University of Veterinary Medicine Hannover
Detection of Y-chromosome bearing bovine sperm using laser-generated gold nanoparticle bio-conjugates
Thesis
Submitted in partial fulfillment of the requirements for the degree -Doctor of Veterinary Medicine-
Doctor medicinae veterinariae (Dr. med. vet.)
by
Roberto Mancini Naples
Hannover 2015
Referee: Prof. Dr. med. vet. D. Rath Detlef Referee: Prof. Dr. Harald Sieme
Day of the oral exam: April 21st, 2015
This thesis was written at the Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics, Neustadt-Mariensee and is
dedicated to my family.
1. Introduction ... 7
2. Literature ... 9
2.1 Sex predetermination in mammals and sperm sex sorting ... 9
2.2 Qualitative sorting: New approach for bovine sperm sex sorting ... 11
2.3 Gene sequence detection in living cells ... 13
2.4 Triplex hybridization ... 16
2.5 Gold nanoparticles (AuNPs) ... 23
2.6 Expected insights ... 28
3. Materials and Methods ... 29
3.1 Bovine Y-chromsome triplex target sites ... 29
3.2 Genomic DNA extraction from blood ... 30
3.3 Southern blot analysis ... 31
3.4 Triplex melting curve analysis ... 32
3.5 Triplex electrophoresis mobility shift assay (EMSA) ... 35
3.6 Sorted sperm nuclear matrix isolation ... 35
3.7 Triplex fluorescence in situ hybridization (T-FISH) ... 37
3.8 Fluorescence in situ hybridization (FISH) ... 39
3.9 Gold nanoparticle synthesis and conjugation to TFOs ... 39
3.10 Gold nanoparticle hybridization and SPR shift analysis ... 40
3.11 Statistical analysis ... 41
3.12 Experimental design ... 41
4. Results... 43
4.1 I Milestone: Bovine Y-chromosome specific triplex target sequences ... 43
4.1.1 Triplex sequence finder and Triplexator ...43
4.2.2 Southern blot ...44
4.3 II Milestone: triplex hybridization analysis ... 46
4.3.1 Triplex melting temperature curve ...47
4.3.2 Triplex electrophoretic mobility shift assay (EMSA) ...48
4.3.3 Triplex fluorescent in situ hybridization ...49
4.4 III Milestone: Gold nanoparticle bio-conjugation – SPR analysis ... 51
4.4.1 Gold Nanoparticle synthesis and conjugation to TFOs ...51
4.4.2 Gold nanoparticle/LNA hybridization to gDNA and sorted sperm nuclear matrix ...52
5. Discussion ... 54
5.1 Sorting technology ... 55
5.2 Triplex forming oligonucleotides and their possible use in sperm chromatin ... 57
5.3 I Milestone: Bovine Y-chromosome specific TTS ... 60
5.4 II Milestone: Triplex hybridization ... 62
5.5 III Milestone: Nanoparticle hybridization and SPR shift ... 67
5.10 Future outlook ... 72
6. Conclusions ... 74
7. References ... 78
Appendix... 78
Abbreviations ... 78
List of tables ... 79
Aknowledgments ... 126
7 1. Introduction
Sex determination in mammals is strictly genetic and not influenced by the environment. The presence of sexual chromosomes is different for female (XX) and male (XY) individuals. During spermatogenesis, male gametes generate two populations of sperm: half bearing a X- and half bearing a Y-chromosome. These two sperm population can be separated by a flow cytometric sorting (CUI & MATTHEWS, 1993; CUI, 1997; JOHNSON et al. 1999) based on the quantitative difference of DNA content between X-and Y-chromosomes. However, the method is insufficiently efficient even with high throughput techniques. A completely different approach focused on qualitative identification of sex-related gene sequences in sperm might be more efficient and still represents an unmet biotechnological need.
This thesis is addressed to investigate the use of a construct as a specific marker for the bovine Y-chromosome in sperm nuclei. This conjugate is made of a triplex forming oligonucleotide (TFO) functionalized to gold nanoparticles (AuNPs).
Here it is hypothesized that TFOs hybridize to sequences enriched on the bovine Y- chromosome, triggering a selective recognition and accumulation. AuNPs will be labeled to the nucleic acids probes and will be necessary for signaling when accumulated after hybridization. Conjugate agglomeration emits a detectable signal that has a significant qualitative deviation from non-agglomerated status.
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The importance of using triplex forming oligonucleotides resides in the necessity of hybridized DNA sequences without denaturation, a process that normally is done using deleterious and toxic compounds. Gold nanoparticles instead, represent a useful and versatile tool that can replace DNA mutagenic molecular dyes as signaling device. AuNPs are inert, do not photobleach and are easy to form bio-conjugates.
