DNA nanotechnology as a tool to manipulate lipid bilayer membranes
Meng, Zhuojun
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Meng, Z. (2017). DNA nanotechnology as a tool to manipulate lipid bilayer membranes. University of Groningen.
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Chapter 2
Stability Study of Lipid-DNA on
the Liposomal Membrane
32
2.1 Introduction
Deoxyribonucleic acid (DNA) is a macro molecule that carries hereditary information of all known living organisms and many viruses. Its double-stranded helix structure was discovered by Watson & Crick in 1953,1 which
has greatly fueled many technologies dealing with DNA and hence revolutionized modern science. In recent years DNA has become a valuable functional building block and tool in nanotechnology and material science due to the unique nature and properties of DNA and DNA hybrid materials. A wide variety of products and applications have been realized using DNA technologies among which is incorporating DNA with a functional group and utilizing its information-carrying capability to develop DNA detection systems. For instance, fluorescent dye-labeled DNA was used as probe monitor in PCR2 or for sequence analysis.3,4 Additionally, coupling DNA
strands with moieties like polymers or nanoparticles changes the morphological structure and introduces new functionalities, which are
33 different from conventional polymers. For instance, DNA conjugated gold nanoparticles were used in DNA microarray technology.5-7 Another
functional moiety chemically conjugated with DNA consists of hydrophobic molecules, such as long alkyl chains, cholesterol, or fatty acids, resulting in amphiphiles, which spontaneously form nanoparticles in solution and enhance the pharmacokinetic behavior and trans-membrane delivery. Their amphiphilic nature arises from the hydrophilic DNA backbone containing charged phosphodiester bonds and the hydrophobicity of attached alkyl chains.8 These nanoparticles can be further functionalized
through hybridization of a modified complimentary DNA or internalization of payloads in the hydrophobic core.9
Our group reported the synthesis and characterization of a family of DNA amphiphiles containing hydrophobically modified nucleobases.10,11
Specifically, 1-dodecyne (C12H22) was attached to a uracil base which was
further attached to the 5’ or 3’ position of a DNA sequence (Fig. 2.1A). In aqueous environment, due to their amphiphilic nature, lipid-DNA self-assembles into micelles whereby the hydrophilic DNA strands shield the hydrophobic lipid core. These DNA micelles can be loaded with cargo by hydrophobic interactions or hybridization with functionalized complementary DNA (Fig. 2.1B). The aggregation properties of lipid-DNA can be relatively easy manipulated by changing the length of the lipid part or the number and position of the modified uracil bases within the DNA sequence. Fig. 2.1C shows three different lipid-DNAs. U2M and U2T are lipid DNA with two modified uracil bases either in the middle or at the terminus and U4T represents lipid DNA with four modified uracil bases at the 5’ end.10
Because of the amphiphilic and sequence specific properties, lipid-DNA can be used for liposome surface modification by insertion of the hydrophobic part into the membrane while the hydrophilic DNA is exposed to the aqueous medium. Compared with existing terminal modifications, our design allows the precise and easy introduction of hydrophobic units at arbitrary positions and numbers in a DNA sequence through conventional solid-phase synthesis. In this chapter, DNA was modified with four lipid
34
chain modified nucleobases at both terminals and it was used to anchor it to phospholipid membranes.
Fig. 2.1 Structure of lipid modified nucleotide and representation of lipid–DNAs. (A) Chemical
structure of the lipid-modified uracil nucleobase. (B) Lipid-DNAs self-assemble to form DNA micelles due to their amphiphilic nature. These self-assembled structures can carry cargo by hydrophobic interaction (1) or by hybridization with a functionalized complementary DNA (2). (C) Schematic representation of the ss and ds lipid–DNA amphiphiles (U2M, U2T, and U4T) and their propensity to undergo Watson-Crick base pairing.10
35
2.2 Results and Discussion
2.2.1 Lipid-DNA design and characteristics
To obtain stable incorporation of DNA into the liposomal bilayer, we use lipid-DNA (U4T-18), which has been designed to contain four modified uracil nucleobases at the 5’ position of a 18-mer oligonucleotide (including the 4 lipid modified uracil bases). CrU4T-18 is complementary to U4T-18 with the lipid anchor at the opposite terminus (i.e. the 3’ position). Cr-ATTO488 is a 14-mer DNA complementary to U4T-18 and was covalently attached an ATTO488 dye to the 3’ end (Table 2.1).
