3. Materials and Methods
3.12 Experimental design
To reach a proof of principle that TFO-targeting and nano-targeting are possible in DNA of bovine sperm nuclei, a study plan of three milestones was organized.
Figure 8 shows the project set up. The 1st milestone’s task was to investigate bovine gene sequences enriched in the Y-chromosome that could possibly be suitable for triplex hybridization. Triplex target sequences were first found through a software analysis and analyzed in a second step for an experimental validation by Southern blotting a method used to search for specific sequences in DNA samples (MARCADET et al., 1989).
The 2nd milestone was the synthesis and screening of different triplex forming oligonucleotides candidates to identify the one characterized by the highest triplex
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hybridization efficiency. The methods used for triplex binding analysis were based spectrophotometric analysis of triplex structure, an electrophoresis mobility shift assay and a triplex in situ hybridization on spermatozoa.
The 3rd milestone was to label the most efficient TFO candidate to laser generated gold nanoparticles and test it as sensor for free DNA and sperm nuclear matrix.
Surface plasmon resonance was tested by spectrophotometry, since, according to the hypothesis, a specific hybridization of the conjugate on XY genome would result with absorbance different from the XX one.
Figure 8: Experimental setup and milestones
43 4. Results
4.1 I Milestone: Bovine Y-chromosome specific triplex target sequences
4.1.1 Triplex sequence finder and Triplexator
The number of sequences found by the programs was about 1000 (table 5 in appendix) when searching for sequences ranging from 15 to 25 bp. Table 1 lists an example of 10 poly-purine target sequences enriched on the Bos taurus Y- chromosome according both to Triplexator and to Daniel Werner’s software.
Table 2: Example of triplex target sites sequences
sequences BLASTn locus
Poly-purine sequences are listed with reference of BLASTn localization and number of hits on the Y-chromosome.
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Eight candidate triplex target sites were chosen as candidates for experimental validation of XY genome enrichment.
In table 6 (in appendix) shows the results on an analysis of Y-chromosome using Triplex inspector software by Dr. Fabian Buske.
4.2.2 Southern blot
The direct validation of the enrichment on bovine Y-chromosome was assessed by Southern blotting with male and female gDNA. Following probes tested were:
Probe1: 5- CTT TCC TTT TCT TCC TTT CT Proge2: 5'-CTC TTC CTT CCT TTC CCT T Probe3: 5'–CTT CCT CCC TTT TCC CTC CCT C Probe4: 5’-CTC CTC CCT CCT CCC T
Probe5: 5’-TTT CCT CTT CCT TTC TCC T Probe6: 5’-CCT CCT CCC TCC TCC CTC C Probe7: 5’-CTT CCT TTC TCC TCC TCT TT Probe8: 5’-CTT CTC TTC CTT TCT CCT CT
Digoxigenin was labeled to oligodeoxynucleotide probe of each probe.
From all probes tested, only probes 1, 2, and 3 showed enrichment on the male genome compared to the female one. Bands of detection were visible gDNA and indicated by black arrows (Figure 9A). Positive bands were also visible for each sequence in female genome (Figure 9, white arrows). Southern blot analyzes showed that the sequences were repeated and enriched in male genome. Probes 4 to 8 did not show a XY genome enrichment and were discharged from the validation
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(data not showed). Probe 6 is shown as representative of a Southern blot that does not identify sequence enrichment on XY genome. Probes that did not show any enrichment were excluded as candidate for triplex hybridization analysis.
Figure 9: Validation of specific detection of triplex target sites on the Y-chromosome by Southern blot A) Southern blot membrane: probe 1, 2 and 3; black arrows represent hybridization of DIG labeled probes on male gDNA, white arrows represent probe hybridization on female gDNA B) Loading control of gDNA samples after restriction digested with PstI used in different southern blots. Probe 6 has a similar pattern for XY and XX genome and no difference in enrichment is detectable.
46 4.3 II Milestone: triplex hybridization analysis
It was decided to test sequences 1, 2, and 3 because they were validated by southern blot analysis as being enriched on the XY bovine genome. The sequences have different content of cytosine. TFO1 has 35% of cytosine, TFO2 has 47% of cytosine content and TFO3 has 60%. The lower the C content is, the higher is the chance for triplex hybridization (G.R. PACK, 1998).
Different batches of TFOs were synthesized using chemically modified tymine LNAs and 5-methyl-cytidine to improve triplex binding affinity. Probe’s sequences and model of triplex binding were designed and listed in table 2.
Table 3: Triplex forming oligonucleotide sequences and triplex binding motif
On the right side, sequences are listed for TFOs and dsDNA target. On the left are probes listed as they were used with chemical modifications: LNA-thymine (Y), and 5-methyl-cytidine (X) were used as substitutes of thymine and of cytosine respectively. Two batches of each probe were ordered: (i) TFO labeled to Alexa 488 for triplex FISH and (ii) TFO DIG labeled for triplex melting temperature analysis and electrophoresis shift assay.
47 4.3.1 Triplex melting temperature curve
To study the hybridization efficiency of triplex candidate probes, first of all the triplex structure was measured by spectrophotometric analysis under controlled temperature (BOUTORINE and ESCUDE, 2007, MCKENZIE et al., 2007, MCKENZIE et al., 2008).