To study and confirm this method, a first approach was performed searching for sequences on the bovine Y-chromosome suitable for triplex hybridization. Second, different TFO probes were designed in order to be used in the trials, testing the triplex binding affinity and efficiency under physiological conditions. Finally, prototype AuNPs were conjugated to oligonucleotide probes and were analyzed for their interaction with bovine DNA and sperm chromatin, searching for eventual hybridization, agglomeration and signaling.
This study contributes to better understand Y-chromosome detection in viable sperm and to improve the use of gold nanoparticles for gene detection in condensed chromatin structure such as sperm DNA. This thesis may represent a basic step for an innovative use in the future of triplex forming oligonucleotides and AuNPs for sperm sorting.
9 2. Literature
2.1 Sex predetermination in mammals and sperm sex sorting
Reproduction is the biological process by which new organisms are produced from predecessors of the same species. Mammals have an XX:XY system of chromosomal sex determination. The male genotype, being XY, can generate two types of sperm: half bearing the X-chromosome and half the Y-one. Sex of the offspring depends therefore on the type of spermatozoa able to fertilize the oocyte.
These two sperm populations can be separated and be used for offspring sex predetermination within artificial insemination protocols (CRAN & JOHNSON, 1996).
In animals sex pre-selection and pre-implantation diagnostics are considered under the economical point of view, especially for farm animals. First, sex selection increases genotype selection intensity. Second, desired traits are correlated with the sex of the animal. It is advantageous to increase the odds of producing offspring with a desired sex especially for farm animals having a long gestation period (MAKKAR et al., 2005).
Currently there are different methods available for sex-predetermination, which can be used on early stage embryos (MAPLETOFT & HASLER, 2005); fetuses (DEVANEY et al., 2011), or artificial insemination using sex-sorted sperm (ESPINOZA-CERVANTES &CORDOVA-IZQUIERDO, 2012).
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The only reproducible and therefore commercially available method for bovine sperm sex sorting is based on flow cytometric analysis of sperm stained with a DNA dye, Hoechst 33324 (RATH and JOHNSON, 2008). The generation of the first offspring using sexed sorted semen by flow cytometry was reported in 1989 in rabbits (JOHNSON et al., 1989) and the first cattle offspring generation was reported in 1993 (CRAN et al., 1993).
Many different methods for sperm separation have been described on the basis of various differences between Y- and X-bearing sperm (SEIDEL, 2012). Sperm sexing has been studied by measuring the difference of the two sperm populations considering several physical or chemical principals, for example sperm density (ERICSSON et al., 1973, KANEKO et al., 1983, KANEKO et al., 1984, GLEDHILL, 1988, MAURO et al., 2014), sperm shape and size (CUI, 1997), DNA content (JOHNSON et al., 1989, JOHNSON et al., 1999); flow fractionation (POPOV and REKESH, 1991), sperm movement (BALLI et al., 2004, YAN et al., 2006), sperm surface charge (KANEKO et al., 1984, ENGELMANN et al., 1988), sperm surface sex specific proteins (HOWES et al., 1997, YANG et al., 2014). There are many concerns about the reliability of the listed methods, first because of a low worldwide reproducibility, second because sex separation does not go beyond the 60-65% in purity.
Currently, the only reliable method to sort sperm is based on flow cytometry, leading to a sex purity of 85-95%. Within this method there is still some concern about the use of Hoechst 33324, which is cancerogenic and mutagenic (DURAND and OLIVE,
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1982). Therefore, it is worthwhile to search for an alternative method of sperm separation that does not require fluorescent DNA dyes, avoiding potential associated risks.
An alternative approach of qualitative sperm sex sorting could be based on DNA sequence variation/differences present between sex chromosomes (RATH et al., 2013). Unavailability of such qualitative method represents an innovative research challenge.
2.2 Qualitative sorting: New approach for bovine sperm sex sorting
The present study focuses on the development of an alternative method for sexing bovine sperm. The possible use of a bio-conjugate that is capable of specifically recognizing bovine Y-chromosomes sequences was examined (BARCHANSKI et al., 2011, RATH et al., 2013). The construct is made by an oligonucleotide probe that recognizes and hybridizes specifically Y-chromosome DNA sequences through
“triplex hybridization” according to Hoogsteen base pair chemistry, in which a single stranded ssDNA molecule can bind to a double stranded dsDNA forming a three stranded DNA. This probe is called TFO (Triplex Forming Oligonucleotide) and hybridizes to poly-purine sequences of the target DNA in a specific manner and without DNA denaturation.
Gold nanoparticles are used as replacement of toxic DNA intercalating dye, for which excitation light is required. Nanoparticles are easy to bind to biomolecules such as
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DNA probes or peptides and they can be detected given their bathochromic characteristics (BOISSELIER and ASTRUC, 2009, LACERDA et al., 2009).