Table 2.1 Sequences of modified DNA.
Name Sequence (5’→ 3’)*
U4T-18 UUUUGCGGATTCGTCTGC
CrU4T-18 GCAGACGAATCCGCUUUU
14mer GCGGATTCGTCTGC
Cr-ATTO488 GCAGACGAATCCGC-ATTO488 *: U represents the lipid-modified uracil base.
U4T-18 can be attached to the liposome surface by insertion of four lipid-modified nucleobases into the lipid membrane while the remaining 14mer DNA part is protruding into the aqueous medium. This DNA unit can hybridize with the DNA part from CrU4T-18 or Cr-ATTO488 (Fig. 2.2A). According to the results from polyacrylamide gel electrophoresis (PAGE), a lower electrophoretic mobility of hybridized lipid-DNA (lane 2) is observed compared to ssDNA controls (lane 1 and lane 3), indicating successful Watson-Crick base pairing (Fig. 2.2B).
After confirming hybridization, the melting temperature (Tm) of the
ds-lipid-DNA (U4T-18+Cr-ATTO488) was determined. The ds-ds-lipid-DNA and ds14mer (14mer+Cr-ATTO488) were heated at 0.5 °C/min while measuring the absorption at 260 nm. Afterwards the first derivative of the curve was calculated and Tm of the ds DNA was taken at maximum slope.
36
The Tm value of lipid-DNA (62.5 °C) is very close to that of 14mer (63.6 °C)
(Fig 2.2C, D). The result indicates that lipid chains have little influence on the melting temperature.
Fig. 2.2 (A) Schematic representation of U4T-18 hybridization with Cr-ATTO488 on the
surface of liposomes. (B) Native PAGE characterization of lipid-DNA (20% TBE gel, 100V, 80min). Lane 1: U4T-18, lane 2: U4T-18 + Cr-ATTO488, lane 3: Cr-ATTO488. (C) Melting curve of dsDNA, U4T-18 + Cr-ATTO488. (D) Melting curve of dsDNA, 14mer + Cr-ATTO488. Melting curve (black squares, left Y-axis) and calculated
derivative
for corresponding sample (red circle, right Y-axis).2.2.2 Characterization of the incorporation of lipid-DNA in liposomal
bilayer.
After synthesis of the nucleobase-modified DNA hybrids and testing their ability for Waston-Crick base pairing, the lipid DNAs were stably anchored into the membrane of DOPC:DOPE:cholesterol lipid vesicles, while the oligonucleotides remained available for hybridization, as demonstrated by a Fluorescence Resonance Energy Transfer (FRET) assay.12
37 Since ATTO488 and rhodamine dyes show energy transfer when there is a sufficiently short distance between them,13 ATTO488 was covalently
attached to the 3’ end of a 14-mer DNA complementary to U4T-18 (Cr-ATTO488) to act as a donor. In parallel, rhodamine-functionalized phospholipid (Rh-DHPE) was incorporated in the liposomal bilayer to function as an acceptor (Fig. 2.3A). To observe the dynamic emission changes of donor and acceptor after adding Cr-ATTO488, fluorescence emission spectra with excitation at 470 nm of Cr-ATTO488 (donor, emission maximum 520 nm) and Rh-DHPE (acceptor, emission maximum 592 nm) were recorded over 30 min (Fig. 2.3B). The fluorescence of donor significantly decreased by adding Cr-ATTO488 and the fluorescence of acceptor slightly increased at the same time, illustrating that FRET is induced by DNA hybridization.
Fig. 2.3 (A) Schematic of FRET assay demonstrating that oligonucleotides anchored into
liposomal bilayers via lipid-DNA remain available for hybridization. (B) Fluorescence emission of Cr-ATTO488 (donor, emission maximum 520 nm) and Rh-DHPE (acceptor, emission maximum 592 nm) followed over 30 min after adding Cr-ATTO488.
