Competitive Assembly To Increase the Performance of the DNA/Carbon-Nanomaterial-Based Sensing Platform

Increasing the rate of target binding on the surface and enhancing the fluorescence signal restoration efficiency are critical to the desirable biomedical application of carbon nanomaterials, for example, single-walled carbon nanotubes (SWNTs). We describe here a strategy to increase the target binding rate and enhance the fluorescence signal restoration efficiency on the DNA-functionalized SWNT surface using a short complementary DNA (scDNA) strand. The scDNA causes up to a 2.5-fold increase in association rate and 4-fold increase in fluorescence signal restoration by its competitive assembly on the nanostructure’s surface and inducing a conformational change that extends the DNA away from the surface, making it more available to bind target nucleic acids. The scDNA-induced enhancement of binding kinetics and fluorescence signal restoration efficiency is a general phenomenon that occurred with all sequences and surfaces investigated. Through this competitive assembly strategy of scDNA, performance improvement of the carbon-nanomaterial-based biosensing platform for both in vitro detection and live cell imaging can be reached

HSV-2 infection of BCBL-1 cells results in release of infectious virions of KSHV.

<p>(<b>A</b>). <b>Real-time DNA-PCR analysis for the viral copy number of KSHV.</b> BCBL-1 cells were infected with HSV-2 or Mock. At 72 hours postinfection, supernatant virus was collected, concentrated, and used for real-time DNA-PCR analysis. Results shown were from three independent experiments performed in triplicate. ** <i>p</i><0.01 for Student's t-test versus mock group. (<b>B</b>). <b>RT-PCR analysis for ORF26 mRNA in BCBL-1 or HEK293 cells treated with supernatant from HSV-2-infected BCBL-1 cells.</b> ORF26 mRNA expression in BCBL-1 cells treated with TPA (<b>Lane 1</b>), HEK293 cells treated for 48 hours with supernatant from TPA-treated BCBL-1 cells for 72 hours (<b>Lane 2</b>), HEK293 cells cultured alone for 27 hours (<b>Lane 3</b>), HEK293 cells infected with HSV-2 for 27 hours (<b>Lane 4</b>), HEK293 cells treated for 27 hours with supernatant from Mock-treated BCBL-1 cells for 72 hours (<b>Lane 5</b>), and HEK293 cells treated for 27 hours with supernatant from HSV-2-infected BCBL-1 cells for 72 hours (<b>Lane 6</b>) were detected by RT-PCR. (<b>C</b>). <b>Western blot for detection of vIL-6 expression in BCBL-1 cells or HEK293 cells treated with supernatant from HSV-2-infected BCBL-1 cells.</b> vIL-6 expression in BCBL-1 cells treated with TPA (<b>Lane 1</b>), HEK293 cells treated for 48 hours with supernatant from TPA-treated BCBL-1 cells for 72 hours (<b>Lane 2</b>), HEK293 cells cultured alone for 27 hours (<b>Lane 3</b>), HEK293 cells infected with HSV-2 for 27 hours (<b>Lane 4</b>), HEK293 cells treated for 27 hours with supernatant from Mock-treated BCBL-1 cells for 72 hours (<b>Lane 5</b>), and HEK293 cells treated for 27 hours with supernatant from HSV-2-infected BCBL-1 cells for 72 hours (<b>Lane 6</b>) were detected by Western blot.</p

Sets of primers of HSV-2 used for RT-PCR.

<p>The oligonucleotides were selected from the sequences with the indicated accession number. The size of each amplified product, its annealing temperature, and numbers of PCR cycles are indicated. F, forward; R, reverse.</p

Elevating NF-κB signaling inhibits HSV-2-induced KSHV replication.

