FIG 4.

In vitro mRNA synthesis and labeled oligonucleotide hybridization. (A) Unlabeled DLPs were incubated with (top) or without (bottom) nucleotides to allow for mRNA production, followed by incubation with a primary VP6-specific antibody and a secondary Alexa 488-conjugated goat anti-mouse IgG. The samples were then incubated with the Atto 565 oligonucleotide probe pool and subsequently imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red) and 561-nm (green) channels are shown. White arrows, DLP/oligonucleotide colocalized particles (pseudocolored yellow); green arrows, DLP signal alone (pseudocolored red). (B) Scatter plot of the number of Atto 565 oligonucleotides colocalized with Alexa 488 signal from samples with (○) and without (□) nucleotides. From samples incubated with the required nucleotides, 7,765 Alexa 488-containing particles colocalized with 4 or more Atto 565 oligonucleotide probes, with an average of ∼20 probes per particle. Conversely, only 45 particles in samples incubated without nucleotides had any significant colocalized oligonucleotide signal. (C) Atto 647N-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with the Atto 565 oligonucleotide probe pool, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 640-nm (red) and 561-nm (green) channels are shown. (D) Probability density of 640-nm channel amplitudes. Detections with amplitudes less than 1,500 were not considered DLPs; detections above 4,000 were considered aggregates or multiple, unresolvable DLPs. (E) Scatter plot of the number of Atto 565 oligonucleotides colocalized with DLPs from samples with and without nucleotides. Of the 1,332 DLPs incubated with nucleotides, 1,291 (96.9%) colocalized with an average of ∼21 Atto 565 oligonucleotide probes (○), while 41 (3.1%) had no significant colocalization of probe. Of the 607 DLPs incubated without nucleotides, 602 (99.2%) had no significant colocalization of probe and 5 (0.8%) did colocalize (□). (F) Atto 488-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with both the Atto 565 and Atto 647N oligonucleotide probe pools, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red), 561-nm (green), and 640-nm (blue) channels are shown. (G) Probability density of 488-nm channel amplitudes derived from image analysis. Detections with amplitudes of less than 400 were not considered DLPs; detections above 1,500 were considered aggregates or multiple unresolvable DLPs. (H) Scatter plot of the number of Atto 565 and Atto 647N oligonucleotides colocalized with DLPs from the samples for panel D. Of a total of 1,126 analyzed DLPs incubated with nucleotides, 1,038 (92.2%) colocalized with both probes (○), 53 (4.7%) colocalized with only the Atto 565 probe (□, black), 14 (1.2%) colocalized with only the Atto 647N probe (△, black), and 21 (1.9%) had no significant colocalization of probe. Of 152 DLPs incubated without nucleotides analyzed, 144 (94.7%) had no significant colocalization of probe, 6 (3.9%) colocalized with only the Atto 565 probe (□, red), and 2 (1.3%), colocalized with only the Atto 647N probe (△, red). Amplitude detection and quantification of the number of labeled probes were performed as described in Materials and Methods.