Gold nanoparticle/LNA hybridization to gDNA and sorted sperm nuclear matrix

4. Results

4.4 III Milestone: Gold nanoparticle bio-conjugation – SPR analysis

4.4.2 Gold nanoparticle/LNA hybridization to gDNA and sorted sperm nuclear matrix

Figure 14: Spectrophotometric analysis of AuNPs-TFO surface plasmon resonance shift. Incubation of different male and female gDNA and AuNPs-TFO1. A)AuNPs/DNA=1,5;

B) AuNPs/DNA=2. Blue curves indicate AuNPs-TFO hybridization to male gDNA, while the red curve indicates the female gDNA. In black, the curve of only AuNPs-TFO. Small inner squares the peak position at different wavelength.

Different concentrations of gold nanoparticles conjugate were tested by co-incubation with male and female genomic DNA extracted from blood lymphocytes with different ratio between AuNPs conjugate and genomic target DNA (data not showed).

Difference in SPR absorbance was recorded when 16,9 µg/mL and 22,5 µg/mL of AuNP/TFO conjugate were incubated with 26 ng/mL of gDNA.

The absorbance peak of the sample AuNPs-TFO1 with male gDNA at a AuNPs/gDNA ratio of 1.5 (Figure 14 A) is of 11nm and with female gDNA is of 4nm.

The shift of the peak is of 7nm. The absorbance peak of the sample AuNPs-TFO1 with male gDNA at a AuNPs/gDNA ratio of 2 (Figure 14 B) is of 8nm while that with

female gDNA is of 4nm. The shift of the peak is of 4nm. The red shift deviation between the samples was not significant. (Analysis was performed by the Center for Nanointegration Duisburg-Essen (Cenide)).

Figure 15: Spectrophotometric analysis of AuNPs-TFO incubated with Y-and X-chromosome bearing sperm nuclear matrix. Blue curve indicates AuNPs-TFO hybridization to Y-chromosome sperm nuclear matrix, the red curve indicates the X-chromosome sperm nuclear matrix. In black, the curve of only AuNPs-TFO(LNA). Small inner square represents the peak position at respective wavelength.

The absorbance peak of the sample AuNPs-TFO1 with Y-chromosome bearing sperm was 534 nm while that with X-chromosome was 533 nm. The absorbance peak of AuNPs-TFO1 alone is of 524 nm. The difference between the peak Y- and X-chromosome bearing sperm hybridized with AuNPs-TFO1 is 1nm. The red shift deviation is not significant. Analysis was performed by the Center for Nanointegration Duisburg-Essen (Cenide).

54 5. Discussion

The aim of this thesis was to test oligonucleotide probes annealed to gold nanoparticles for specific recognition of the Y-chromosome in bovine sperm chromatin. The investigation was conceived because of the need of a method for gene detection in viable sperm that could represent an alternative method to current sperm sorting by flow cytometry. The oligonucleotide probes were needed for specific gene recognition and gold nanoparticles were used as a marker signal. The bio-conjugate was thought to hybridize to enriched target sequences (poly-purine sequences) on the Y-chromosome without DNA denaturation. The first part of the thesis described how the research for target sequences was carried out. After that, triplex hybridization was experimented using wet chemistry to understand the suitability of triplex formation in bovine chromatin sperm. In the last part of the thesis a prototype of the bio-conjugate was synthesized. The focus of conjugate analysis was a first study of the interaction between AuNPs/TFO and target DNA (free in solution or condensed in sperm chromatin).

This new approach aims to have the following characteristics:

• Sequence specific! Nucleotide base pairing chemistry is used to trigger gene detection. Hoogsteen chemistry allows both functions of sequence specificity and physiologic environment feasible to sperm viability.

• Reversible! Triplex forming oligonucleotides have a half-life approximately of 10 h (BRUNET et al., 2005). Labeling would be temporary and it is expected

not to interfere with embryo development. In case of Y-chromosome specific probes, the X-chromosome one would not be affected.

• Detectable at low concentrations! Small concentrations of TFOs trigger a specific accumulation because of enriched target sequences on the bovine Y-chromosome.

• Bio-compatibile! Gold nanoparticle concentration below 10µg/ml do not affect bovine sperm membrane integrity, morphology and motility (TAYLOR et al., 2014).

5.1 Sorting technology

Sperm sorting by flow cytometry is a system commercially applied in cattle industry to separate Y- from X-bearing chromosome sperm populations and dead sperm population from viable ones. Sex sorting is currently based on the relative difference between Y- and X-sperm populations expressing an unbalanced Hoechst uptake relative to the different DNA content of sex chromosomes.

Different experimental methods for sex sorting have been described based on speculated differences of sperm density (ALEAHMAD et al., 2009), surface charge characteristics (KANEKO et al., 1984), sex specific proteins on the sperm membrane.