Figure 10 shows the melting temperature curves of each probe. TFO1 (Figure 10 A) has a melting temperature of 44 °C and its target dsDNA is melted at 63°C. TFO2 (Figure 10 B) has a melting temperature of 33 °C and its target dsDNA is melted at 63°C. TFO3 (Figure 10 C) has a melting temperature of 30°C and its target dsDNA is melted at 55°C. Analyses were performed by Center for Nanointegration Duisburg-Essen (CENIDE).
Figure 10: Spectrophotometric analysis of triplex/duplex melting points. TFO1, TFO2 and TFO3 were mixed to DNA target at ~15°C. Melting curves were analyzed by increasing the temperature up to 80 °C. Black arrows indicate triplex melting temperature and green arrows show the dsDNA melting point.
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4.3.2 Triplex electrophoretic mobility shift assay (EMSA)
Electrophoresis mobility shift assay identified unbound from hybridized TFO. TFOs are listed in table 1 and digoxigenine was labeled on the oligodeoxynucleotide probe.
Digoxigenine was used as specific marker for TFOs and therefore to detect exclusively the probes and not the target gDNA.
Figure 11: Validation of triplex electrophoresis mobility shift assay (triplex-EMSA): left images show the loading control for each dsDNA/TFO sample. On the right column the EMSA membrane chemiluminescence is represented. A) TFO1 triplex hybridization are indicated by the black arrows, yellow arrows indicate free and unbounded triplex forming probes B) TFO2 triplex hybridization, only free and unbounded are visible (yellow arrows) C) TFO3 triplex hybridization are indicated by the black arrows, yellow arrows indicate free and unbounded triplex forming probes.
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The left side of the Figure 11 shows the loading control of digested gDNA and TFO samples. Image was taken under fluorescence detection of the electrophoretic gel incubated with propidium iodide. The pictures on the right side instead represent the chemiluminescence originating uniquely from the TFOs (arrows) transferred on the nylon membrane.
EMSA results in figure10 line A show that TFO1 hybridization are visible on male gDNA (black arrows) and not on the female gDNA. Free and not hybridized TFO1 is visible on bottom (yellow arrow). On line B, TFO2 hybridization shows no positive hybridization in any of the samples. Line C shows TFO3 hybridization. A positive hybridization in all the samples of male and female gDNA was found.
4.3.3 Triplex fluorescent in situ hybridization
Direct localization of triplex probes hybridized to sex sorted sperm nuclear matrix was performed by incubating TFOs on slides with bovine sperm demembranized heads, with no DNA denaturation protocol.
A fluorescent duplex in situ hybridization was made as control for each TFO to have a correct interpretation of the hybridization based on the presumption that hybridization triplex were to have the same location of a hybridization duplex.
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Figure12: Fluorescent microscope images of Triplex fluorescent in situ hybridization.
A) FISH (fluorescence in situ hybridization of TTS1; B) triplex FISH using probe TFO1-Alexa488; C) triplex FISH using probe TFO1, 2 and 3 together, all labeled to Alexa488.
Table 4: Triplex fluorescent in situ hybridization rate in sorted sperm.
A B
X-pop Y-pop P-value tfo1 tfo2 tfo3
tfo1 6.3±1.03% 16± 1.4% 0.8.9x10-7 tfo2 0.00034
tfo2 3.5±0.54% 8± 0.9% 0.00283 tfo3 0.00042 1
tfo3 5±0.89% 11±1.63% 0.37215 tfo1+2+3 0.02x10-14 0.02x10-14 0.02x10-14 tfo1+2+3 15±2.04% 36±1.86%* 0.02x10-14
Chi-square for triplex fluorescent in situ hybridization. A) Comparison of hybridization of each treatment group for X-and Y-sperm population B) Comparison of hybridization between each treatment group relative to Y-chromosome bearing sperm population.
Triplex hybridization for TFO1 was up to 16% of the total Y-chromosome bearing sperm and 6% of the total X-chromosome ones. The difference between Y- and X- population was not significant. TFO2 had a positive triplex hybridization up to 8% of the total Y-chromosome bearing sperm and 3% of the total X-chromosome ones. The
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difference between Y- and X- population was not significant.TFO3 had a positive triplex hybridization up to 11% of the total Y-chromosome bearing sperm and 5% of the total X-chromosome ones. The difference between Y- and X- population was not significant. All TFOs together result in a positive triplex hybridization up to 36% of the total Y-chromosome bearing sperm and 15% of the total X-chromosome ones. In this last case the difference between Y- and X- population was statistically significant (p<0.05).
4.4 III Milestone: Gold nanoparticle bio-conjugation – SPR analysis
4.4.1 Gold Nanoparticle synthesis and conjugation to TFOs
Figure 13: Disc centrifuge analysis of fragmented laser generated AuNPs:
A) Number and size of AuNPs ranged from 5.4 to 7.6 nm.B) TEM image of AuNPs.
AuNPs solution was fragmented for 30 min with laser pulses of 25 mJ. AuNPs of the mother solution showed a broad distribution of 6.5 ± 1.1 nm in diameter. Analysis was performed by Center for Nanointegration Duisburg-Essen (CENIDE).
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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
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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
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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
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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
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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
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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
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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
The closest analysis of intrinsic triplex DNA formation in male gametes was