Meanwhile, as demonstrated by the increase in the maximum intensity ratio I592/I520 (acceptor/donor peak) (Fig. 2.4D), hybridization only
occurred upon mixing of Cr-ATTO488 with U4T-18-grafted Rh-DHPE-containing vesicles, positioning both dyes sufficiently close to each other to achieve FRET (Fig. 2.4A), whereas for vesicles containing non-complementary lipid-DNA, CrU4-18, (Fig. 2.4B) or no lipid-DNA at all (Fig.
38
Fig. 2.4 Anchoring of lipid-DNA in the membrane and hybridization on the vesicle surface
leads to Fluorescence Resonance Energy Transfer (FRET) upon hybridization of donor-modified complementary DNA with DNA-functionalized, acceptor-containing vesicles. (A) FRET is achieved when complementary Cr-ATTO488 DNA hybridizes with U4T-18 and brings the donor close to the acceptor, rhodamine, positioned in the membrane. If hybridization is not possible, either due to mismatch of the two DNA strands (B) or the absence of membrane-grafted DNA (C) FRET does not occur. (D) Fluorescence spectra of systems capable of FRET (red) and non-FRET controls, either due to DNA mismatch (blue) or absence of membrane-grafted DNA (green).
Disruption of vesicles by addition of Triton X-100 to a final concentration of 0.3% (v/v) resulted in a drop in FRET in the U4T-18 vesicles hybridized with Cr-ATTO488 (Fig. 2.5A vs Fig 2.4D), confirming that FRET was indeed caused by bringing the donor in close vicinity to the acceptor dye located in the liposomal membrane. As expected, in two control non-FRET systems in which DNA hybridization could not occur, either due to absence of DNA in the membrane (Fig. 2.5B) or the presence of non-complementary DNA (Fig. 2.5C) energy transfer from donor to acceptor
39 was prevented. Therefore, similar spectra were observed before and after liposomal disruption.
Fig. 2.5 To further investigate the engraftment of the lipid-DNA hybrids into the membrane,
FRET liposomes were disrupted with Triton X-100 at a final concentration of 0.3 % (v/v). Fluorescence spectra of FRET liposomes before and after adding Triton X-100 (A). Similar spectra were observed in control experiments before and after liposomal disruption, either due to the absence of DNA on the membrane (B) or the presence of non-complementary DNA (C).
2.2.3 Temporal stability of lipid-DNA in the liposomal membrane.
To study whether the incorporation of U4T-18 in the membrane is stable overtime, FRET (U4T-18/Cr-ATTO488/Rh-DHPE) liposomes were incubated with non-FRET (NF) liposomes (Fig. 2.6A) at different ratios (1:1, 1:5 and 1:10).40
Fig. 2.6 Measurement of stability of lipid-DNA in liposomes over time. FRET
(U4T-18/Cr-ATTO488/Rh-DHPE) liposomes were incubated with non-FRET (NF) liposomes (A) at different ratios (1:1, 1:5 and 1:10), and the relative Rh-DHPE/ATTO488 (IA/ID) emission intensity ratio
was monitored over 24 h after mixing (B). Fluorescence spectra of Cr-ATTO488/Rh-DHPE pair in FRET liposomes mixed with NF liposomes at different ratio (v/v): 1:1(red line), 1:5(blue line), 1:10(green line) (C). Solid and dashed lines represent the spectra of the mixed systems before and after adding Triton X-100, respectively.
The relative Rh-DHPE/ATTO488 (IA/ID) emission intensity ratio of the
three systems was monitored over 24 h after mixing (Fig. 2.6B). If lipid-DNA redistributes from FRET liposomes to NF liposomes, a decrease in relative fluorescence of acceptor peak would be observed. After 24 h, some of the acceptor intensity had dropped, but the relative fluorescence IA/ID of
the mixture remained at a similar value as that during the initial measurement before non-FRET liposomes were added. The results demonstrate that the lipid–DNA is stably anchored in the liposomes over at last 24 hours. Fig. 2.6C shows the fluorescence spectra of Cr-ATTO488/Rh-DHPE pair in FRET liposomes mixed with NF liposomes at different ratio before and after liposomal disruption.