Antibodies synthesized by different immunological approaches were used to recognize specifically X-bearing sperm and were reported by Howes et al., 1997 (HOWES et al., 1997). Yang et al. (2014) also showed that rabbit antibodies can be used to enrich X-bearing bovine sperm, leading to a sorting purity of 73%. A

qualitative sperm sorting was described for isolation of non-apoptotic sperm fraction using magnetic activated cell sorting (MACS) (NADALINI et al., 2014). Magnetic sperm sorting was developed to remove defective spermatozoa using magnetic nanoparticles labeled to ubiquitin or lectin PNA (ODHIAMBO et al., 2014). All the above mentioned methods are always based on sex differences between sperm populations, but up to now there is no method available based on identification of mammalian sperm through live, genetically based sperm sorting. Above all, these methods lack of reproducibility and therefore cannot be considered reliable.

The tested setup of sperm gene nano-targeting provides the basis for detection of live Y-chromosome bearing sperm in theirs physiological environment. Under these conditions the advantage of the method is related to the Y-chromsome’s uniqueness.

In the 1960 it was proposed that the mammalian Y-chromosome evolved from an ordinary autosome (OHNO, 1967). Ohno speculated that the Y-chromosome lost all but one gene involved in sex determination named SRY and the most prominent features are eight massive palindromes, at least six of which contain testis related genes (SKALETSKY et al., 2003). Lahn and Page (1997) describe a non-recombining region of the human Y-chromosome (NRY) and identified two groups of genes: the first group expressed in many organs; these housekeeping genes have X homologs that escape X-chromosome inactivation. The second group, consisting of Y-chromosomal gene families expressed specifically in testes (LAHN and PAGE, 1997). Bellot et al. (2014) explained that gene content of the Y-chromosome became specialized through selection in order to store homologous X–Y gene pairs. Beyond

its roles in testis development and spermatogenesis, the Y-chromosome is essential for male viability, and has unappreciated roles in male phenotypic differences, in health and disease (BELLOTT et al., 2014). Male-specific region (MSY) comprises 95% of the chromosome's length and was described to be dominated by repeated sequences (JANGRAVI et al., 2013). Differently expressed genes in X and Y sperm probably needed for sperm maturation were recently characterized (CHEN et al., 2014).

5.2 Triplex forming oligonucleotides and their possible use in sperm chromatin According to Hoogsteen hydrogen bonds, synthetic RNA or DNA molecules can bind to the major groove of a duplex DNA in a sequence-specific manner without DNA denaturation (HOOGSTEEN, 1963, RICCIARDI et al., 2014), representing a suitable method for vital cell genotyping. For this reason, triple helix DNA attracted interest as method for intracellular gene targeting (ANTONY et al., 2001, BOUTORINE et al., 2013). Gene targeting strongly depends on availability of binding sites and/or probe accessibility. Triplex forming oligonucleotides follow strict rules: They bind to a poly-purine sequence if that sequence faces the major groove of the dsDNA and if that site is not jet occupied by histones or protamines or other intra-molecular triplexes.

Specific binding is favored by acidic condition and mostly for a poly-pyrimidine probe sequence with a parallel orientation. TFOs cannot bind to a site if already occupied by a triplex DNA or to poly-purine sites that are in the minor groove. A study of sperm chromatin structure is necessary to understand if a triplex structure is intrinsic to sperm DNA, to find out possible obstacles to triplex targeting. The main feature that

can influence or hinder triplex binding is intrinsic sperm chromatin structure and its architecture composed of protamines, nucleosomes and non-B DNA structure.

It was described that triplex formation takes place to some extent in somatic cell chromatin (BRUNET et al., 2005) and that 50% of somatic chromosomal target can be covered by TFOs in living cells (BRUNET et al., 2005).

Sperm chromatin has a compact pseudo-crystalline state and details of its structural organization are still poorly understood (AUSIO et al., 2014). Each sperm has different pattern of chromatin condensation. Human spermatozoa treated with DTT show different patterns of de-condensation (BEDFORD et al., 1973), therefore it is logic to suppose and expect that triplex binding is different for each sperm.

Protamines have a crucial role in the packaging and protecting of sperm DNA (ZINI, 2013, JODAR and OLIVA, 2014), and may represent an obstacle to TFOs accessibility. According to some sperm chromatin structure models, protamines bind to DNA on the minor groove (CORZETT et al., 2002, BALHORN, 2007, HAMMOUD et al., 2009). Other models describe that P1 and P2 families bind DNA on its major groove (POGANY and BALHORN, 1992, BIZZARO et al., 1998). Most likely both minor and major are bound to protamines as described by D’Auria et al. (D'AURIA et al., 1993). Moreover, TFOs do not form triplexes on nucleosomes, except at sites located towards the extremities of the nucleosomal DNA fragments (BRUNET et al., 2005). In mammalian sperm, approximately 3-4% of DNA is bound to nucleosomes (HAMMOUD et al., 2009, VAVOURI and LEHNER, 2011, RAJESWARI, 2012) and

this may mean that 3-4% of DNA is not possible to be detected through triplex hybridization.

