SAGE-Hindawi Access to Research
Journal of Nucleic Acids
Volume 2011, Article ID 586935, 7 pages
Branched RNA: A New ArchitectureforRNA Interference
Anna Avi˜ n´ o,1SandraM. Ocampo,1Jos´ e CarlosPerales,2and RamonEritja1
1Institute for Research in Biomedicine (IRB Barcelona), Institute of Advanced Chemistry of Catalonia (IQAC-CSIC),
Networking Centre on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Baldiri Reixac 10, 08028 Barcelona, Spain
2Department of Physiological Sciences, School of Medicine, University of Barcelona (Campus Bellvitge), Feixa Llarga,
L’Hospitalet de Llobregat, 08907 Barcelona, Spain
Correspondence should be addressed to Ramon Eritja, email@example.com
Received 16 October 2010; Accepted 14 January 2011
Academic Editor: Arthur van Aerschot
Copyright © 2011 Anna Avi˜ n´ o et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The
branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing
experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an
innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described
to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable
In recent years, siRNAs have generated tremendous interest
in therapeutics . Nevertheless, the transition of siRNAs
from the laboratory to the clinical practice has encoun-
tered several obstacles. Briefly, siRNA duplexes are rapidly
degraded in serum by exonucleases and endonucleases .
The polyanionic phosphodiester backbone of siRNA suffers
from difficult cell uptake , and oligonucleotides may
have off-target effects, either by stimulating the immune
system  or by entering other endogenous gene regulation
pathways . Several chemical modifications have been
proposed in the literature to address these drawbacks [2–4].
Most of these modifications are based on modified nucle-
osides and changes on backbone linkages [6, 7]. Thus,
changes in sugar moiety influences sugar conformation,
and, therefore, overall siRNA structure. Modifications of the
2?-OH by F or OMe as well as LNA [8, 9] are well toler-
ated and improve binding affinity and nuclease resistance.
Base modifications that stabilize base pairs (5-bromouracil,
5-methylcytosine, 5-propynyluracil, and others) have also
been proposed [7, 10]. Terminal conjugates, especially at the
termini of the sense strand, have been modified with a large
number of lipids to achieve improved cellular uptake .
In addition to these modifications, siRNA architecture
is also crucial in the design of effective and specific siRNA.
The architecture itself can be altered by chemical synthesis.
In addition to the canonical siRNA architecture of 21-nt
antiparallel, double-strand RNA with 2-nt 3?-overhangs
, several forms of siRNA have been described. Blunt-
ended siRNA , 25/27mer Dicer-substrate or asymmetric
siRNA  are among the siRNA structures formed by two
strands. Moreover, functional siRNA can also be formed by
one single RNA strand. This is the case in small hairpin RNA
(shRNA), where the two strands are linked by a single loop
, or RNA dumbbells , made by closing the open end
of the hairpin. This last structure retains RNAi activity while
providing complete protection from nucleases . Finally,
siRNA can also comprise three strands, namely, two 9–13nt
sense strands and the intact antisense strand. This structure
is known as small internally segmented interfering RNA 
(sisiRNA). Some of these modifications have reduced off-
target effects and increased potency (Figure 1(a)). Another
architecture not yet explored in siRNA is the branched RNA
structure obtained from a central building unit and several
branching points that enable the strand growth.
Several strategies can be used to prepare branched RNA
structures. Although the synthesis of these compounds is
2Journal of Nucleic Acids
dT or RNA, DNA nucleosides
Figure 1: Duplex RNA architectures for RNA interference. (a)
Described previously in the introduction: (i) canonical siRNA
antiparallel duplex; (ii) small internally segmented interfering RNA
(sisiRNA); (iii) small hairpin RNA (shRNA); (iv) dumbbell siRNA.
(vi) two-stranded RNA.
complex and tedious, commercially available synthons have
improved the complexity and yields of these structures.
The assembly of branched nucleic acids on a solid
support can be achieved by convergent or divergent strate-
gies. In the former, synthesis of branched oligonucleotides
containing two or more identical strands can be achieved by
branching derivatives1,3-diaminopropanol, pentaerythritol,
the commercially available symmetric doubler [18–20], or
by a ribonucleoside bisphosphoramidite  as synthons.
In contrast, in the divergent approach two or more dis-
tinct strands are prepared with synthons with orthogonal
protecting groups or from commercial sources [20, 22].
In addition, 2?-O-silylribonucleosides have been used to
synthesize asymmetric double oligonucleotide strands .
Synthetic branched oligonucleotides have been applied
for several purposes. Initially, most of the interest in this
area was focused on the study of branched oligoribonu-
cleotides as splicing intermediates of eukaryotic mRNAs
[24–26]. Moreover, branched oligonucleotides show high
affinity for single-strand oligonucleotides to form alternated
have been used as building blocks in the synthesis of
new nanostructures [30–34]. Multilabelled oligonucleotides
containing branching points have been described to increase
the sensitivity of hybridization experiments .
