) Cell-specific aptamer-functionalized
siRNA. Cell-internalizing aptamers are covalently fused or non-
covalently assembled to siRNA agents. A linker (for example: 2-
4-nucleotides, sticky sequence, or biotin-streptavidin connector)
connects the aptamer and siRNA. (D) Cell-specific aptamer-
functionalized therapeutic nanocarrier. siRNAs are encapsulated
into a nanocarrier functionalized by cell-internalizing aptamers.