Supplementary information
Gold
N
anocluster
M
ediated
D
elivery of siRNA to
I
ntact
P
lant
C
ells for
E
fficient
Gene Knockdown
Huan Zhang
1,
2,
#
, Yuhong Cao
3
,
#
, †
, Dawei Xu
2
, Natalie S. Goh
2
,
Gozde S. Demirer
2,
$
,
Stefano
Cestellos
-
Blanco
3
,
Yuan Chen
4
, Ma
r
kita P
.
Landry
2
,
5
,
6*
, Peidong Yang
3
,
7
,
8
,
9*
1.
Department of
Chemistry,
College of Chemistry and
Material
s
Science,
Jinan University,
Guangzhou, Guangdong 510632, China
2.
Department of Chemical and
B
iomolecular Engineering, Univer
sity of California, Berkeley, CA
94720, USA
3.
Department of Materials Science and Engineering, University of California, Berkeley, CA 94720
4.
Plant Gene Expression Center, United States Department of Agriculture
-
Agricultural Research
Service, and
Department of Plant and Microbial Biology, University of California Berkeley, Albany,
California 94710
5.
California Institute for Quantitative Biosciences, QB3, University of California, Berkeley, CA 94720,
USA
6.
Chan Zuckerberg BioHub, San Francisco, CA 9415
8, USA
7.
Department of Chemistry, University of California, Berkeley, CA 94720
8.
Chemical
Sciences Division,
Lawrence Berkeley National Laboratory, Berkeley,
CA
94720
9.
Kavli Energy NanoScience
Institute,
University of California, Berkeley,
CA
94720
†
Current address
:
CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National
Center of Nanoscience and Technology, 11 Zhongguancun Beiyitiao Haidian District, Beijing, China
,
100190
$
Current address: Department of Plant Biology and Genome Center, University of California, Davis,
451 Health Sciences Drive, Davis, CA 95616, USA
*
_
Corresponding authors, Email: p_yang@berkeley.edu and landry@berkeley.edu;
#
_
H.Z. and Y.C.
c
ontributed equally to this work.
Materials and Methods
Chemicals and materials
Lipoic acid, N
,
N'
-
Dicyclohexylcarbodiimide
(DCC),
Dichloromethane
(DCM)
,
Polyethyleneimine (PEI) and Tetrachloroauric
(III) acid
(HAuC
l
4
.3H
2
O) were
purchased from Sigma
-
Aldrich.
Slide
-
A
-
Lyzer
d
ialysis
f
lask
s were
purchased
from
ThermoFisher Scientific. DNAs and RNAs were purchased from Integrated DNA
Technologies.
RNase
was purchased from New England BioLabs.
Preparation of PEI
-
lipoic acid
s
PEI
-
lipoi
c acid conjugates were synthesized by coupling lipoic acid to 800
,
2.5
K
, or
25
K
PEI through carbodiimide chemistry
as
previously reported
35
. In brief, PEI and
DCC
were
first dissolved in ice
-
cold DCM under nitrogen. After 5 h
ours
for
activation,
the reaction mixture was filtered.
Next,
the lipoic acid solution was added dropwise to
the filtrate. The mixture was stirred overnight under nitrogen atmosphere at room
temperature. The product was purified through ice
-
cold diethyl ether preci
pitation and
dried in vacuo before use.
Preparation of PEI functionalized gold nanocluster (PEI
-
AuNCs)
A solution of 800, 2.5
K
, and 25
K
PEI
-
lipoic acid ligands were prepared in a
clear
glass
vial. To
synthesize
the
PEI
-
AuNCs, 30 μL of HAuCl
4
·3H
2
O stock sol
ution (100 mM)
was added along with DI water to bring the final volume of the reaction to 6 mL
with a
molar ratio of ligand to gold
of
6
:1
. Then, 30 μL of 2 M aqueous NaOH solution was
added to make the reaction mixture basic. The mixture was heat
ed
at 9
5
°C
and left
stirring for 24
h
ours
.