(Figure 1(b)) and evaluated their capacity to inhibit the
tumour necrosis factor (TNF-α) protein, which is involved
in the apoptosis, inflammation, and immunity processes
. We reasoned that branched siRNA could provide a
new RNA architecture for RNA interference activity. Given
that the use of symmetric branching units is compatible
with most of the modifications described to enhance the
inhibitory capacity of siRNA, the molecules described here
may provide a starting point for further modifications.
2.1. Oligonucleotides. The following RNA sequences were
obtained from commercial sources (Sigma-Proligo, Dhar-
macon): sense or passenger scrambled 5?-CAGUCGCGU-
UUGCGACUGG-dT-dT-3?, antisense or guide scrambled
5?-CCAGUCGCAAACGCGACUG-dT-dT-3?, antisense or
guide anti-TNF-α: 5?-GAGGCUGAGACAUAGGCAC-dT-
dT-3?, and sense or passenger anti-TNF-α: 5?-GUGCCU-
AUGUCUCAGCCUC-dT-dT-3?. RNA monomers in capital
letters, dT represents thymidine. The anti-TNFα siRNA
was previously described to efficiently downregulate murine
TNFα mRNA .
Figure 2 refers to the branched RNA structures synthe-
sized in this study. DB stands for the symmetric doubler
phosphoramidite obtained from commercial sources (Glen
Research). Guanosine was protected with the dimethy-
laminomethylidene group, cytidine with the acetyl group,
and adenosine with the benzoyl group. t-Butyldimethylsilyl
(TBDMS) group was used for the protection of the 2?-OH
function oftheRNAmonomers.The phosphoramiditeswere
dissolved in dry acetonitrile (0.1M), and a modified cycle
was used with increased coupling time to 10min. Oligori-
bonucleotide 1 was synthesized on a CPG solid support with
a symmetric branching unit of two arms containing two
DMT-protected hydroxyl groups, as described in . Olig-
oribonucleotides 2 and 3 were synthesized using standard
low-volume polystyrene thymidine columns. After the solid-
phase synthesis, the supports were treated with concentrated
aqueous ammonia-ethanol (3:1) for 1h at 55◦C. After
filtration of the supports, the solutions were evaporated
to dryness. The residue was dissolved in 85μL of 1M
tetrabutylammonium fluoride (TBAF) in tetrahydrofuran
(THF) for 12h. Then, 85μL of 1M of triethylammonium
on a NAP-10 column using water as eluent. The compounds
were purified by HPLC under the following conditions.
Column: Nucleosil 120–10C18(250 × 4mm); 20-min linear
gradient from 15% to 100%B (DMT ON conditions); flow
rate 3mL/min; solution A was 5% acetonitrile in 0.1M
aqueous triethylammonium acetate (TEAA) buffer and B
70% acetonitrile in 0.1M aqueous TEAA. The purified
products were analyzed by MALDI-TOF mass spectrometry.
2.2. Thermal Denaturation Studies. The thermal melting
curves for duplexesof the oligoribonucleotides 1–3 and their
unmodified RNA complementary strands (guide strand)
were performed following the absorption change at 260nm.
Samples were heated from 20◦C to 80◦C, with a linear tem-
perature ramp of 0.5◦/min in a JASCO V-650 spectropho-
tometerequippedwith a Peltiertemperature control. Sample
concentration of the samples was around 2μM. All the
Journal of Nucleic Acids3
R = COCH2CH2CONH-LCAA-CPG n = 3
R=PO3H-Thy n = 5
R = PO3H-Thy n = 5
3?TNFα5?: 3?TTG CAU GCG CCU UAU GAA GCU 5?
Figure 2: Schematic representation of the chemical structure of the branching units of the oligonucleotides described in this study.
measurements were repeated three times and conducted in
15mM HEPES 1mMMg(OAc)2and 50mM KOAc pH 7.4.
2.3. Cell Culture, Transfection, and Cellular Assays. HeLa
cells were cultured under standard conditions (37◦C, 5%
CO2, Dulbecco’s Modified Eagle Medium, 10% fetal bovine
serum, 2mM L-glutamine, supplemented with penicillin
(100U/mL) and streptomycin (100mg/mL)). All in vitro
experiments were performed at 40–60% confluence. HeLa
cells were transfected with 250ng of a plasmid expressing
murine TNF-α using lipofectin (Invitrogen), following the
were transfected with 100nM double strand concentration
of siRNA against TNF-α, using oligofectamine (Invitrogen).
