II Milestone: Triplex hybridization

Several methods are used to analyze various aspects of triplex structures and confirm the formation of triple-stranded structures (RAJESWARI et al., 1992). Simple, conventional methods like UV absorption and calorimetric melting have been used for thermodynamic characterization of inter-molecular triplexes (RAJESWARI et al., 1992, JAIN and RAJESWARI, 2002, MCKENZIE et al., 2007, MCKENZIE et al., 2008, RAJESWARI, 2012). NMR and IR spectroscopy and X-ray analysis provide more detailed information on triplex formation (CEROFOLINI et al., 2014). In this

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thesis a triplex hybridization efficiency was assessed by three different methods (i) melting temperature measured by spectrophotometry, (ii) electrophoresis mobility shift assay to test the probe binding under an electric field, and finally (iii) a triplex in situ hybridization.

Tm (temperature of melting) is used to define the temperature of mid-transition for nucleic acids and their thermal denaturation. This experiment is a common tool to determine the stability of DNA structures (MERGNY and LACROIX, 2003). All nucleic acids absorb light at 260nm wavelength, due to the heterocyclic ring structure associated with each of the four bases (YAKOVCHUK et al., 2006).

Spectrophotometric analysis of the DNA structure is based on the fact that the optical density of a double helix is different from the absorbance of the same DNA molecule if the two helixes are separated one from the other. The same principle applies to a triple-stranded DNA, which has an absorbance lower than a TFO separated from the duplex target. The absorbance is related to the energy held by the molecular structure. If that molecule is melted, the absorbance is higher because there is more free energy. The temperature needed to melt a triplex DNA is much lower than the one needed for a duplex because Hoogsteen hydrogen bonds are weaker then Watson and Crick bonds.

Result indicate different tm for each probe, and is in relation to the amount of cytosine content for each TFO (HEGARAT et al., 2014). Highest tm is given by TFO1, with a lower concentration of cytosine (35%). These results are consistent with the repeated observations made by Plum et al. (PLUM et al., 1990, PLUM et al., 1995).

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The electrophoresis mobility shift assay (EMSA) is based on the different migration rate of duplex and triplex DNA in a non-denaturing gel in an electric field. Two dimensional electrophoresis has proved to be the method of choice for testing triplex formation in inter-molecular triplexes by labeling exclusively the TFO with radioactive nucleotides (MOSES et al., 1997).

Here a new method was developed to evaluate triplex hybridization binding as an adaptation of what is known in literature on electrophoretic gel shift (BOUTORINE and ESCUDE, 2007, BOUTORINE et al., 2013). It was decided to analyze the triplex electrophoresis shift using TFO probes linked to digoxigenin (DIG). DIG is often used in southern blot assays and can be detected via immuno-chemiluminescence. Use of DIG labeled probes allows low amounts of marked oligonucleotide and therefore raising the sensibility of the technique.

MgCl2 concentration is an important feature for hybridization. The range described in literature varies from 1 to 20 mM (ESCUDE et al., 1996, TORIGOE et al., 2001), but also depends on the buffer used (PBS, Hepes, NaCl, TBE, TE) (TORIGOE et al., 2001, TORIGOE et al., 2012). Experiments assessed in this thesis were made at MgCl2 concentration of 5, 10, and 20 mM in PBS buffer. Salt solutions are important not only for pH equilibrium, but also for molecular kinetics (FUNASAKI, 1979). Future experiments will also take into consideration a comparison of the buffers influence on triplex binding affinity because of a double influence. Salt solution is also influencing

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nanoparticle agglomeration, higher MgCl2–concentration increases AuNPs agglomeration and therefore a red shift in the SPR (ZABETAKIS et al., 2012).

The triplex electrophoresis mobility assay (EMSA) in figure 11 shows a positive result of TFO1 binding to male gDNA. TFO2 presents no triplex hybridization. TFO3 has a double band for male and female gDNA in all the groups. Different band intensity is visible at different MgCl2 concentration. The location of the band representing TFO3 is the same for XX and for XY gDNA, this may be interpreted as a triplex hybridization available on sequences present on the autosomes and not on the Y-chromosome or to off-targets hybridization.

Hybridization consistency is comparable to cytosine content for each probe. If TFO1 is seen as the most consistent probe, it can assume that about 30% of cytosine content is better for a triplex hybridization targeting. These findings are consistent with previous studies (ALUNNI‐FABBRONI et al., 1994, PLUM et al., 1995).

Why are the other bands not visible as shown on the Southern blot assay? First, it is possible that the TTS are there, but do not face the major groove of DNA. Then it is impossible to generate a triplex hybridization. Second, it could be due to a low number of repetitions and to a low sensitivity of the acquisition method.

Triplex FISH results demonstrate that the TFO1 has a higher efficiency for triplex formation. This is consistent with the results from the triplex electrophoresis mobility shift assay. Alone, the TFO1 has a positivity of 16% for sperm Y-chromosome

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carriers and 6% in those carrying X-chromosome. A percentage is lower for the TFO2 and 3, respectively of 8% and 11% in Y-population and 3 and 5% in X-one.

Triplex hybridization difference is not significant between Y- and X-bearing chromosome sperm population when TFO1, 2, and 3 are used separately. On the contrary, when used together, TFOs have a triplex hybridization significantly higher in Y-sperm population compared to the X- population, respectively 36% in Y and 15% in X.

It was not possible to detect 100% of Y-sperm population and this is due to different possibilities. First, not all triplex target sites may be available for TFO binding. This could be due to different patterns of methylation of protamine and / or to a different conformation of chromatin in the sperm. A second possibility may be represented by the buffer used, that would not guarantee the maximum efficiency of the links between TFO and DNA target. A higher hybridization rate can be achieved using TFO1, 2, and 3 together, as it is shown in fig 13, indicating improved efficiency can still be generated by increasing numbers of TTS. The idea of screening for only one target revealed inconvenient for gene detection purposes.

Taking together the result of EMSA and triplex FISH, there is consistency of the fact that TFO1 is the probe with a higher efficiency, and this is also consistent to the fact that low content of cytosine increases triplex hybridization (HEGARAT et al., 2014).

More probes with such characteristics have to be found.

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