41 Moreover, lipids were mixed with U4T-18 at different molar ratios (5000, 1000, 100, 62.5). The final concentration of Cr-ATTO488 and lipid-mixture (DOPC+DOPE) were kept at 7.32 µM and 0.45 mM, respectively, in all FRET experiments. The results show the I592/I520 ratio increased markedly with
higher U4T-18 densities in the membrane (Fig. 2.7, Table 2.2). These results demonstrate that when more lipid DNA is incorporated into the membrane more DNA strands can be attached to this vesicle surface by hybridization (Table 2.2).
Fig. 2.7 U4T-18/Rh-DHPE fluorescence spectra of FRET liposomes mixed with Cr-ATTO488 at
different lipid/U4T-18 ratios. The inset shows a zoom-in of the acceptor Rh-DHPE peak. Solid lines and dashed lines represent the spectra of the FRET system before and after adding Triton X-100, respectively. Lipids were mixed with U4T-18 at different molar ratios (5000, 1000, 100, 62.5).
Table 2.2 The acceptor/donor fluorescence intensity ratios (I592/I520) at different lipid/U4T-18
ratios.
Lipid : U4T-18 ratio
U4T-18:liposome
ratio I592/I520 FRET system
5000 8 0.22
1000 38 0.24
100 380 0.31
42
2.3 Conclusion
In conclusion, we proposed a powerful new approach employing lipid-DNA which contains four lipid chains modified nucleobases to tightly anchor the nucleotide to the lipid membrane. The incorporation and stability of lipid-DNA on the liposomal membrane were proved by FRET. FRET was achieved when the hybridization occurred between Cr-ATTO488 and U4T-18, which brought the donor (Cr-ATTO488) close to the acceptor (rhodamine) that was positioned in a U4T-18 functionalized membrane. Meanwhile, the I592/I520 (acceptor/donor peak) ratio increased markedly
with higher U4T-18 densities in the membrane, and disruption of vesicles by addition of Triton X-100 resulted in a drop of FRET vesicles system, confirming that FRET was indeed caused by bringing the donor in close vicinity to the acceptor dye located in the liposomal membrane. Finally, the lipid–DNA remained stably anchored in the liposomes for at least 24 hours.
2.4 Experimental Section
2.4.1 Materials
Cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids (Alabaster, USA) (Fig 2.8A-C, purity >99%) and used without further purification. Headgroup-labeled phospholipid, Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (Rh-DHPE) was purchased from Invitrogen (Amsterdam, Netherlands), and used as received (Fig.
2.8D). The DNA-dye conjugate Cr-ATTO488 was purchased from
Biomers.net GmbH (Ulm, Germany). Trition X-100 (10% in water), and Tris/HCl buffer were purchased from Sigma-Aldrich (St. Louis, United States). Anhydrous CHCl3 was purchased from Acros Organics (Geel,
Belgium) and stored over molecular sieves. For all experiments, ultrapure water (specific resistance > 18.4 MΩ cm) was obtained by a Milli-Q water purification system (Sartorius).
43
Fig. 2.8 Structures of lipids: (A) DOPC, (B) DOPE, (C) Cholesterol; fluorescent lipids: (D)
Rh-DHPE.
2.4.2 Synthesis and characterization of amphiphilic oligonucleotides
The synthesis of 5-(dode-1-cynyl) deoxyuracil and 5-(dode-1-cynyl) deoxyuracil phosphoramidite were reported previously (Fig. 2.9).10,11 Inshort, the modified uracil phosphoramidite was dissolved in CH3CN to a
final concentration of 0.15 M in the presence of 3 Å molecular sieves and the prepared solution was directly connected to a DNA synthesizer (ÄKTA oligopilot plus, GE Healthcare (Uppsala, Sweden)). Oligonucleotides were synthesized on a 10 μmol scale using standard β-cyanoethylphosphoramidi -te coupling chemistry. Deprotection and cleavage from the PS support was carried out by incubation in concentrated aqueous ammonium hydroxide solution for 5 h at 55 °C. Following deprotection, the oligonucleotides were purified by using reverse-phase chromatography, using a C15 RESOURCE RPCTM 3 mL reverse phase column (GE Healthcare) through a custom gradient elution (A: 100 mM triethylammonium acetate (TEAAc) and 2.5% acetonitrile, B: 100 mM TEAAc and 65% acetonitrile). Fractions were
44
desalted using centrifugal dialysis membranes (MWCO 3000, Sartorius Stedim). Oligonucleotide concentrations were determined by UV absorbance using extinction coefficients. Finally, the identity and purity of the oligonucleotides was confirmed by RPC-HPLC (Fig. 2.10) and MALDI-TOF mass spectrometry (Fig. 2.11).
