In this chapter, we explore some common and simple methods
for the assessment of nanocomplex formation. Techniques utilizing
fluorescence (dye displacement), agarose gel electrophoresis to
determine DNA/RNA binding and protection from serum
nucleases, transmission electron microscopy for a visual determination of the ultrastructural morphology of the nanocomplexes, and
nanoparticle tracking and analysis to assess the effect of the interactions on the size, charge, and stability of the nanocomplex. These
methods all play significant roles in the elucidation of the mass/
charge ratios required for optimal nucleic acid:nanocarrier interaction, the structural features of the resulting nanocomplexes, their
stability, and their ability to bind and enter cells. Hence, these
preliminary studies will provide valuable information as to the
potential of the nanocarriers for further gene therapeutic
investigations.
2 Materials
All reagents, nanoparticles, and the nucleic acid stock solution can
be made up in ultrapure water (preferably 18 MΩ) or sterile HEPES
buffered saline (HBS) at physiological pH 7.4.
2.1 Molecular
Biology Reagents
and Materials
1. Nucleic Acid Stock: 0.25 or 0.5 μg/μL in ultrapure water
or HBS.
2. Ethidium Bromide: 10 mg/mL in ultrapure water (see Note
1).
3. 5% Sodium Dodecyl Sulfate (SDS): 5 g SDS in 100 mL H 2 O.
4. 0.5 M ethylenediamine tetra-acetic acid (EDTA), pH 8: 18.6 g
EDTA in 100 mL distilled water (see Note 2).
5. Ultrapure Agarose (Molecular Biology Grade).
6. Fetal Bovine serum (FBS)/RNase A/DNase.
7. Flat-bottom black multiwell plate for fluorescence
measurements.
8. Formvar/carbon-coated grids for electron microscopy.
2.2 Buffers
and Solutions
1. HEPES Buffered Saline (HBS): 20 mM HEPES and 150 mM
NaCl, pH 7.4.
2. Gel Loading Buffer: 25 mg bromophenol blue, 25 mg xylene
cyanol, 4 g sucrose in 10 mL H 2 O (see Note 3).
3. 10Â Electrophoresis Buffer: 0.36 M Tris–HCl, 0.3 M
NaH 2 PO 4 , 0.1 M EDTA, pH 7.4–7.5 (see Note 4).
2.3 Equipment
1. Electrophoresis apparatus (mini-sub type) with power supply.
2. Gel Documentation System/Gel Imager.
Nucleic Acid:Nanoparticle Interactions
45
for the assessment of nanocomplex formation. Techniques utilizing
fluorescence (dye displacement), agarose gel electrophoresis to
determine DNA/RNA binding and protection from serum
nucleases, transmission electron microscopy for a visual determination of the ultrastructural morphology of the nanocomplexes, and
nanoparticle tracking and analysis to assess the effect of the interactions on the size, charge, and stability of the nanocomplex. These
methods all play significant roles in the elucidation of the mass/
charge ratios required for optimal nucleic acid:nanocarrier interaction, the structural features of the resulting nanocomplexes, their
stability, and their ability to bind and enter cells. Hence, these
preliminary studies will provide valuable information as to the
potential of the nanocarriers for further gene therapeutic
investigations.
2 Materials
All reagents, nanoparticles, and the nucleic acid stock solution can
be made up in ultrapure water (preferably 18 MΩ) or sterile HEPES
buffered saline (HBS) at physiological pH 7.4.
2.1 Molecular
Biology Reagents
and Materials
1. Nucleic Acid Stock: 0.25 or 0.5 μg/μL in ultrapure water
or HBS.
2. Ethidium Bromide: 10 mg/mL in ultrapure water (see Note
1).
3. 5% Sodium Dodecyl Sulfate (SDS): 5 g SDS in 100 mL H 2 O.
4. 0.5 M ethylenediamine tetra-acetic acid (EDTA), pH 8: 18.6 g
EDTA in 100 mL distilled water (see Note 2).
5. Ultrapure Agarose (Molecular Biology Grade).
6. Fetal Bovine serum (FBS)/RNase A/DNase.
7. Flat-bottom black multiwell plate for fluorescence
measurements.
8. Formvar/carbon-coated grids for electron microscopy.
2.2 Buffers
and Solutions
1. HEPES Buffered Saline (HBS): 20 mM HEPES and 150 mM
NaCl, pH 7.4.
2. Gel Loading Buffer: 25 mg bromophenol blue, 25 mg xylene
cyanol, 4 g sucrose in 10 mL H 2 O (see Note 3).
3. 10Â Electrophoresis Buffer: 0.36 M Tris–HCl, 0.3 M
NaH 2 PO 4 , 0.1 M EDTA, pH 7.4–7.5 (see Note 4).
2.3 Equipment
1. Electrophoresis apparatus (mini-sub type) with power supply.
2. Gel Documentation System/Gel Imager.
Nucleic Acid:Nanoparticle Interactions
45