Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.

Materials The HEp-2 epithelial cell line was obtained from American Type Culture Collection (ATCC). RSV type A2 virus and anti-F protein monoclonal antibody were a gift from the laboratory of James Crowe, Vanderbilt University Medical Center. Oligonucleotides were synthesized by Biosearch Technologies. Fetal bovine serum, gentamicin/amphotericin B, OptiMEM media, quantum dot nanocrystals, cholera toxin B, dextran, LIVE/DEAD viability kit, Mitotracker Red, and secondary detection antibodies were from Invitrogen Corporation. Lab-Tek II chambered coverglass was from Nunc Inc. Gold nanoparticles were from Ted Pella, Inc. Cytoseal 60 was from Richard Allan Scientific. Goldenhance gold colloid detection kit was from Nanoprobes, Inc. Cytochalasin D and Dynasore were from Tocris Bioscience. Brefeldin A was from Epicentre Biotechnologies. Genistein was from Ascent Scientific. Accutase was purchased from Innovative Cell Technologies. All other chemicals were purchased from Sigma-Aldrich at the highest purity available.

Synthesis and characterization of hAuNP Hairpin

DNA used for coupling to the AuNP surface were synthesized with the following sequences: RSV: 5′-(SH)-TTT TTT TTT TCG ACG AAA AAT GGG GCA AAT ACG TCG-(Cy5)-3′, GAPDH: 5′-(SH)-TTT TTT TTT TCG ACG GAG TCC TTC CAC GAT ACC ACG TCG-(CAL Fluor Red 590)-3′. Bolded regions indicate sequences encoding specificity for target mRNAs, and regions in parenthesis indicate modifications of 5′ thiol and 3′ fluorophore conjugations. The RSV sequence chosen is based on a consensus sequence found in RSV genomic RNA, and has been targeted successfully using antisense therapies directed against RSV replication. 27 The GAPDH targeted sequence is based on exon 6 of the GAPDH coding region. Sequences for complementary targets for evaluation of hAuNP hybridization specificity were as follows: RSVcomp: 3′-TTT TTT TAC CCC GTT TAT TTT TT-5′, GAPDHcomp: 3′-TTT TTT GGT ATC GTG GAA GGA CTC TTT TT-5′. A negative control target sequence with similar C-G content of RSVcomp was synthesized as follows: 3′-AGT ATT GGA AGT CGT TCG AAA TTG ATC ACT CGC A-5′. DNA concentration was verified by UV absorbance at 260 nm. Prior to coupling of hairpin DNA to AuNP, lyophilized DNA was resuspended in 100 mM DTT, 0.2 mM phosphate buffer (pH = 8.3) and incubated for one hour at 25°C to reduce residual 5′ disulfide bonds. A solution of 450 nM DNA and 2.2325 nM 15 nm AuNP was prepared in nuclease-free water and incubated for 4 hours at 25°C protected from light. After 4 hours, the solution was buffered to 10 mM phosphate buffer (pH 7.0), and brought to 0.1 M NaCl, and 0.1% Tween-20. The hAuNPs were then incubated for an additional 4 hours at 25°C. The NaCl concentration was then adjusted to 0.2 M and hAuNP were incubated for an additional 4 hours at 25°C. Finally, the NaCl concentration was adjusted to 0.3 M and incubated for a final 4 hours at 25°C. The solution was then centrifuged at 13,200 rpm for 30 min and hAuNPs were resuspended in PBS. The supernatant was reserved for further analysis. The process of centrifugation and resuspension was repeated thrice. hAuNP concentration was measured by absorbance at 260 nm and 520 nm, using the extinction coefficient for 15 nm AuNP ε 520 = 3.64 × 10 8 M −1 cm −1 . Reserved supernatant was also measured for absorbance at 260 nm to determine unbound DNA concentration, enabling calculation of the number of hairpin DNA molecules per AuNP. To determine fluorescence spectra of hAuNP, a solution of 2 μM of RSVcomp, GAPDHcomp, or negative control sequence and 1.4 nM hAuNP specific for RSVcomp or GAPDHcomp was incubated for 1 hour at 70°C and subsequently at 25°C for 12 hours, protected from light. Emission was scanned using a Cary Eclipse fluorescence spectrophotometer from 500–800 nm in 1 nm increments. In a separate experiment, 1.4 nM hAuNP targeting RSV or GAPDH RNA as described above were incubated with increasing concentrations of complementary target RNA (RSVcomp, GAPDHcomp, or negative control sequence) as indicated at 37°C in PBS for 6 hours, and measured for fluorescence emission intensity using a SpectraMax M5 microplate reader (Molecular Devices) configured for hAuNP fluorescence. As a blank, a AuNP surface functionalized with a 10 thymine spacer with identical fluorophore on the 3′ end was used to correct for intrinsic background fluorescence of the quenched hAuNP. For measurement of hydrodynamic diameter, 0.25 nM solutions of AuNP, or hAuNP specific for RSVcomp or GAPDHcomp were resuspended in nuclease-free water and measured using dynamic light scattering (DLS) on a Malvern Nano ZS. Resistance of hAuNP to DNAse I degradation was assayed fluorimetrically by incubating a solution of 1.6 nM hAuNP in PBS with 50 mg/mL bovine serum albumin and 0.38 mg/L DNAse I for 120 minutes as previously described. 21 Fluorescence emission resulting from degradation of DNA and thus liberation of the fluorophore from the quenching effect of the AuNP surface was monitored throughout the duration of the reaction using a Biotek Synergy HT configured for reading hAuNP-specific emission in 1 min. increments.