Moreover, gold is inert and does not have a toxic effect in cells (BOISSELIER and ASTRUC, 2009).
The concept behind the research is that oligonucleotide hybridization leads to a gold nanoparticle accumulation on a certain region of Y-chromosome that changes the reflected light upon interaction with the target DNA (MCKENZIE et al., 2007).
Sperm nano-targeting is a new method that aims to be used in viable sperm for live imaging and for sperm separation. The method is based on genetic detection and besides sexing, it can be applied to analyze sperm chromosome linked diseases (GUSELLA et al., 1983, HOSTIKKA et al., 1990, DENG et al., 2014).
The key point of this new method is the DNA identification with no denaturation suitable for specific detection in viable and physiologic conditions leading to gene detection maintaining sperm viability. In figure 1 the new approach for sperm nano- targeting is explained in a schematic model.
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Figure 1: Nanoparticle-based sperm sorting. Optical detection principle based on the shift of the plasmon resonance peak upon regional accumulation of AuNPs. (A) Design of a nano- bioconjugate with a hybridizing triplex forming oligonucleotide. (B) Distribution of AuNPs conjugate in X- and Y-chromosome bearing sperm. Selective triplex formation at the Y- chromosome causes nanoparticle aggregation. (C) Aggregation results in a bathochromic shift (change of spectral absorption) to be detected.
2.3 Gene sequence detection in living cells
Live gene detection of single cell requires high affinity and specificity, good cellular penetration, ability to reach the nucleus, stability, low toxicity and no distortion of cellular mechanisms, simple detection, and high signal/background ratio. Attention is mainly paid to fluorescent DNA recognizing probes in living somatic cells and direct imaging. Different classes of dsDNA-targeting systems have been proposed (GHOSH et al., 2006, WANG et al., 2008, MAYA-MENDOZA et al., 2012):
(i) Antibodies recognizing palindromic sequences (CERUTTI et al., 2001) or poly-purine sequences (BURKHOLDER et al., 1991).
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(ii) Fluorescent cofactors coupled with DNA using DNA-methyltransferase provides a method for sequence-specific labeling of large DNA fragments (PLJEVALJCIC et al., 2003).
(iii) Zink finger proteins transcription factors or nucleases enzymes characterized by stability guaranteed through zinc ions identify specific sequences (PORTEUS and CARROLL, 2005)
(iv) TALEN (transcription activator–like effector nucleases) generally coupled to oligonucleotides that recognize short specific sequences (RNA, DNA, molecular beacons) (LEI et al., 2012, SANJANA et al., 2012).
(v) CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats), a recent tool currently used for gene editing as well. A holoenzyme constituted by a caspase9 nuclease coupled to a guide-RNA that triggers the protein specifically at the target sequence (CHEN et al., 2013, ANTON et al., 2014). The inconvenient feature is that CRISPR/Cas9 is a big molecule and cannot be delivered inside the cell without plasmid transfection.
(vi) Triplex forming oligonucleotides (VAN DAELE et al., 2008, DOLUCA et al., 2014, RICCIARDI et al., 2014), modified RNA that hybridize target DNA forming a three stranded DNA. Triplex binding chemistry takes place according to Hoogsteen base pairing, different from the canonical Watson and Crick model applied for B-DNA structure. Hoogsteen base pair applies the N7 position of the purine base (as a hydrogen bond acceptor) and C6 amino group (as a donor), which bind the Watson-Crick (N3–N4) of the
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pyrimidine nucleotide. TFOs represent a validate alternative for gene targeting that does not require gene transfection or other molecular carriers, because these are small molecules compared to enzymes or nucleases
All these systems rather deliver one or two different fluorophores in a binary probe (donor and acceptor) that in close proximity change in different spectrum, such as molecular beacon (CLEGG, 1995, TYAGI and KRAMER, 1996).
Direct observation of dsDNA in living cells is generally investigated in somatic cells (fibroblast or Hela cells) and limited by the incomplete information about the real structure of DNA chromatin, its movements and rearrangements (BOUTORINE et al., 2013).
Noninvasive visualization of Y-chromosome in living sperm is a challenge.
Mammalian Y-chromsome analysis was developed primarily for genetic studies and for diagnostic screening but not in living cells (KUNKEL et al., 1977, CHARCHAR et al., 2012, DULIK et al., 2012). Y chromosomes is the basis for sex determination in mammals, but its structure rich of repeats has hampered sequencing and associated evolutionary studies (CORTEZ et al., 2014). There is a well-known difference in DNA sequences of X- and Y-chromosomes (GVOZDEV, 2008) important for sex determination. Gender identification is usually performed by the polymerase chain reaction (PCR) amplification of Y-chromosome copies of the differently present sex related genes (SULLIVAN et al., 1993, CHEN et al., 2014, ALLWOOD and HARBISON, 2015). The Y-chromosome is different from other chromosomes. It lacks
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of recombination (YAMATO et al., 2007), the Y-chromosome contains the testis determining factor SRY as well as dozen identified important genes related to spermatogenesis. All well characterized Y-genes have close relatives on the X, but their location and expression varies in different species. Genes on the Y- chromosome represent a small and random subset of genes from the X, caught in various stages of degradation and loss (GRAVES, 1995).