An aqueous solution of PEI
-
AuNC
with yellow
color was formed.
The PEI
-
AuNC
solution was further purified by dialysis.
The
purified
PEI
-
AuNC
solution was stored at 4°C
until use
.
AuNC
Characterization
T
he size
distribution, polydispersity index
,
and surface charge of the prepared
nanomaterials were determined using
a
Zetasizer
(NanoZS, Malvern). For morphology
characterization, the samples were examined by transmission electron microscope
(TEM
,
FEI TECNAI 12
and
F
EI F20 UT T
ECNAI
). A
NanoDrop Microvolume UV
-
Vis
Spectrophotometer
(ThermoFisher Scientific) was used to characterize
the
UV
-
Vis
spectrum of the 800, 2.5K
,
and 25K PEI
-
AuNCs and siRNA (120 ng) loaded PEI
-
AuNCs (800, 2.5K
,
and 25K).
siRNA loading
capacity me
asurements
and protection gel assay
D
uplex siRNA
was
obtained
by mixing two fully complementary RNA oligonucleotides
(sense and antisense
RNA
strands in Table S1) in PBS buffer
and
heating to 95
º
C
for
5 minutes, follow
ed
by cooling to room temperature (
20
o
C
)
with
in 30 minutes.
To
determine the loading capacity of PEI
-
AuNCs,
the
pre
-
hybridized siRNA duplex was
incubated with PEI
-
AuNCs at various mass ratios (4
:
1, 8:1, 20:1 and 40:1 for 800
-
PEI
AuNCs; 3:1, 1.5:1, 1:1.5 and 1:3 for 2.5K
-
PEI AuNCs; 3:1, 1:1 and 1:1.
5 for 25K
-
PEI
AuNCs) in DI water
for 10 minutes
. The prepared samples were characterized by 10%
native page gel electrophoresis and the band intensity was analyzed and normalized
to the free siRNA band (120 ng) to determine how much siRNA was loaded onto t
he
various AuNCs.
RNase Digestion
For the siRNA protection assay,
106
n
g of free siRNA or siRNA loaded onto PEI
-
AuNCs (at various PEI
-
AuNC
s
:s
iRNA
mass ratios of 1:10, 1:20, 1:50 and 1:100) were
incubated with RNase H (10 μg/ml, New England BioLabs, M0297L
)
at 37
°C
for 30
minutes
. The samples were next
characterized by 20%
native
page
gel
electrophoresis. The band intensity represents intact RNA, which was analyzed with
Image J software.
Plant growth and Maintenance
Transgenic mGFP5
Nicotiana benthamiana
(
Nb
,
obtained from the Staskawicz Lab,
UC Berkeley) seeds
were germinated and grown in SunGro Sunshine LC1 Grower
soil mixture within
a
growth chamber
(740 FHLED, HiPoint, Taiwan)
.
The plants were
grown in 4
-
inch pots
under LED light and a 14/10 light/dark photoperiod at 23°C and
60% humidity. Plants were allowed to mature to 3
-
4 weeks of age within the chamber
before experimental use.
Leaf Infiltration
A
tiny puncture
hole was introduced
into the
Nb
plant leaf with a
10 μ
L
pipette tip
on
the leaf abaxial surface
prior to infiltration of leaves. Infiltrations were performed by
gently push
ing ~100
μ
L of fluid
into the leaf tissue with a 1 mL capacity needle
-
less
syringe
.
Internalization
of
Cy3 labeled PEI
-
AuNCs
into
mGFP5
Nb
plant leaf cells
quantified
through
colocalization analysis
100 ng of
C
y3
-
modified ssDNA was loaded onto
0.4
μ
g 2.5K
-
PEI
AuNCs and infiltrated
into mGFP5
Nb
leaves with a final concentration of 200
nM Cy3 DNA. The plants w
ere
kept in the
plant growth
chamber for
the specified
incubation time
s
before preparing
infiltrated leaves for confocal imaging. A small leaf section
at the infiltration site
was
cut and mounted between a glass slide and
a
#1 thickness cover slip. Water was
ad
ded to keep the leaf sections hydrated during imaging. A Zeiss LSM 710 confocal
microscope was used to image the plant tissue with 488 nm and 543 nm laser
excitation for GFP
(
collection
window: 490
-
520 nm)
and Cy3
(
collection
window: 530
-
600 nm)
signal col
lection
,
respectively. Images were obtained at 20x magnification.