<p>(<b>A</b>). <b>Transfection of IKK₂EE promoted the binding activity of P65 of NF-κB pathway in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Cells were collected and nuclear proteins were extracted for ELISA-based NF-κB activity assay. Excess competitor oligonucleotides were coincubated with the nuclear protein of MIGRI-transfected and HSV-2-infected BCBL-1 cells for competition assays (HSV-2+MIGRI+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus Mock+MIGRI group, respectively; ^##<i>p</i><0.01 and ^&<i>p</i><0.01 for Student's t-test versus HSV-2+MIGRI group, respectively. (<b>B</b>). <b>Transfection of IKK₂EE promoted HSV-2-induced activity of NF-κB in BCBL-1 cells.</b> BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were co-transfected with the plasmid IKK₂EE and an NF-κB-dependent firefly luciferase reporter construct (0.4 µg) together with a Renilla luciferase construct pRL-TK (0.02 µg) for 12, 24 and 48 hours. Whole-cell extracts were prepared, and the firefly as well as the Renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean ± s.e.m is shown. ** <i>p</i><0.01 and ^##<i>p</i><0.01 for Student's t-test versus Mock+MIGRI and HSV-2+MIGRI groups, respectively. (<b>C</b>). <b>Transfection of IKK₂EE inhibited ORF26 mRNA expression in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Cells were collected and ORF26 mRNA expression in BCBL-1 cells was measured by RT-qPCR. Relative quantities of ORF26 expression represented on the y-axis. Results shown were from three independent experiments performed in triplicate. ** <i>p</i><0.01 for Student's t-test versus Mock+MIGRI group; ^#<i>p</i><0.05 and ^##<i>p</i><0.01 for Student's t-test versus HSV-2+MIGRI group, respectively. (<b>D</b>). <b>Transfection of IKK₂EE inhibited vIL-6 expression in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (<b>E</b>). <b>Real-time DNA-PCR analysis for the viral copy number of KSHV.</b> BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI. At 24 and 48 hours postinfection, supernatant virus was collected, concentrated, and used for real-time DNA-PCR analysis. Results shown were from three independent experiments performed in triplicate. ** <i>p</i><0.01 and ^##<i>p</i><0.01 for Student's t-test versus Mock+MIGRI and HSV-2+MIGRI groups, respectively.</p

Inhibition of NF-κB signaling enhances HSV-2-induced KSHV replication.

<p>(<b>A</b>). <b>HSV-2 infection elevated the P65 level of NF-κB pathway in BCBL-1 cells.</b> BCBL-1 cells were infected by HSV-2 or Mock for 12, 24 and 48 hours. Then cells were collected and nuclear proteins were extracted for ELISA-based NF-κB activity assay. Excess competitor oligonucleotides were coincubated with the nuclear protein of HSV-2-infected BCBL-1 cells for competition assays (HSV-2+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. ** <i>p</i><0.01 and ^##<i>p</i><0.01 for Student's t-test versus Mock and HSV-2 groups, respectively. (<b>B</b>). <b>HSV-2 infection increased NF-κB activity in BCBL-1 cells.</b> BCBL-1 cells adsorbed with HSV-2 or Mock for 1 hour, then were transfected with an NF-κB-dependent firefly luciferase reporter construct (0.4 µg) together with a Renilla luciferase construct pRL-TK (0.02 µg) for 12, 24 and 48 hours. Whole-cell extracts were prepared, and the firefly as well as the Renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean ± s.e.m is shown. ** <i>p</i><0.01 for Student's t-test versus Mock group. (<b>C</b>). <b>A pharmacologic inhibitor of IKK–NF-κB activation, Bay 11-7082, inhibited the binding activity of P65 of NF-κB pathway in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 12, 24 and 48 hours. Then cells were collected and nuclear proteins were extracted for ELISA-based NF-κB activity assay. Excess competitor oligonucleotides were coincubated with the nuclear protein of DMSO-pre-treated and HSV-2-infected BCBL-1 cells for competition assays (HSV-2+DMSO+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. ** <i>p</i><0.01 for Student's t-test versus Mock+DMSO group; ^##<i>p</i><0.01 and ^&<i>p</i><0.01 for Student's t-test versus HSV-2+DMSO group, respectively. (<b>D</b>). <b>Bay 11-7082 inhibited HSV-2-induced activity of NF-κB in BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 1 hour and then were transfected with an NF-κB-dependent firefly luciferase reporter construct (0.4 µg) together with a Renilla luciferase construct pRL-TK (0.02 µg) for 12, 24 and 48 hours. Whole-cell extracts were prepared, and the firefly as well as the Renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean ± s.e.m is shown. ** <i>p</i><0.01 and ^##<i>p</i><0.01 for Student's t-test versus Mock+DMSO and HSV-2+DMSO groups, respectively. (<b>E</b>). <b>Bay 11-7082 enhanced ORF26 mRNA expression in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 12, 24 and 48 hours. ORF26 mRNA expression in BCBL-1 cells was measured by RT-qPCR. Relative quantities of ORF26 expression represented on the y-axis. Results shown were from three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus Mock+DMSO group, respectively; ^#<i>p</i><0.05 and ^##<i>p</i><0.01 for Student's t-test versus HSV-2+DMSO group, respectively. (<b>F</b>). <b>Bay 11-7082 enhanced vIL-6 expression in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 12, 24 and 48 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (<b>G</b>). <b>Real-time DNA-PCR analysis for the viral copy number of KSHV.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection. At 24 and 48 hours postinfection, supernatant virus was collected, concentrated, and used for real-time DNA-PCR analysis. Results shown were from three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus Mock+DMSO and HSV-2+DMSO groups, respectively.</p

Expression of KSHV lytic cycle RNA and protein in HSV-2-infected BCBL-1 cells.