Recent genomic analysis of the bovine Y-chromosome revealed that 40% is composed of repeated sequences and that at least six multi-copy protein coding gene families located on the male-specific region (MSY) (MATTHEWS and REED, 1992, YUE et al., 2014).
2.4 Triplex hybridization
The canonical secondary structure of DNA is a B-form double helix and consists of A- T and G-C base pairs (TATEISHI-KARIMATA et al., 2014). Other unusual DNA structures are possible (GHOSH and BANSAL, 2003, JAIN et al., 2008). Under certain conditions, repetitive DNA motifs have the potential to adopt non-B structures, such as hairpins, triplexes (three stranded DNA), Z-DNA (left-handed double helical structure), G-quadruplexes (four stranded DNA in sequences rich in guanine) (LUEDTKE, 2009), and i-motifs (four stranded DNA in sequences rich in cytosine) (DU and ZHOU, 2013, DAY et al., 2014). Triple helix DNA, is a different geometric structure named H-DNA, where H stands for Hoogsteen, who theorized in 1963 the triple helix DNA (Figure 2) (HOOGSTEEN, 1963).
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H-DNA is structured by 3 strands of DNA, possible only in poly-purine and poly- pyrimidine sequences. The third strand binds to the duplex exclusively on its major groove and hybridizing to the poly-purine tracts (GHOSH and BANSAL, 2003).
There are two types of triplex: Intra-molecular, where hybridization takes place within the same DNA molecule, and Inter-molecular, between two different molecules of dsDNA or ssRNA (FRANK-KAMENETSKII, 2002).
Figure 2 Triplex DNA structure chemistry: In blue a dsDNA (Watson and Crick base pairing) and in green a triplex forming oligonucleotide (TFO). On the right a triplex DNA (Hoogsteen pairing).
Triplex DNA can already be formed in living cells by native DNA or by external oligonucleotides (GOOBES et al., 2002). The genome is characterized by a high number of triplex target sites (TTS), especially in the promoter regions (LI et al., 2011). Non-B DNA structures are generally unstable in vitro and under physiological conditions, and therefore their intracellular stability may be enhanced by specific proteins (COLLIER and WELLS, 1990). Non-B structures may be involved in DNA replication and transcription processes, used as specific recognition signal for
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regulatory DNA binding proteins (AMIRHAERI et al., 1988, VAN HOLDE and ZLATANOVA, 1994). Triplex DNA structures have also been related to diseases as Friedrich ataxia, caused by an intra-molecular triplex formation that negatively influences the FXN gene (WELLS, 2008).
Hoogsteen pairs have quite different chemical properties from Watson-Crick base pairing. Watson and Crick base paring is strictly defined (A hybridize to T and G to C). Hoogsteen base paring has different patterns (an example is show in figure 3).
• A purine TFO motif (Figure 3A) permit guanines (G) and adenines (A) in an anti- parallel orientation respectively to the target double helix.
• A pyrimidine TFO motif (Figure 3B) permit cytosines (C) and thymines (T) in a parallel orientation respect the DNA.
• A purine-pyrimidine TFO mixed motif (Figure 3C) permit guanines (G) and thymines (T) either in parallel or anti-parallel orientation respect the DNA.
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Figure 3: Different pattern of triplex hybridization. In red an example of triplex forming oligonucleotides, and in black the target double helix DNA. Nucleotide base pairings are shown in parallel triplex and antiparallel binding.
Duplex DNA undergoes helical distortions upon triplex binding to accommodate the third strand (because of electrostatic repulsions) (CHIN and GLAZER, 2009). In Hoogsteen bonds one base is rotated 180° in respect to the other. In some DNA sequences Hoogsteen base pairs exist as transient entities that are present in thermal equilibrium with standard Watson–Crick base pairs. The detection of these transient species require the use of Nuclear Magnetic Resonance (NM) (WU and BAUM, 2010).
Purine and pyrimidine motifs have different behavior. The formation of a pyrimidine motif triplex requires an acidic environment and is extremely unstable in physiological conditions. It is possible instead that purine motif probes hybridize a duplex at pH 7.