There are multiple potential triplex forming sequences in the human genome (BURKHOLDER et al., 1991, ZAIN et al., 2003). Most annotated genes in both mouse and human genomes contain at least one unique TFO binding site, and these sites are enriched in the promoter regions (JAIN et al., 2008, TOSCANO-GARIBAY and AQUINO-JARQUIN, 2014). A database of triplex target sites has been developed for human and mouse genome (JENJAROENPUN et al., 2015). As no data on triplex target sites were available for the bovine genome this thesis is the first report offering information of bovine triplex target sites. There is evidence that H-DNA is a common structure already present in the mammalian genome (LYAMICHEV et al., 1986, VASQUEZ et al., 2000, JAIN et al., 2008). Physiologically expressed proteins have a specific recognition activity for triplex DNA involved in gene expression (MUSSO et al., 1998, HURLEY, 2002), genetic reparation or associated with clinical outcomes (NELSON et al., 2012, WANG and VASQUEZ, 2014).

The closest analysis of intrinsic triplex DNA formation in male gametes was performed in grasshopper spermatids. Grasshopper spermatids show a triplex DNA structure during maturation. The triplex formation disappears while histones are replaced by protamines (CERNA et al., 2008). Use of monoclonal antibodies for triplex DNA would also be useful to study the concentration of triplex structure in cells

and gametes in physiological or pathological conditions, or perhaps to speculate a difference of triplex DNA content between XY and XX genome.

5.3 I Milestone: Bovine Y-chromosome specific TTS

The first milestone’s task was to find poly-purine sequences on the bovine Y-chromosome. According to triplex binding principles only a poly-purine sequence can be targeted by three different oligonucleotide sequences: a purine motif (GA); a pyrimidine motif (CT); and a mixed motif (GT) (BUSKE et al., 2012). In this thesis, a pyrimidine motif probe was chosen in order to avoid cross-hybridization and off-targets.

An ideal triplex target sequence should have the following characteristics:

• Composed of at least 20 bases to be fairly specific and enriched on the Y-chromosome. Likelihood of a candidate sequence to have high number of copies in the genome decreases with its length. The same sequence highly repeated would lead to a higher signaling capability.

• Target sequences should not be too far from each other otherwise nanoparticles would be too distant from each other in order to have a plasmon resonance shift.

• Low off-target rate to reduce background and false positive hybridization.

The software Triplexator is the informatics tool used to search for such TTS. The program found about 1000 different sequences (table in appendix). A non-B DNA motif search tool was described in 2012 (CER et al., 2012), useful to find every kind of non-B DNA structure, such as repeats, slipped motifs, G-quadruplex, inverted repeats, cruciform motifs, mirror repeats, triplex motif, Z-DNA motif and last, A-phased repeats. The tool is useful but it is not possible to analyze an entire chromosome sequence. The only use of the software could lead to false positive results because of the redundant annotation of sequences loaded on the Pubmed database (KOONIN EV). Therefore, here eight sequences were selected to investigate their specificity for the Y-chromosome by a Southern blot analysis. Three sequences showed enrichment on XY genome compared to the XX one (Figure 7, pp. 41). Number of bands and signal intensity on male genome was higher compared to the female one. No absolute XY specificity was established for any of the probes.

Southern blot was used to check for the presence of the triplex target sites and for their enrichment on male genome, but the results do not give any indication on their affinity for triplex hybridization. Southern blot results validated the software analysis regarding the sequence annotation but no data are available on the absolute number of sequence repeats.

A third check of the sequence specificity was performed searching in the NCBI database. The BLASTn results indicate with a score of 100% (low-level error) that the sequences are enriched in the Y-chromosome. Database analysis is consistent with the results of the Southern blot assay, in which positivity was seen also in female genome.

Other software can be used to map genomes to provide TTS sequences. An integrative web tool was described to find G-quadruplets and other regulatory DNA elements in various genome regions (JENJAROENPUN and KUZNETSOV, 2009), with which it was possible to create a catalog of TTS for human genome (JENJAROENPUN et al., 2015) for both therapeutic and detection purposes.

Milestone I was achieved finding 3 triplex target sites highly enriched on the bovine Y-chromosome, considering sequences about 20 bases long. Higher specificity can be achieved searching for longer probes. The search of sequences is based on the compromise that sequences too long would be less enriched and would because of the distance negatively affect gold nanoparticle agglomeration. For Y-chromosome bearing sperm detection, the best sequence target has to be enriched on the Y-chromosome and not on the X-Y-chromosome. No matter the length of it and its presence on the autosomes.

5.4 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

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).

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

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

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

Im Dokument Detection of Y-chromosome bearing bovine sperm using laser-generated gold nanoparticle bio-conjugates (Seite 52-0)