<p>(<b>A</b>). <b>Rta mRNA expression in HSV-2-infected BCBL-1 cells.</b> Rta mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 3, 6, 12, 24, 48, 72 and 96 hours was quantitated by RT-qPCR. Relative quantities of Rta expression represented on the y-axis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031652#s2" target="_blank">Results</a> shown were from three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus mock group, respectively. (<b>B</b>). <b>ORF26 mRNA expression in HSV-2-infected BCBL-1 cells.</b> ORF26 mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 3, 6, 12, 24, 48, 72 and 96 hours was measured by RT-qPCR. Relative quantities of ORF26 expression represented on the y-axis. Results shown were from three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus mock group, respectively. (<b>C</b>). <b>RT-PCR analysis for ORF57 and vIL-6 mRNA in BCBL-1 cells infected with HSV-2.</b> ORF57 and vIL-6 mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 24, 48, 72 and 96 hours was detected by RT-PCR. β-Actin was readily detectable in all samples indicating the presence of amplifiable cDNA. (<b>D</b>). <b>Rta protein expression in BCBL-1 cells infected by HSV-2.</b> BCBL-1 cells were infected with HSV-2 or mock for 24, 72 and 96 h and whole-cell lysates were subjected to Western blot with Rta antibody. The membrane was stripped and reprobed with anti-actin antibody as a loading control. Results shown are from a representative experiment of three independent experiments with similar results. (<b>E</b>). <b>Immunofluorescence assay staining of BCBL-1 cells infected with HSV-2 (original magnification, 40×).</b> KSHV lytic protein ORF59 expression in normal BCBL-1 cells (untreated) and BCBL-1 cells infected with mock or HSV-2 for 24 and 72 h was detected by immunofluorescence assay staining with ORF59 MAb. (<b>F</b>). <b>Quantification of results in</b> (<b>E</b>). Quantitative and statistic methods were described in the text. ** <i>p</i><0.01 for Student's t-test versus mock group.</p

HIV-1 Tat cooperates with HSV-2 to induce KSHV lytic cycle replication.

<p>(<b>A</b>). <b>Effect of Tat on ORF26 mRNA expression in HSV-2-infected BCBL-1 cells.</b> Real-time quantitative PCR was performed to detect KSHV ORF26 mRNA expression in Mock-treated and pcDNA3.1-transfected BCBL-1 cells (<b>Mock+pcDNA</b>), Mock-treated and pTat-transfected BCBL-1 cells (<b>Mock+Tat</b>), or HSV-2-infected and pcDNA3.1-transfected BCBL-1 cells (<b>HSV-2+pcDNA</b>), HSV-2-infected and pTat-transfected BCBL-1 cells (<b>HSV-2+Tat</b>) for 6, 24, 48 and 72 hours. The results shown were from three independent experiments performed in triplicate. ** <i>p</i><0.01 for Student's t-test versus HSV-2+pcDNA group. (<b>B</b>). <b>Effect of Tat on vIL-6 protein expression in HSV-2-infected BCBL-1 cells.</b> Cell lysates were collected from HSV-2-infected and pcDNA3.1-transfected BCBL-1 cells, or HSV-2-infected and pTat-transfected BCBL-1 cells for 48 and 72 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (<b>C</b>). <b>Effect of Tat on Rta promoter activities in HSV-2-infected BCBL-1 cells.</b> BCBL-1, B95-8 and Vero cells were infected with Mock and then co-transfected with pcDNA3.1, p50-Luc and Renilla vector pRL-TK (<b>Mock+pcDNA3.1</b>), infected with HSV-2 and then co-transfected with pcDNA3.1, p50-Luc and Renilla vector pRL-TK (<b>HSV-2+pcDNA3.1</b>), or infected with HSV-2 and then co-transfected with pTat, p50-Luc and Renilla vector pRL-TK (<b>HSV-2+Tat</b>). Luciferase activities were measured, normalized to Rellina inner control, and presented as fold increase (<i>n</i>-fold). All data points were the averages of three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus HSV-2+pcDNA3.1 group, respectively. (<b>D</b>). <b>Effect of Tat on HSV-2 replication in BCBL-1 cells.</b> BCBL-1 cells were infected with Mock and then transfected with pcDNA3.1 (<b>Mock+pcDNA</b>), infected with HSV-2 and then transfected with pcDNA3.1 (<b>HSV-2+pcDNA</b>), or infected with HSV-2 and then transfected with pTat (<b>HSV-2+pTat</b>) for 24, 48 and 72 hours. Whole cell lysate were subjected to Western blot with the indicated antibody.</p

Primers used for RT-qPCR detection of KSHV genes (F, Forward; R, Reverse).