Purine motifs are highly inhibited by monovalent cations, especially K+ ions
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(SORENSEN et al., 2004). Because of the phosphate residues in the backbone, the DNA is negatively charged and metal cations play a crucial role in the stabilization of multi stranded DNA structures. Triplex formation requires Mg2+, whereas it is inhibited by K+ ions. Hoogsteen hydrogen bonds formed in the presence of magnesium ions were described to be more stable (BESCH et al., 2004).
Several modifications of both nucleotides and backbone are required to have efficient TFOs (TORIGOE et al., 2001, SUN et al., 2004, KAUR et al., 2008, MCKENZIE et al., 2008, TORIGOE et al., 2012, HEGARAT et al., 2014).
• Locked nucleic acids (LNAs) are bases, in which the ribose ring is constrained by a methylene linkage between the 2'Oxygen- and 4'Carbon. This bridge results in a locked C3'-endo sugar reducing thus the conformational flexibility of ribose and increasing the local organization of the phosphodiester backbone (SUN et al., 2004, VESTER and WENGEL, 2004). LNAs stabilize nucleic acids molecules by up to 10°C, and seems to be the most promising nucleic acid analogue with application in diagnostic and oligonucleotide-based therapeutics (PETERSEN and WENGEL, 2003). The high affinity hybridization properties of LNA encompass dsDNA recognition as well, and allow formation of dsDNA/LNA triplexes at physiological pH (JEPSEN and WENGEL, 2004, WERONIKA et al., 2014). As such, incorporation of five or seven LNA monomers in a 15mer TFO was described to raise the melting temperature of the triplex to duplex transformation from 33 to about 60 °C and at pH 6,8 (MCKENZIE et al., 2007). The use of a TFO consisting of a chimera of DNA- LNA in 3:1 ratio promotes the resistance of TFO to a physiological pH,
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reaching a greater thermal stability (MCKENZIE et al., 2008). Furthermore, the use of LNA ensures resistance to nucleases. LNAs may be mixed with residues in the DNA or RNA oligonucleotide when desired, significantly increasing the properties of hybridization (melting temperature) of oligonucleotides (KAUR et al., 2008).
• UNA (unlocked nucleic acids) demonstrate the potential to improve nucleic acids stability. The incorporation of UNA residues at certain positions of dsDNA was found to be energetically favorable or at least did not affect triplex stability (KOTKOWIAK et al., 2014).
• The 5-methylcytosine increases binding strength at physiologic pH compared to the unmodified oligonucleotides (LIN et al., 1994, BESCH et al., 2004, MCKENZIE et al., 2008). One of the factors determining the triplex stability is the pKa values of cytosine (G.R. PACK, 1998). A large number of cytosine analogues with elevated pKa values have been developed to improve triplex binding. 5-methylcytosine has a slightly higher pKa than C. (SORENSEN et al., 2004, SUN et al., 2004, MCKENZIE et al., 2008, FOX and BROWN, 2011).
• Use of natural polyamines like spermine, spermidine, putrescine improve triplex hybridization binding (FLORIS et al., 1999, WANG et al., 2012).
• Partial substitution of natural guanine by 8-aza-7-deaza-guanine (PPG) in a purine motif TFO was demonstrated to improve target site binding (ROGERS et al., 2014).
• Twisted intercalating nucleic acid (TINA) is a nucleic acid molecule described to stabilize Hoogsteen triplex DNA formation because of its ability to twist
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around a triple bond and to increase its intercalation within dsDNA (GECI et al., 2007, SCHNEIDER et al., 2010).
• Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities through triplex hybridization. PNAs enhance triplex formation in physiological conditions (KUHN et al., 2002, STENDER et al., 2014).
Triplex forming oligonucleotides were described as inhibitors for gene expression (DAHMEN and KRIEHUBER, 2012). They also can position DNA-reactive agents to specific locations in the genome, induce targeted mutagenesis (RESHAT et al., 2012) or can be used as marker probes (JOHNSON III and FRESCO, 1999, SCHWARZ-FINSTERLE et al., 2007). There is evidence that a synthetic RNA strand binds duplex DNA and influence transcription and expression (HOYNE et al., 2000).
A TFO can hybridize in a living cell to generate mutagenesis and influence gene expression in a specific manner. TFO are also used in genome targeting and editing in vitro and in vivo (REZA and GLAZER, 2014).
It was reported that triplex formation stimulates DNA repair mechanisms in human cells because of the electrostatic repulsion DNA caused be the TFO. Subsequent metabolism of the triplex structure may lead to mutagenic and re-combinatory activity at the TFO binding site (WANG et al., 1996).
