Supporting Information
Synthesis
HBsAg (18.8mg/0.00074 moles) in PBS at pH 7.4 was blocked with NEM
(4.8mg/0.0385moles) at a 50:1 molar ratio (NEM: HBsAg) for an hour followed by
treatment with sSMCC (3.22mg/0.0074moles) for 2 Hrs at a 10:1 molar ratio of
(sSMCC: HBsAg); Tangential Flow Filtration (TFF) was used to remove excess small
molecules.
The
activated
HBsAg
(1c)
was
reacted
with
5’-thio
ISS
(55.3mg/0.0074moles) for two hours at a 10:1 molar ratio of (5 ‘-thio- ISS: HBsAg) to
produce the conjugate HBsIC-ISS (1d). The excess oligonucleotide was removed via
TFF and the final product was formulated in PBS.
Tangential Flow Filtration (TFF)
TFF was performed to remove small molecules and excess ISS from the activated
HBsAg (1c) and conjugate HBsIC-ISS (1d) respectively. A Cole Parmer 77200-60 (Cole
Parmer, USA) pump was employed. The TFF membrane used was PXC300C50 300KD
MWCO regenerated cellulose UF membrane (Millipore, USA). The 20nm fluorescent
nanobeads used for the control experiment were obtained from Duke Scientific.
Absorbance Units
5' thio-ISS-ODN Removal From HBsIC-ISS Via UF
Using a 300KD MWCO Cellulose Membrane
3
2.5
2
Series1
1.5
Series2
1
Series3
0.5
0
0
5
10
15
Diavolumes
Series 1 A215, Series 2 A260, Series 3 A280
1
DC protein concentration Determination for HBsIC-ISS (1a):
The DC protein concentration assay is a Lowry based method. The DC Protein
assay kit (Bio-Rad, USA) was used. A protein standard curve was generated with Bovine
Serum Albumin (Pierce, USA) as a standard. The absorbance was recorded at 750nm
using a HP8453 U.V. spectrophotometer.
ISS Content Determination for HBsIC-ISS (1d)
HBsIC-ISS (1d) and HBsAg (1a) were diluted in PBS to a concentration of 0.071
mg/ml of protein (the protein concentrations were determined using the DC assay). The
spectrophotometer was blanked with HbsAg solution (0.071mg/ml of protein) and the
absorbance of HBsIC-ISS sample which arises from the DNA component (ISS
component) was measured at 260nm to be 0.646; this procedure of blanking the
spectrophotometer with HbsAg solution (0.071mg/ml of protein) eliminates the
absorbance at 260nm arising from the protein component of HBsIC-ISS conjugate. Using
a previously determined extinction coefficient of 25.6 (at 260nm) for DNA (ISS), the
concentration of DNA in the HBsIC-ISS. conjugate sample was determined to be 0.0252
mg/ml.
A control experiment was performed: the spectrophotometer was blanked with
HbsAg solution (0.071mg/ml of protein) and the absorbance of a control sample which
consisted of a non-covalent mix of 0.071 mg/ml (protein concentration) HBsAg and
0.02524 mg/ml of DNA (ISS) was measured; the control sample had an absorbance of
0.646 at 260nm which was identical to that of the test sample.
The DC protein concentration of 0.071mg/ml was used to compute the number of
moles of HBsAg protein subunits in the HBsIC-ISS sample: 2.8*10-6 moles (MW of
2
HBsAg subunit = 25,400 Dalton). The number of moles of DNA (ISS) in the HBsIC-ISS
conjugate was determined from the above UV experiment to be 3.4*10-6 moles (MW of
ISS = 7,500 Dalton).The ISS/protein subunit ratio thus determined was 1.2.
HPLC determination of the A215/A260 ratio
The ratio of the protein absorbance at 215nm to the absorbance arising from the
oligonucleotide at 260nm (A215/A260 ratio) was determined by HPLC using a
Phenomenex BioSEp-SEC-3000, 300X4.6mm column at a flow rate of 0.347ml/min,
(Sample concentration100µg/ml; 17µl injection) using a UV Detector set at 215nm and
260nm, the elution buffer employed was a mixture of 90%PBS and 10% MeOH. The
samples 1a and 1d eluted at the void volume because of their large size. The A215/A260
ratio was 13.1 for the control 1a sample and 5.9 for the oligonucleotide modified 1d
sample.
3
215nm
260nm
A215/260 for HBsAg
4
215nm
260nm
A215/260 for HBsICISS
Capillary Zone Electrophoresis (CZE)
Two Capillary Zone Electrophoresis methods were used to analyze HBsAg-ISS.
An Agilent CE (Agilent, USA) machine was used for measurements. The first method
was used to distinguish the HBsAg-ISS conjugate from HBsAg. This method used a
34cm PVA coated capillary with an ID of 50µm (Agilent, USA). The analysis was
performed at 25°C and a voltage of –14KV. A second method was used to distinguish
HBsAg-ISS conjugate from ISS, the method used a 50cm PVA coated capillary with and
5
ID of 50µm (Agilent, USA). The second analysis was performed at 25°C and a voltage of
–15KV.
Size Exclusion HPLC
An Agilent 1100 series HPLC (Agilent Technologies, USA) with a UV DAD
module was used. For SEC analysis of the HBsAg-ISS conjugate two columns were
connected in series, TSK G6000 PW, 7.5mm x 300mm followed by TSK G4000 SW,
7.5mm x 300mm (Phenomenex, USA). An isocratic flow at 0.5ml/min of phosphate
buffered saline (Pierce, USA) was used to elute samples.
SDS-PAGE
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was
carried out for sample analysis. Samples were first reduced by heating at 95°C for 5min
in the presence of Bond Breaker (TCEP solution from Pierce, USA). 4-12% Nu Page gels
were used in a Xcell II electrophoresis Mini-Cell bath (Invitrogen, USA). Molecular
weight standards used for comparison were Precision Protein Standards (Bio-Rad, USA).
For DNA specific staining the DNA Silver Staining Kit (Amersham, USA) was used.
Coumassie staining was done with a Gel Code Coumassie Blue Stain (Pierce).
Light Scattering
A NICOMP Model 380 Particle Sizing Systems, Santa Barbara CA was used for
making light scattering measurements. A 20 nm Nanosphere size standard from Duke
Scientific (product number 3020A) was used as a control. The HBsAg and HBsICISS
samples were diluted to a final concentration of 40 µg/mL with water before making
measurements.
6
20 nm Standard
HBsAg
HBsIC-ISS (1d)
HBsIC-ISS (1d)
7
Transmission Electron Microscopy
Negatively stained TEM Image of HBsIC-ISS (1d) at 165000 Magnification
8