4 Journal of Nucleic Acids
RNA 3, (5 TNFα3 )4-(DB)2-DB-dT
Solid support = controlled pore glass
Solid support = polystyrene
RNA 1, (5 TNFα3 )2-DB
RNA 2, (5 TNFα3 )2-DB-dT
Figure 3: Outline of the synthesis of branched RNA oligonucleotides 1, 2, and 3 with two and four arms with identical sequence. (a)
Assembly of the RNA sequence by standard solid-phase methods; (b) removal of protecting groups and release of the RNA molecule from
solid support; (c) addition of the symmetrical branching phosphoramidite.
0510 15 20
Figure 4: HPLC profile of DMT-containing oligonucleotide 2
with two arms. Truncated sequences without DMT groups had
a retention of less than 5min. Fraction eluting between 7-8min
contained oligonucleotides with a single DMT group. The last
fraction contained the desired sequence with two DMT groups.
Previously, siRNA duplex annealing was performed by
mixing modified (1, 2, 3) and unmodified passenger strands
(unm) with the appropriate amount of the corresponding
unmodified guide strand.
TNF-α concentration was determined from cell culture
supernatant by enzyme-linked immunosorbent assay kit
(Bender MedSystems) following the manufacturer’s instruc-
tions. The inhibitory capacity of the siRNA duplexes is
expressed as double strand concentration for comparative
purposes. A 100mM double strand concentration is equiv-
alent to a 50nM concentration of two-branched siRNA (1 or
2) and to 25nM of four-branched siRNA (3).
3.1. Oligonucleotide Synthesis. In order to prepare branched
RNA for RNA interference, the potential steric hindrance
of the branching unit with RISC must be considered.
As the passenger strand is removed from the siRNA
duplex upon binding to RISC, we introduced the branch-
ing modification at the protruding 3?-end of the sense
strand. This position has been demonstrated to allow the
introduction of a large number of modifications with-
out affecting the inhibitory capacity of siRNA [6, 7].
We thus designed branched oligonucleotide sequences 1–3
of the passenger strand of a siRNA directed against TNF-
α (Figure 2). Sequence 1 was synthesized using a con-
trolled pore glass (CPG) solid support containing a sym-
metric doubler , as shown in Figure 3. Sequences 2
and 3 with two or four strands, respectively, were syn-
thesized on a low-volume polystyrene support (LV200)
functionalized with dimethoxytrityl- (DMT-) thymidine.
The commercially available symmetric doubler phospho-
ramidite was used to introduce two and four branches
on the 3?-position of the starting thymidine (Figure 3).
Sequences were assembled using standard protocols for
RNA synthesis. The 2?-OH function of ribonucleosides was
protected with the t-butyldimethylsilyl (TBDMS) group.
Coupling yields, determined by the absorbance of the DMT
Journal of Nucleic Acids5
05 10 15 20
Figure 5: HPLC purification of oligonucleotide 3 with four arms. (a) HPLC profile of DMT-containing oligonucleotide 3. The last fraction
contained the desired sequence with four DMT groups; (b) analytical HPLC of purified oligonucleotide 3after removal of DMT groups.
Figure 6: Inhibitory capacity of branched anti-TNF-α siRNAs.
The inhibitory capacity of the siRNA duplexes are expressed as
double strand concentration for comparative purposes. A 100-nM
double strand concentration is equivalent to a concentration of
100nM of unmodified siRNA duplex, 50nM of siRNA 1, and 2 and
25nM of siRNA 3. Transfection of siRNAs was carried out using
oligofectamine. Values are represented as the average ±ES, n = 3
and are compared to a scrambled sequence.∗∗∗P < .001, ANOVA
Test, Bonferroni post-test.
cation released in each synthesis step, were more efficient
(98%) on low-volume (LV200) polystyrene supports than
on CPG support (95%). After assembly of the sequences,
the DMT-containing oligonucleotides were released from
the supports with ammonia, and the resulting compounds
were treated with fluoride to remove the TBDMS groups.
HPLC analysis of the resulting products is shown in Figures
4 and 5. Several peaks were observed for the synthesis of the
two-branch RNA sequences (1 and 2; Figure 4). Truncated
sequences without DMT groups eluted between 3–5min.
A fraction containing oligonucleotides with a single DMT
group was eluted next, and the last fraction contained the
desired sequence with two DMT groups. Mass spectrometry
analysis (Table 1) and electrophoresis analysis confirmed the
mass and size of the desired branched oligoribonucleotides.
Figure 5 shows the HPLC profile of the mixture obtained
in the synthesis ofthe four-branch RNAsequence (3).In this
case, three peaks in the DMT-containing area were observed.
Although resolution of these peaks was not as good as in
the previous case, the last eluting peak corresponded to the
desired tetra-DMT compound (3). The purified compound
had the expected molecular weight, was homogeneous
by analytical HPLC (Figure 5), and showed the correct
migration in polyacrylamide gel electrophoresis (PAGE).