Moreover, if a TFO is labeled to a mutagenic compound, triplex binding can induce mutagenesis on duplex DNA, primarily at the binding site itself, but also in the surrounding sequence. TFO induces DNA double strand breaks and activation of apoptosis (ROGERS et al., 2014). The mutations in these cases are base
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substitutions or deletions, similar to those mutations that take place during repairing of duplex brakes by the non-homologous and joining pathway (NHEJ) (ALAM et al., 2014). Hégarat et al. (2011) targeted with a TFO the IGF-I P1 promoter, which contains a purine/pyrimidine (R/Y) sequence that is central for transcription (HEGARAT et al., 2014). Research in TFO has improved substantially since 2010 due to new chemical modifications (ØSTERGAARD and HRDLICKA, 2011, HEGARAT et al., 2014, ROGERS et al., 2014).
2.5 Gold nanoparticles (AuNPs)
Gold nanoparticles (AuNPs) have a size between 1 and 100 nm. They have unique versatile characteristics and have been investigated and used in many applications for technology and biotechnology, for example as therapeutic agents (BABAEI and GANJALIKHANI, 2014), drug delivery (BROWN et al., 2010, STUCHINSKAYA et al., 2011, BAKSHI, 2014), electronic conductors and catalysts (PITA et al., 2013, LI et al., 2014), inert bio-markers as replacement of fluorochromes, and toxic dyes (KLEIN et al., 2010, TORABI and LU, 2014).
Different aspects of AuNPs make them interesting for the scientific community: they are inert to cell function and easily synthesized by different methods; they are tunable and have unique optical and electronic properties. These characteristics are always due to the elemental compositions of the particles (DANIEL and ASTRUC, 2004, NEUMEISTER et al., 2014). Gold nanoparticles and nanotechnology in general are key components of future technologies in biotech industry, and in a wider range of
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products from cancer detection systems (LONGO, 2014) to building artificial brains (MAYNARD, 2014).
The zeta potential is what mostly characterizes AuNPs and their optical properties when used as markers. Zeta potential refers to the electro-kinetic on the nanoparticle surface and is made of surface plasmons, oscillating electrons of the AuNPs. Surface plasmon resonance (SPR) leads to an optical absorption in the visible/infrared spectrum (BENETTI et al., 2013).
Interaction between AuNPs and light is strongly influenced by environment, size, and shape because of the change in the zeta potential. For example, for small (about 30nm) and monodisperse gold nanoparticles the SPR causes an absorption of light in the blue-green portion of the spectrum (~450 nm), while red light (~700 nm) is reflected, yielding a red color. As particle size increases, the wavelength of SPR related absorption shifts to longer and redder wavelengths. Red light is then absorbed, and blue light is reflected. The plasmon related absorption peak undergoes very large shifts in its wavelength, from 650 to 900 nm when differently dispersed in a medium or on a substrate. AuNPs potentially exhibit tremendous optical applications of detection if related to the absorbance (LI et al., 2014).
Aggregation is also a phenomenon that causes a shift in SPR. The aggregation of AuNPs of appropriate sizes induces interparticle surface plasmon coupling, resulting in a visible color change from red to blue (SAHA et al., 2012).
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Fig .4: Example of gold nanoparticle bathocromic effect. A) Gold nanoparticles of same size and shape but differently agglomerated, reflect a different visible light. B) Different sized gold nanoparticles reflect different wavelengths of visible light.
AuNPs become molecular markers, when labeled to probes that guarantee specific binding and accumulation on target molecules (protein, DNA, drugs) causing the color change (WANG et al., 2014). The color change during AuNP aggregation provides a practical absorption-based colorimetric sensing for any molecular target that leads to AuNP agglomeration or dispersion (ROSI and MIRKIN, 2005, LIU et al., 2007, JIANG et al., 2010). Such sensing represents a valid alternative to photo bleaching problems related to fluorochromes, and a more suitable solution for biology excluding toxicity. Moreover, AuNPs can serve as excellent fluorescence quenchers for FRET-based assays (Fluorescence Resonance Energy Transfer) for sensing small organic molecules (CHEN and CHANG, 2004, HUANG and CHANG, 2006, JAIN et al., 2007). AuNPs provide non-toxic carriers for drug and gene delivery applications due to their characteristic promoting up take by tumor cells (GHOSH et al., 2008, WANG et al., 2014).
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AuNPs cell internalization was obtained especially in cancer cells or tissue (GIBSON et al., 2007, SALMASO et al., 2009, TAYLOR et al., 2010, HAINFELD et al., 2014).
Uptake in tumor cells of LNA functionalized AuNPs was reported by Huo et al. (HUO et al., 2014). Microglial cells are also able to take up AuNPs, if these are labeled with cell penetrating peptides (CPPs) (HUTTER et al., 2010).
Gold nanoparticles are conventionally generated through chemical reduction methods (CRM) and several protocols are reported and reviewed (DANIEL and ASTRUC, 2004). Unfortunately, these procedures are known to be inefficient, since a significantly high excess of ligands and toxic residues remain in the AuNPs (ALKILANY and MURPHY, 2010).