Fig. 2.9 Synthesis of 5-(dode-1-cynyl) deoxyuracil 2 and 5-(dode-1-cynyl) deoxyuracil
phosphoramidite 3.
45
Fig. 2.11 RPC HPLC analysis of purified lipid-DNAs: (A) U4T-18, (B) CU4T-18 and (C) CrU4T-18.
Numbers beside the elution peaks represent the buffer B contents when lipid-DNAs were eluted.
2.4.3 Preparation and characterization lipid-DNA liposomes
An appropriate amount of freeze-dried lipid-DNA was mixed with DOPC:DOPE:Cholesterol (50:25:25 mol% in chloroform), to obtain the required lipid:lipid-DNA ratio. Afterwards, chloroform was removed by evaporation under an air stream and then under vacuum overnight. An aqueous buffer (100 mM NaCl, 20 mM Tris, pH 7.5) was added to the flask and the solution was vortexed and freeze-thawed 5 times. Subsequently, the dispersion was extruded 21 times, using an extruder and 100 nm polycarbonate membranes (Whatman), to obtain unilamellar vesicles. After extrusion, external buffers of each sample were removed by size exclusion chromatography. The column was filled with Sephadex G-75 (GE Healthcare Life Sciences) and equilibrated with buffer (100 mM NaCl, 20 mM Tris, pH 7.5). Lipid-DNA liposomes were used within one day. All liposomal formulations had an average diameter of around 130 nm as determined by DLS (ALV/CGS-3 ALV-Laser Vertriebsgesellschaft mbH, Langen, Germany). The ratio between lipid and U4T-18 was 500:1, unless stated otherwise.
46
2.4.4 Calculation of lipid-DNA/liposome ratio.
The amount of lipid-DNAs per liposome was calculated using the equation:
where Φ is the number of lipids per liposome which can be calculated from geometrical considerations:
where Souter and Sinner are the outer and inner surface area of the spherical
liposomes. Assuming the thickness of the lipid bilayer is 5 nm.14,15 α is the
average cross-sectional area of the lipid headgroups, which is assumed to be (2*80+65)/3=75 Å for DOPC:DOPE(2:1 molar ratio).16 Router is the
averaged radius of spherical liposomes, which was determined by DLS.
2.4.5 Characterization of lipid-DNA incorporation in liposomes
measured by Fluorescence Resonance Energy Transfer (FRET) assay
Fluorescence emission spectra of Cr-ATTO488 (donor) and Rh-DHPE (acceptor) in the 500–700 nm region were recorded with excitation at 470 nm using a SPECTRAMAX M2 (Molecular Devices) fluorescence spectrophotometer. Measurements were carried out at constant temperature of 25.0 °C, using a 100 mM NaCl, 20 mM Tris, pH 7.5 buffer.2.4.6 FRET assay via DNA hybridization
U4T-18 was incorporated in Rh-DHPE/(DOPC+DOPE) (3:97 molar ratio) liposomes to obtain U4T-18 liposomes with a lipid to U4T-18 ratio of 500:1. Subsequently, an aliquot of these liposomes was mixed with a small amount of Cr-ATTO488 such that [U4T-18] = [Cr-ATTO488] = 0.906 μM and with a final lipid (DOPC+DOPE) concentration of 0.45 mM. Then, U4T-18 and Cr-ATTO488 were hybridized using an Eppendorf Mastercycler (Germany). The protocol consisted of heating the mixture 15 min to 40 °C
47 and slowly cooling to 4 °C over a period of 140 min. Afterwards, the emission spectra of Cr-ATTO488/Rh-DHPE pair were measured.
Author contributions
Meng Z designed and conducted the experiments, performed data analysis and wrote the manuscript. Liu Q and de Vries JW synthesized lipid-DNA. Herrmann A supervised the project.
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