<p>Primers used for RT-qPCR detection of KSHV genes (F, Forward; R, Reverse).</p

Either miR-498 or miR-320d regulates KSHV replication in BCBL-1 cells without HSV-1 infection.

<p>(<b>A</b>)<b>. Transfection of miR-498 or miR-320d mimic inhibited KSHV RTA expression in TPA-stimulated BCBL-1 cells.</b> Western blot was used to detect the expression of RTA in BCBL-1 cells which were transfected with miR-498, miR-320d mimics or Neg. Ctrl. for 12 h, and stimulated with TPA for another 24 h. The relative level of RTA was determined by quantitative densitometry. Numbers labeled above the RTA band were the relative intensities of the bands compared to GAPDH. The relative level of RTA in the TPA+Neg. Ctrl. group was considered to be 1 for comparison. (<b>B</b>)<b>. Transfection of miR-498 or miR-320d mimic inhibited KSHV ORF21/57 mRNA expression in TPA-stimulated BCBL-1 cells.</b> ORF21 and ORF57 mRNA in BCBL-1 cells treated as in A was quantitated by RT-qPCR. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 for Student’s t-test versus TPA+Neg. Ctrl. group. (<b>C</b>)<b>. Transfection of miR-498 or miR-320d mimic inhibited KSHV progeny virions release in TPA-stimulated or unstimulated BCBL-1 cells.</b> Real-time DNA-PCR was used to detect the viral genome copy number in the supernatant of BCBL-1 cells, which were transfected with miR-498, miR-320d mimics or Neg. Ctrl. for 12 h, and treated with TPA for another 24 h (left columns) or without TPA (right columns). *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 for Student’s t-test versus Neg. Ctrl. group. (<b>D</b>)<b>. Transfection of LNA-498 or LNA-320d enhanced KSHV RTA expression in TPA-stimulated BCBL-1 cells.</b> Western blot was used to detect the expression of RTA in BCBL-1 cells transfected with LNA-498, LNA-320d or LNA-Scr. Ctrl. for 12 h, and stimulated with TPA for another 24 h. The relative level of RTA was determined by quantitative densitometry. Numbers labeled above the RTA band were the relative intensities of the bands compared to GAPDH. The relative level of RTA in the TPA+LNA-Scr. Ctrl. group was considered to be 1 for comparison. (<b>E</b>)<b>. Transfection of LNA-498 or LNA-320d enhanced KSHV ORF21/57 mRNA expression in TPA-stimulated BCBL-1 cells.</b> ORF21 and ORF57 mRNA in BCBL-1 cells treated as in D was quantitated by RT-qPCR. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 for Student’s t-test versus TPA+LNA-Scr. Ctrl. group<b>.</b> (<b>F</b>)<b>. Transfection of LNA-498 or LNA-320d enhanced KSHV progeny virions release in TPA-stimulated BCBL-1 cells.</b> Real-time DNA-PCR was used to detect the viral genome copy number in the supernatant of BCBL-1 cells treated as in D. ***<i>P</i><0.001 for Student’s t-test versus TPA+LNA-Scr. Ctrl. group.</p

Bioinformatics GO and pathway analysis based on miRNAs target genes.

<p>(<b>A–B</b>)<b>. GO analysis of the predicted target genes of miRNAs.</b> Only the top 23 significant GO terms for differentially up-(A) or downregulated (B) miRNAs were listed. The vertical axis was GO category and the horizontal axis was fold enrichment, which equaled (Count/Pop.Hits)/(List.Total/Pop.Total) and represented the significant level of GOs [Count: the number of differentially expressed (DE) genes associated with the listed gene ontology term; Pop.Hits: the number of background population genes associated with the listed gene ontology term; List.Total: the total number of DE genes; Pop.Total: the total number of background population genes<b>].</b> (<b>C–D</b>)<b>. KEGG pathway analysis based on miRNAs target genes.</b> The top significant pathways targeted by differentially up-(C) or downregulated (D) miRNAs were listed. The vertical axis was pathway category, the horizontal axis was enrichment score, which equaled [-log10(P value)] and represented the significant level of pathways.</p