AuNPs synthesized by pulsed laser ablation in liquid (Figure 5) is an alternative to conventional synthetic methods and it provides ligand-free NPs avoiding chemical residues (BARCIKOWSKI et al., 2007, BARCIKOWSKI and COMPAGNINI, 2013, REHBOCK et al., 2014). Laser generation of AuNPs also allows control of size and shape and their subsequent conjugation with organic molecules (REHBOCK et al., 2013). Pulsed laser systems irradiate solid state gold. Particle size can be tuned by variation of specific laser parameters (femtosecond, picosecond or nanosecond pulses). Functionalization with molecules like nucleic acids and proteins can be achieved in situ (direct addition of the functional agent to the medium prior to the laser process) or ex situ (the functional agent is mixed with the particles in a second synthesis step) (PETERSEN et al., 2009, PETERSEN et al., 2009).
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Figure 5: Gold nanoparticles synthesis and conjugation. A) Process and course of laser- generated gold nanoparticles in water. B) Ex situ conjugation of AuNPs to TFO, separated synthesized TFO and AuNPs are diluted in a same tube. C) AuNP/TFO bio-conjugate.
Strategies that use DNA hybridization to control the placement of nanoparticles in one and two dimensions have been demonstrated (PINTO et al., 2005, NYKYPANCHUK et al., 2008, LI et al., 2014). Interaction of AuNPs and DNA was described to have a bi-exponential kinetic curve and reveal the presence of three kinetic steps. The first is a diffusion step with the formation of an unspecific precursor complex made of target DNA and AuNPs. The second step involves binding between hydrophilic groups and the DNA grooves and finally, the third step has been interpreted as a consequence of a conformational change of the first complex forming a more compacted form of DNA/AuNPs structure triggered by specific DNA hybridization (PRADO-GOTOR and GRUESO, 2011).
28 2.6 Expected insights
This research aims to find an innovative method to detect Y-chromosome bearing bovine sperm using AuNPs triggered by specific oligonucleotide hybridization.
The project started up considering flow cytometry sperm sorting issues, mainly represented by Hoechst dye 33342, which has a known potential risk of mutagenesis. A different signaling method may be found using different sensors.
Gold nanoparticles represent a valid, reliable, and innovative tool for molecular detection because they are inert, they show no photobleaching, they are easy to synthesize, and they are easy to be labeled with molecular probes guaranteeing target specificity. For instance AuNPs can be triggered by oligonucleotides to be used as sensor for gene targeting. The challenge in this thesis is to investigate oligonucleotides functionalized AuNPs, able to detect genes of highly condensed sperm DNA.
Moreover, it is expected to elucidate new aspects of biotechnology: (i) to understand if intermolecular triplexes are possible in a highly condensed chromatin configuration of the sperm nucleus; (ii) to investigate gold nanoparticle/bio-conjugate interactions within the bovine sperm chromatin; (iii) to provide assumptions on sperm chromatin structure and detection in bovine sperm for qualitative sorting; (iv) to validate the use of triplex hybridization with sperm chromatin.
29 3. Materials and Methods
3.1 Bovine Y-chromsome triplex target sites
Specific triplex target sites on bovine Y-chromosome were found using two software programmes. One is Triplexator (BUSKE et al., 2012) and the second one was written by Dr. D. Werner from University of Essen. Dr. Werner’s program was used to search for sequences and double checked with Triplexator. Both software programs were used to analyze the sequence of Bos taurus Y-chromosome deposited from GenBank accession no. CM001061.2. Ten sequences were taken for experimental validation through Southern blotting.
Triplexator identified triplex target sites in double-stranded sequences able to accommodate a third strand and also assesses the compatibility of potential TFO/TTS pairs according to the canonical triplex formation rules (BUSKE et al., 2012).
A list of TTS sequences was with the promt: >triplexator -l 15 -e 0 –m R–mrl 4 –mrp 4 –of 0 –o results.tts –ds ychrbull.
With such promt it was set up a research in the database for TFOs with characteristics: not smaller than 15 bases (-l 15), no error rate (-e 0), a TFO made of poly-pyrimidine sequences (-m R), with multiple repeats of 4 bases (-mrl 4 –mrp 4), avoiding off-targets (-of 0). List of TTS was made with the name of results.tts and analysing a FASTA file containing the entire Bos taurus Y-chromosome sequence (GenBank accession no. CM001061.2).
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Triplex inspector is a different software suite able to calculate possible off-target sites (non-specific sequences that differ from the real target for about 5% of the nucleotides). Off target sites were analyzed for 20 sequences and listed in appendix, table 6 (BUSKE et al., 2013).
