Lineamientos académicos de RSU en la USTA

In the course of nanophage analyses by agarose gel electrophoresis, it was observed that the standard protocol for in vitro disassembly, heating the virions for 10 min at 70°C in a buffer containing 1% SDS, was not efficient in releasing DNA from the nanophage particles (data not shown). This indicated that the nanophage particles could be more stable to heat/SDS treatment than the full length helper virions, even though both are composed of identical coat proteins (pVIII, pVII, pIX, pIII and pVI) and were assembled within the same E. coli cell. To test this hypothesis, a time-course experiment was used to monitor disassembly of approximately equal number (2× 1012) full-length helper phage and nanophage at 70 °C in the presence of 1% SDS; one sample was also incubated at 100 °C. Disassembly of the helper (full-length) phage and nanophage virions was monitored through release of ssDNA, which was separated by agarose gel electrophoresis and visualized directly by staining with ethidium bromide (Figure 3.3 A). To identify the ssDNA remaining encapsidated inside the virions that resisted heat/SDS treatment, the virion proteins were stripped off the ssDNA by soaking the gel in an alkaline buffer (NaOH), followed by neutralization and re-staining of the gel by ethidium bromide (Figure 3.3 B).

When the full-length (helper) virions were analyzed, untreated samples did not contain any free ssDNA and all ssDNA was contained within the virion (Figure 3.3, compare the corresponding untreated sample lanes in gels A and B). At the first time-point (5 min) of incubation in SDS buffer at 70 °C, all full-length virion ssDNA was detected as a free form, and none within the virion (Figure 3.3 A). When the nanophage were subjected to the same analysis, some free DNA was observed at the time points 5 min – 20 min, however a large proportion was encapsidated and were only detected after the in situ disassembly (Figure 3.3, compare the 70 °C-treated sample lanes (5-20 min) in A vs. B). The amount of virion-encapsidated DNA decreased gradually between 5 min and 20 min time points, but was only completely eliminated by incubation at 100 °C. Conversely, the free DNA amount sharply increased at 100 °C, confirming that this is the only treatment that disassembles all nanophage virions (Figure 3.3, compare lane 12 in A vs. B). An unexpected free DNA band was detected in all heat-treated nanophage samples; this band did not increase in the intensity upon heating at 100 °C,

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hence this represents a virion population that has the same level of stability as the full- length virions. This band is not present in all nanophage samples (data not shown). A faint band of the same size was also detectable in the full-length phage preparation upon treatment at 70 °C (Fig. 3.3A). The full-length phage were purified from the same culture as the nanophage preparation, by the low-PEG precipitation. This is consistent with the helper phage giving rise to shorter particles or “miniphage” if a replicating phage in the culture undergoes duplication of the origin of replication (La Farina et al. (1987); data not shown). These shorter phage would have been enriched for in the nanophage fraction that was obtained by high-PEG precipitation, but not in the low-PEG fraction that poorly precipitates relatively short miniphage. Unequivocal identification of this DNA band requires further analysis by sequencing or Southern Blotting using appropriate probes. It is interesting that these miniphage particles, despite being relatively short, do not demonstrate high resistance to heating in SDS that the nanophage possess.

Overall, this experiment has demonstrated that the nanophage have superior resistance to heating in the presence of ionic detergent SDS in comparison to the full-length phage, given that a good proportion of the particles remains intact during prolonged incubation at 70 °C in the presence of 1% SDS. From the standpoint of understanding filamentous phage structure and physical properties, these findings indicate that the site of action for SDS is likely along the filament, and that perhaps imperfections of pVIII packing due to mechanical bending and twisting, which is observed in the full- length phage, but not the nanophage electron micrographs (Bennett, 2010), may play a role in this. From the technological standpoint, this property may be of interest to nanotechnology or diagnostic applications that involve harsh conditions, such as introduction of chemical modifications or use at high temperature in detergent- containing environment. Further investigation into stability under other conditions (e.g. pH extremes and organic solvents) would provide further information that would be valuable for the nanophage applications.

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Figure 3.3 Resistance of nanophage to heating in SDS vs. full-length (helper) phage. A. Free DNA, released from the virions by heat/SDS treatment. Virion-

encapsulated DNA cannot be detected, as it is not exposed to ethidium bromide. B. Same gel as in A, except that the DNA packaged inside the intact (undamaged) virions (in addition to the free DNA) was now visualised by in-gel virion disassembly, followed by staining in ethidium bromide.

Annotation: Treatments for the samples in each lane are indicated above the gel. Arrowheads along the sides of the gel point to the relevant bands of phage DNA: intact full-length or intact nano, the virion-encapsidated DNA inside the SDS-resistant virions; disassembled full-length and disassembled nano, DNA from the SDS/heat- disassembled virion fraction.

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