Combining QD-FRET and microfluidics to monitor DNA nanocomplex self-assembly in real-time.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships.

Protocol

A. Biotinylation of DNA

Plasmid DNA were covalently biotinylated with guanine-specific biotin labels as described by the manufacturer (Mirus Bio, Madison, WI) but scaled to have ~1-2 biotin labels per DNA. Plasmid DNA (pEGFP-C1, 4.9 kb, Clontech, Mountain View, CA) was labeled in this protocol.

Dissolve den class="Chemical">sired amount of pDNA into TE buffer of DNase-free anpan>d RNase-free (molecular biology-grade quality) pan> class="Chemical">water to make a 1μg/μL DNA solution.

Conduct the labeling reaction un class="Chemical">sing the following reactionpan> mixtures. Add the LabelIT reagent last.

For 100μg Dn class="Chemical">NA reactionpan>:

Incubate the reaction at 37 °C for 1 hour.

Purify the labeled sample by n class="Chemical">ethanol or pan> class="Chemical">isopropanol precipitation following standard protocols.

Note: Gel filtrationpan> based columnpan>s may lead to high UV absorbance or fluorescence background, which may affect the Dn class="Chemical">NA quantification or fluorescence characterization. Note: The level of biotinylation may be determined by HABA-based tests.

B. Labeling of the Cy5-Cationic Polymer

Chitosan (390 kDa, 83.5% deacetylated, Vanson, Redmond, WA) was used as a model cationic n class="Chemical">polymer in this study. The free primary pan> class="Chemical">amines on the chitosan polymer backbone were labeled with Cy5-NHS (Amersham Biosciences, Piscataway, NJ).

To facilitate complete conjugation of n class="Chemical">Cy5 dye, calculate the required amount of pan> class="Chemical">Cy5-NHS such that the molar ratio of Cy5 : primary amines is 1 : 200.

Adjust pH of the chitosan solution (in 25mM n class="Chemical">acetate buffer) to ~6.5 by additionpan> of pan> class="Chemical">NaOH. Note that the NHS reaction is more efficient at basic pH, but the solubility of chitosan here limits the working pH range.

While stirring, slowly add the calculated amount of n class="Chemical">Cy5-NHS (1 mg/ml pan> class="Chemical">DMSO) to the chitosan solution in a drop-by-drop manner.

Agitate the mixture in the dark at room n class="Gene">temperature overnpan>ight.

To purify, dialyze with 10k MWCO Slide-a-Lyzer (Pierce) for 2 hr against 1% n class="Chemical">acetate buffer at room n class="Gene">temp in the dark.

Replace buffer and dialyze another 2 hr at room n class="Gene">temp in the dark.

Replace buffer and dialyze overnight at 4 °C in the dark.

Store purified labeled n class="Chemical">polymer at -20 °C.

Note: In this study, a standard curve is constructed by measuring the emission intensity of Cy5-NHS ester at 670 nm. Characterize the labeling density by measuring the obtained emission at 670 nm from Cy5-labeled chitosan in the standard curve. Absorbance may also be used to determine the labeling efficiency but was not performed here.

C. Preparation of QD-labeled DNA and Cy5-Polymer

The molar ratio of pDNA to QD was kept in excess (pDpan> class="Chemical">NA : QD ≈ 1 : 2) to ensure complete conjugation of QDs to pDNA. The number of QDs labeled onto each pDNA can be estimated through TEM imaging or other equivalent facilities. In our study, the number of QDs per pDNA is estimated to be ~1-3 by TEM and single molecule spectroscopy.1 Use Millipore Milli-Q gradient water (>18.0 MW, 0.2um filtered) during the preparation.

Calculate the required amount of chitosan for 10μg pDNA according to den class="Chemical">sired N/P ratio, the theoretical ratio of protonpan>ated pan> class="Chemical">amines in the chitosan solution to the negative phosphates in the DNA solution.

Add streptavidin-functionalized 605QDs (Qdot 605 n class="Gene">ITK, Invitrogen, Carlsbad, CA) into the n class="Chemical">biotinylated pDNA solution.

Incubate the solution at room n class="Gene">temperature inpan> the dark for 15 minpan>.

Add the QD-labeled Dn class="Chemical">NA into 50 mM pan> class="Chemical">sodium sulfate solution to make the final volume 200μL.

Dilute n class="Chemical">Cy5-chitosan, according to depan> class="Chemical">sired N/P ratio, with Milli-Q water to make the final volume 200μL.

Note: Keep the reaction in dark to prevent possible photobleaching. Important: Be cautious to use the Qdot 605 ITK™ streptavidin conjugate (the ITK series), as Quantum dots in this catalog are designed for the purpose of FRET. The regular Qdot series are conjugated with a PEG layer to prevent non-specific binding, especially for cellular labeling. However, this additional coating enlarges the donor-acceptor distance, resulting in reduced energy transfer efficiency.

n class="Chemical">Si wafer is piranpan>ha cleanpan>ed anpan>d pan> class="Chemical">baked at 200 °C for 5 min.

For the den class="Chemical">signated master thickness of 25μm, pan> class="Gene">spin coat the negative photoresist (SU-8 2025, Microchem, Newton, MA) on Si wafer at 2000 rpm for 30 sec.

Soft n class="Chemical">bake the wafer onpan> a hotplate with a ramp of 65 °C/hr to 95 °C.

Expose to UV light (365nm) for 250mJ/cm2 through a mask film (CAD/Art Services, Bandon, OR) containing the den class="Chemical">sign of microchanpan>nels.