Cell culture

HEp-2 cells were cultured at 37°C, 5% CO 2 in Opti-MEM supplemented with 2% FBS, 2% L-Glutamine, and 1% gentamicin/amphotericin B. For infection of HEp-2 with RSV, cells in log phase growth were counted using a hemacytometer with trypan blue exclusion. Cells were plated 24 hrs. prior to incubation with virus at varying multiplicities of infection (MOIs) as previously described. 26 Incubation of cells with RSV at the appropriate concentration was conducted with OptiMEM for at least 1 hour prior to assays.

Flow cytometric analysis

HEp-2 cells infected at an MOI ranging from 0 to 1 were incubated with 0.5 nM RSV and GAPDH-targeted hAuNP for 6 hours, rinsed thrice with PBS to remove unbound/uninternalized hAuNP, and detached using Accutase. Cells were assayed using a Becton Dickinson LSR II flow cytometer, monitoring emission of hAuNP using appropriate filter settings for CAL Fluor 590 or Cy5. In a separate experiment, cell viability was measured in similarly-treated cells using the LIVE/DEAD single color fixable green kit (Invitrogen Corp.) according to the manufacturer’s instructions. Data was analyzed using FlowJo 7.5 (Treestar Software). Pharmacologic and environmental inhibition of endocytosis All solutions for inhibition of HEp-2 endocytosis were sterilized with Pall Supor 0.22 μm filters prior to usage in cell culture assays. HEp-2 cells were seeded in 96-well microplates at a density of 5 × 10 4 cells per well. A subset of these wells was selected for RSV infection the following day, and cells were incubated for an additional 48 hrs. Cells were rinsed three times in serum-free DMEM, and were either untreated or pretreated with 100 μL volumes of DMEM containing nystatin/progesterone, sodium azide/2-deoxy-D-glucose, chlorpromazine, chloroquine, brefeldin A, dynasore, FBS, nocodazole, heparin, amiloride, genistein, cytochalasin D, or no additives at specified concentrations for 1 hour. The range of working concentrations of reagents was determined using values reported in the literature, or, when possible, empirically using known tracers of endocytosis: transferrin (clathrin-dependent), cholera toxin B (lipid-raft-dependent), or dextran (caveolar-depedent). An acceptable working concentration for hAuNP internalization assays was determined when the reagent inhibited internalization of the tracer by > 15% in HEp-2 cells as measured by microplate spectrophotometry. This concentration was used as the middle concentration in a range of 3 concentrations, to evaluate dose-dependent effects. Media was then aspirated and replaced with DMEM containing the same additives supplemented with RSV or GAPDH-targeted hAuNPs, and cells were incubated for 4 hrs. Following this period, cells were rinsed thrice in HBSS, fixed for 15 min. at room temperature with 4% paraformaldehyde in PBS, and analyzed for hAuNP fluorescence using a Molecular Devices Spectramax M5 microplate reader configured for hAuNP detection. As several solutions used to dissolve endocytosis study reagents were made in DMSO, cells were incubated with hAuNPs suspended in DMEM with the highest concentration of DMSO used (0.5%) to monitor solvent-induced effects. Cells were incubated in parallel with endocytosis study reagents, DMEM, and 1 μM calcein-AM as a viability indicator to ensure reagents did not induce significant (> 5%) cell death in any case as determined by fluorescence microscopy and automated image analysis using Image Pro Plus 5.1.