3.2. Thermal Denaturation Studies. The melting tempera-
tures of the branched siRNA duplexes formed by annealing
of equimolar amounts of sequences 1–3 with unmodified
passenger strand are shown in Table 1. Duplex 1 had the
lowest melting temperature, which was 3.5◦C lower than
the unmodified duplex (Table 1). Duplex 2 melted 1.5◦C
lower than the unmodified duplex. In contrast, duplex 3
had similar melting temperatures as the unmodified duplex.
The small decrease in melting temperatures of the two-
branched siRNA structures is possibly due to a steric effect
in the branching point that holds the two duplex strands in
close proximity. The four-stranded architecture had a larger
separation between strands as a result of the introduction of
3branching units,thustheresulting duplexesshowed greater
similarity to the unmodified duplex. Thus we believe that
the small destabilizing effect observed in the two-branched
RNA duplexes could be optimized in further experiments by
adding a linker between the branching unit and the RNA
strands, as described by Grimau et al. .
3.3. Cell Culture, Transfection, and Cellular Assays.. Tumor
necrosis factor (TNF-α) was selected as a target for RNA
interference studies. This protein is a major mediator of
apoptosis as well as inflammation and immunity, and it has
6 Journal of Nucleic Acids
Table 1: Mass spectrometry data on modified passenger strand and melting temperatures of siRNA duplexes formed by oligonucleotides
1, 2, and 3 and the linear unmodified control sequences. Buffer conditions: 15mM HEPES, 1mM magnesium acetate, 50mM potassium
acetate pH 7.4.
been implicated in the pathogenesis of a wide spectrum of
human diseases. Consequently, inhibition of this protein is
of particular relevance. Modified oligoribonucleotides (1–3)
were annealed with equimolar amounts of the unmodified
guide, and the resulting duplexes were used to inhibit the
expression of TNF-α gene. HeLa cells were transfected first
with the murine TNF-α plasmid using lipofectin, and 1h
later they were cotransfected with the siRNA duplex using
oligofectamine. After 24h, cellular TNF-α production was
analyzed by enzyme-linked immunosorbent assay (ELISA).
The inhibitory capacity of the siRNA duplexes is shown
in Figure 6. To compare the efficiency of each siRNA to
inhibit TNF-α, we normalized the data taking in account the
number of strands of each siRNA. Thus, a 100nM double
strand concentration is equivalent to 100nM of unmodified
siRNA duplex, 50nM of siRNA 1, and 2, and 25nM of
siRNA 3. Figure 6 shows that the inhibitory capacity of the
branched structures was maintained similar to that of the
unmodified duplex. This result indicates that the branched
siRNAsdescribedhere are compatiblewith RNAinterference
machinery, and thus the RISC complex binds to branched
shown in Figure 1. Two-stranded RNA duplexes (1 and 2)
were more efficient than four-stranded ones (3). In addition,
siRNA1 showed slightly greater efficiency at inhibiting TNF-
α than siRNA duplex 2. This small difference may be related
to the lower melting temperature of the former (Table 1).
For several years, research has focused on chemical mod-
ifications and delivery technologies to improve the phar-
macokinetic properties of siRNA. Many of the chemically
modified siRNA with interesting inhibitory capacity contain
one or multiple modifications in the sugar, nucleobases,
and phosphate linkages or at the 3?- or 5?-ends. In addi-
tion to these modifications, duplex architecture of siRNA
itself is also relevant, and several modifications have been
reported to show satisfactory inhibitory capacity. Here we
demonstrate that branched siRNA is compatible with RNAi
and that, when transfected with cationic lipids, siRNA has
similar inhibitory capacity than unmodified duplex siRNA.
Although the potency of branched siRNA containing two
or four strands was not increased, we consider it a suitable
starting point for further development. Given that the
method described here is compatible with most of the
RNA modifications described to date, these compounds
may be further functionalized to obtain more potent siRNA
derivatives. In addition, they offer an internal mid position
that could be suitable for attachment to delivery systems.
In this regard, optimization of the branching approach for
the synthesis of asymmetric branched siRNAs may lead to
the development of siRNA for the combined inhibition of
multiple targets. These asymmetric siRNA duplexes carrying
two RNA sequences attached or bound to an appropriate
delivery system will insure the 1:1 ratio of two RNA
sequencesfor the combined inhibition oftwo genesthat may
improve the treatment of a particular disease.
This paper was supported by the Spanish Ministry of
Education (Grants BFU2007-63287, CTQ2010-20541) and
the Generalitat de Catalunya (2009/SGR/208). CIBER-BBN
is an initiative funded by the VI National R&D&i Plan 2008–
2011,Iniciativa Ingenio 2010,Consolider Program,and CIBER
Actions and financed by the Instituto de Salud Carlos III with
assistance from the European Regional Development Fund.
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