Post-exposure n class="Chemical">bake the wafer onpan> a hotplate with a ramp of 65 °C/hr to 95 °C.

Develop the wafer un class="Chemical">sing pan> class="Chemical">SU-8 photoresist developer.

The patterned wafer is hard n class="Chemical">baked onpan> a hotplate with a ramp of 65 °C/hr to 200 °C. Maintain the wafer at 200 °C for at least 5 hours, then gradually cool the wafer down to room n class="Gene">temperature.

D. Fabrication of the SU-8 Masters Using Standard Photolithography

Important: Gradual ramping during the n class="Chemical">SU-8 master baking process is necessary, otherwise the pan> class="Chemical">SU-8 structure may detached from the silicon wafer or cracks on the SU-8 structure may be induced by stress-release.

The n class="Chemical">SU-8 master is placed inpan> a weighinpan>g boat.

Mix n class="Chemical">silicone elastomer anpan>d curing agent (pan> class="Chemical">Poly(dimethylsiloxane), PDMS, Sylgard 184, Dow Corning, Midland, MI)  in a 10 : 1 ratio.

Pour the PDMS mixture onto the n class="Chemical">SU-8 master anpan>d leave the weighing boat in a vacuum depan> class="Chemical">siccator to remove bubbles.

Cure the PDMS at 65 °C for 1-2 hours.

Peel the PDMS strip from the n class="Chemical">Si master mold.

Punch channel inlets and outlets of the fluidic device.

Clean the PDMS strip and cover glass with n class="Chemical">ethanol anpan>d then air-dry.

Treat the cleaned PDMS strip and cover glass with n class="Chemical">oxygen plasma (20W for 1min).

Immediately bond the PDMS strip with cover glass.

Leave the bonded microfluidic chip in the oven at 95 °C for overnight.

E. Replica Molding of PDMS from the masters and Bonding to the Cover Glass

Important: Plasma treatment and overnight baking are essential to enhanced bonding strength.

Fill the microfluidic channel with n class="Chemical">water (to ensure there is no bubbles within the microfluidic chanpan>nel), before loading the reagents to ensure smooth flow during the experiment.

Load the QD-labeled Dn class="Chemical">NA anpan>d pan> class="Chemical">Cy5-labeled chitosan solutions into two individual glass syringes, through the tubing described in the video.

Connect the tubing with the two inlets of microfluidic devices. Be cautious not to introduce any air during the process. Set the flow rate at 20nL/min (PHD-2000 syringe pump, Holliston, MA), under laminar flow conditions.

Check the microchannels under the microscope.

When the flow is stable (~15 to 20 minutes), QD-mediated FRET should be observed in the center of the channel.

Take fluorescence pictures (Cooled CCD, Qimaging, BC, Canada) at different locations along the channel.

Analyze the fluorescence images with ImageJ and OriginLab.

F. Monitor the formation of DNA Nanocomplexes In the Microfluidic Device

Figure 1. QD-FRET provides a senn class="Chemical">sitive indicationpan> of the onpan>set of DNA Nanpan>ocomplexes self-assembly

Quantum dot-mediated fluorescence resonance energy transfer (QD-FRET) can provide a quantitative and highly sensitive indication of polyplex stability in either extra- or intra-cellular environments, allowing for unambiguous detection of the onset of interactions between DNA and the gene carrier. The FRET pair, 605QD and Cy5, was chosen based on maximizing spectral overlap between the donor and acceptor and minimizing potential cross-talk.  For this pair, the Förster distance is 69.4Å.3

Self-assembly of the QD-FRET DNA nanocomplexes. Anionic plasmid DNA (pDNA) and the cationic gene carrier were labeled with QD (energy donor) and Cy5 (energy acceptor), respectively. QD-FRET nanocomplexes were formed through electrostatic complex coacervation. Upon excitation at 488 nm, QD-FRET-mediated Cy5 emission indicated formation of a compact and intact nanocomplex. The residence time (tR) can be calculated according to the distance (x) which measures from where the two streams meet to the position of reaction under investigation, and the mean flow speed (v). Due to the nature of laminar flow, mixing only takes place at the interface (center of each image), allowing precise calculation of mass transport as a function of tR. Temporal resolution can be adjusted by varying the applied flow rates. (Inset) FRET-mediated signal was observed immediately at the interface when the two streams met, indicating that binding was rapid, occurring within a few milliseconds according to the applied flow rates. Scale Bar: 100μm.

n class="Chemical">Signpan>ificanpan>ce of our work:

This is the first atn class="Gene">tempt to monpan>itor pan> class="Chemical">polymeric DNA nanocomplex self-assembly kinetics in real-time (millisecond resolution) through QD-FRET responses within a simple microfluidic chip.

QD-mediated FRET provides a highly senn class="Chemical">sitive anpan>d quanpan>titative indicationpan> of the onpan>set of molecular interactionpan>s anpan>d throughout the self-assembly process, whereas microfluidics offers a well-conpan>trolled microenvironpan>ment to spatially anpan>alyze the process during the DNA nanpan>ocomplex synthen class="Chemical">sis.

The integration of microfluidics and nanophotonics suggests a new and interesting approach to investigate any type of complexation reactions.

The resulting QD-FRET n class="Chemical">polymeric DNA nanpan>ocomplexes could be readily applied for establishing structure-functionpan> relationpan>ships.1,2

Disclosures

The authors have nothing to disclose.

DNase-free and RNase-free water	75 μL
10X Labeling Buffer A	20 μL
1μg/μL DNA	100 μL
LabelIT reagent	5 μL
Total Volume	200 μL