Confocal microscopy of hAuNP Uninfected HEp-2 cells or infected

HEp-2 cells (5 × 10 4 ) in Lab-Tek II chambered coverglass were incubated with 0.5 nM hAuNP for 6 hours. Prior to live cell imaging, cells were rinsed thrice and resuspended in PBS. Imaging was conducted using a Zeiss LSM 510 Meta laser scanning confocal microscope configured for hAuNP-specific emission. SYTO-13 was used as a nuclear counterstain, and in some experiments, Mitotracker Red live cell mitochondrial tracking reagent was used according to the manufacturer’s instructions. Cell viability was monitored in cultured cells processed in parallel using trypan blue to ensure > 95% viability throughout imaging experiments. For immunofluorescence and immunohistochemical analysis of hAuNP-treated cells, HEp-2 were fixed with 4% paraformaldehyde in PBS for 15 minutes at 25°C, then rinsed thrice with PBS containing 50 mM glycine. Cells were stained for RSV-specific F protein as previously described. 26 For detection of gold colloids in fixed cells, the Goldenhance LM gold labeling kit was used according to the manufacturer’s instructions. Samples were mounted in Cytoseal 60 and imaged using confocal microscopy.

Materials The HEp-2 epithelial cell line was obtained from American Type Culture Collection (ATCC). RSV type A2 virus and anti-F protein monoclonal antibody were a gift from the laboratory of James Crowe, Vanderbilt University Medical Center. Oligonucleotides were synthesized by Biosearch Technologies. Fetal bovine serum, gentamicin/amphotericin B, OptiMEM media, quantum dot nanocrystals, cholera toxin B, dextran, LIVE/DEAD viability kit, Mitotracker Red, and secondary detection antibodies were from Invitrogen Corporation. Lab-Tek II chambered coverglass was from Nunc Inc. Gold nanoparticles were from Ted Pella, Inc. Cytoseal 60 was from Richard Allan Scientific. Goldenhance gold colloid detection kit was from Nanoprobes, Inc. Cytochalasin D and Dynasore were from Tocris Bioscience. Brefeldin A was from Epicentre Biotechnologies. Genistein was from Ascent Scientific. Accutase was purchased from Innovative Cell Technologies. All other chemicals were purchased from Sigma-Aldrich at the highest purity available.