The same imaging parameters and quantification analyses were applied to samples
imaged
at
different time points.
Quantitative PCR (qPCR) experiments for gene silencing
Water (control), free siRNA,
siRNA loaded
PEI
-
AuNCs (total volume of 100 μL
and a
final concentration of
100 nM siRNA
)
were infiltrated into plant leaves and left on the
benchtop at
room temperature (
20
o
C
)
for
1 day prior to extraction of
total RNA
of the
treated leaves
with
a
n
RNeasy plant mini kit
(QIAGEN). Two
-
step qPCR was
performed to quantify GFP gene silencing
using the
total RNA
extracted
from leaves
:
an
iScript cDNA synthesis kit
(Bio
-
Rad)
was used
to reverse transcribe total RNA i
nto
cDNA, and PowerUp SYBR green master mix
(Applied Biosystems)
was used
for
qPCR. The target gene in our qPCR was mGFP5
(GFP transgene inserted into
Nb
)
as the gene target for RNAi
-
based silencing
, and
respiratory burst oxidase homolog B
(
NbrbohB
)
was th
e target
gene
for toxicity assessments, with
EF1
(elongation factor
1) as the
invariant
housekeeping gene
standard
36
. Primers (see detailed sequences
in Table S1) for these genes were ordered from IDT and used without further
purification. An annealing temperature of 60 ̊C was used for qPCR, which was run for
40 cycles. q
PCR data was analyzed by the ddCt method
37
to obtain the normalized
GFP
gene expression
-
fold change with respect to the EF1 housekeeping gene and
control. For each sample, qPCR was performed
for
3 technical replicates
(3 reactions
from the same
isolated RNA batch), and the entire experiment
consisted
of
3
independent infiltrations and RNA extractions from different plants
(3 biological
replicates).
Quantitative Western blot experiments and data analysis
Infiltrated plant leaves were harvested
3
days post
-
infiltration
and ground in liquid
nitrogen to
obtain
dry frozen powders. The frozen powders were then transferred to a
tube with pre
-
cooled
lysis buffer containing 10 mM Tris/HCl
(pH 7.5), 150 mM NaCl, 1
mM EDTA, 0.1% NP
-
40, 5% glycerol, and 1%
cocktail. After lysing
on ice for 1 h
our
,
the tube was centrifuged at 10,000 rpm for 20 minutes and the supernatant containing
whole proteins was
collected into
a new tube. After quantification of the total extracted
proteins by a Pierce 660 nm Protein Ass
ay
(Thermofisher, 22660), 0.
5
μg of
normalized total proteins from each sample were analyzed by 12% SDS
–
PAGE and
blotted to a PVDF membrane. The membrane was then blocked for 1 hour using 7.5%
BSA in
T
BST (PBS containing 0.1% Tween
20) buffer and rinsed 3
times in PBST
buffer, followed by overnight incubation at 4°C with the primary GFP antibody as
required (1:2000 dilution, Abcam, ab290). After extensive washing, the corresponding
protein bands were probed with a goat anti
-
rabbit horseradish peroxidase
-
con
jugated
antibody (1:5000 dilution, Abcam, ab205718) for 30 min
utes
. After 3 washes
with
TBST
, the membrane was then developed by incubation with chemiluminescence
(Amersham ECL prime kit) and imaged
with a
ChemiDoc™ XRS+ System (BIORAD).
The
intensity of GFP bands were quantified with Image
J software. To correct for
variability in protein expression across different plants and leaves, the GFP extracted
from each leaf sample was normalized by the total protein recovered from that leaf
tissue.