Flow cytometric analysis

The population of RSV-infected cells (MOI = 1) exhibiting RSV-specific hAuNP fluorescence was 95.3%, which is close to the theoretical fraction of cells that would be infected with RSV at this MOI (> 90%) ( Figure 2B ). RSV-specific hAuNP fluorescence in infected HEp-2 cells was dependent on the MOI ( Figure 2C ), consistent with measurements conducted with RSVcomp in buffer. The fluorescence of GAPDH-specific hAuNP, however, was similar in RSV-infected and uninfected cells. Neither RSV- or GAPDH-specific hAuNP were toxic to HEp-2 cells in culture when compared to untreated cells at the same MOI, although cell death attributable to increased RSV infection was observed as expected ( Figure 2D ). Flow cytometric analysis collectively indicated that hAuNP were specific for mRNA targets within cells, were efficiently taken up by cells in culture without the use of transfection reagents or cell permeabilization, and were not cytotoxic to cells. Furthermore, hAuNP exhibited target-dependent increases in fluorescence, which may enable the use of hAuNP for the quantitative determination of intracellular mRNA content. The consistent emission signal of GAPDH-targeted hAuNP in RSV-infected and uninfected cells suggests that hAuNP can be used for mRNA normalization and assay standardization as an internal control, similar to how GAPDH primers are utilized in RT-PCR. Furthermore, in addition to its role as an mRNA loading control, GAPDH hAuNP fluorescence emission in uninfected and infected cells serves as a control for the adverse effects of RSV on cells, which in response to infection exhibit marked changes in membrane permeability and integrity, in addition to cell death. 26 The consistent GAPDH-targeted hAuNP signal, therefore, also validates that hAuNP efficiently enter cells irrespective of viral infection, which will enable studies of mRNA localization for any molecular biology application which interrogates cellular mRNA dynamics in health and disease.

Immunofluorescence analysis

Emission from RSV-specific hAuNP was detected in RSV-infected cells, but not in uninfected cells ( Figure 4A-B ), consistent with flow cytometric analysis. Distribution of emission was associated with perinuclear patches in the cytoplasm ( Figure 4A ). Immunohistochemical staining of HEp-2 cells exposed to hAuNP, regardless of their target sequence, indicated that hAuNP were present throughout the cytoplasm in a perinuclear distribution, in RSV syncytia (multinucleated, fused cells) of infected cells ( Figure 4C , asterisk), or in normal uninfected cells ( Figure 4D ). The customized fluorescence emission profiles of hAuNP enabled multiplexed analysis of intracellular mRNAs and proteins ( Figure 5 ), with preservation of mRNA spatial localization afforded by hAuNP. RSV F protein was detected on cell membranes, in areas distinct from RSV mRNA localization ( Figure 5A–C ), whereas colocalization of RSV mRNA was frequently observed within several regions containing GAPDH mRNA ( Figure 5D ) and mitochondrial organelles ( Figure 5E ). Studies collectively indicate that hAuNP are nonspecifically-internalized by cells, but exhibit mRNA-specific emission, enabling imaging with high signal to background ratios. Uptake of hAuNP occurs irrespective of the presence or absence of target mRNA. Targeted hAuNP were observed to colocalize in HEp-2 cells with molecular beacons targeted toward the same transcript and introduced into cells using streptolysin O-based reversible membrane permeabilization as previously published ( Supplementary Figure 3 ). 14 hAuNP did not penetrate the nuclear compartment following translocation ( Supplementary Figure 4 ). As the design of the hAuNP construct provides for spatial localization of mRNA, new insights into RNA trafficking within the cell may be obtained. By customization of fluorescence emission from hybridized hAuNP, imaging can be extended toward multiple mRNA targets. Furthermore, multiplexed imaging of mRNAs and proteins such as subcellular organelles is possible, using a number of methods such as live cell organelle dye-labeled homing peptides, fluorescent protein encoding, and conventional immunofluorescence techniques. 39 – 42 This strategy for imaging specific RSV-associated sequences simultaneously with an internal control RNA such as GAPDH will enable the quantitation of distinct RSV mRNAs in the cell throughout the time course of RSV infection and replication, for development of targeted therapies and diagnostic tools. In addition, the amenability of hAuNP to customization will enable the imaging of multiple biological and disease-relevant genomic sequences within the cell in other applications. Ongoing work is directed toward the multiplexed imaging of cancer-specific mRNAs in tumor specimens.

Supplementary Material 1_si_001