An additional BLAST analysis (The Basic Local Alignment Search Tool), which directly approximates alignments that optimize a measure of local similarity was also performed to check the sequence specificity on NCBI’s database (National Center for Biotechnology Information) (ALTSCHUL et al., 1990). On the webpage http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=OGP__9 913__10708 it is possible to check if the sequences listed by Triplexator belong to the bovine genome and if they are located on the sex chromosomes or autosomes.
3.2 Genomic DNA extraction from blood
Genomic DNA was isolated from white blood cells of female and male cattle. First, 10 mL of blood were diluted 1:1 in PBS, and centrifuged at 800 x g for 20 minutes. Pellet was washed 1x with 20mL of distilled water and vortexed for 1 minute to cause hemolysis . Thereafter 20 mL of 2x PBS were added to stop the hemolysis and centrifuged at 800 xg for 20 minutes. Hemolysis step was repeated at least 2 or 3 times until hemoglobin tracks disappeared. Cells were treated with a lysis buffer (50 mM Tris-HCl pH 8, 100 mM EDTA, 100 mM NaCl, 1% SDS) and 1mg/mL protein kinase. Each sample was incubated at 55 °C over night. The sample was centrifuged for 15 min at 20000 x g and the supernatant was mixed with 1 ml of saturated 6M
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NaCl, and centrifuged for 15 min at 20000 x g. The pellet was washed with 1 ml of ethanol 100% and centrifuged for 15 min at 20000 x g. A second washing step was performed diluting the pellet in 1ml of 70% ethanol at 37°C for 2 hours on a thermo- shaker. After centrifugation for 15 min at 20000 x g the pellet, containing the DNA, was dried in air and diluted in distilled water. gDNA concentration and purity were measured by spectrophotometry at 260 nm wavelength (NanoDrop ® ND-1000 Spectrophotometer).
3.3 Southern blot analysis
Genomic DNA from male and female cattle were digested as follows: 400ng of gDNA were incubated with 5 U of PstI (New England Biolabs, INC.), 0.1x BSA, 1x Pst buffer (New England Biolabs, INC.) for 2 hours at 37°C.
Southern blot was performed according to Current Protocols in Molecular Biology, 2003 (Unit 2.9A Southern Blotting) (MARCADET et al., 1989, SOUTHERN, 2006).
Electrophoresis in 0.8% agarose (TAE buffer) was performed of 1µg and 4µg respectively of bovine male and female gDNA for ~2 hours at 100V. The gel was first treated with 0.25M HCl for 10 minutes and washed with distilled water. A second treatment with denaturation buffer (1,5M NaCl and 0.5M NaOH) was made for DNA denaturation for 20 minutes, and washed with distilled water. The gel was finally washed for 20 minutes in neutralization buffer (0.5M of TRIS HCl pH 7 and 1.5M of NaCl) to neutralize the denaturation reaction. Gel was finally incubated with 20 x SSC for 10 minutes.
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The blotting was performed via upward capillary transfer of DNA from agarose gel onto a nylon membrane, using a 20x SSC buffer. DNA immobilization was achieved by UV irradiation (120x103 µJoule for 10 sec). The membrane was incubated at 42
°C for 16 hours with digoxigenine (DIG) labeled probes in hybridization buffer (DIG Easy Hybridization buffer by Roche) at concentration of 25 ng/ml. Probes for hybridization were synthesized by Eurofins MWG Operon (Eiserberg – Germany).
Washing steps after hybridization were made with high stringency to increase binding affinity. The digoxigenin-based detection system (Boehringer Mannheim) was assessed by incubating the membrane for 10 minutes in detection buffer (100ml 1M Tris HCl pH 9 and 33.3ml 3M NaCl) and CDP-Star® Chemiluminescent Substrate at a concentration of 0.25 mM. Membrane was then ready for chemiluminescence detection. Chemiluminescence and fluorescence detection were made using FUSION® image system acquisition.
3.4 Triplex melting curve analysis
Melting temperature curve is an analysis of the dissociation-characteristics of double- stranded DNA during heating and the same principle is valid for triplex stranded DNA analysis. The measurement is made using spectrophotometry, detecting the relationship between DNA absorbance and its structure. The physical principle is based on the fact that DNA structure has a lower absorbance (a value that correlates the light absorbed by a chemical substance) when in triplex formation. Rising the temperature, the triplex DNA structure melts and the third strand detaches from the target dsDNA corresponding to a different absorbance. A further increase of the
33
temperature will melt the dsDNA. Two variations of the absorbance at two different temperatures were recorded, one for triplex melting and a second for the duplex melting.
dsDNA target (listed in table 1) were designed in a way that 2 molecules of the same TFO could hybridize to one molecule of target dsDNA, as represented in figures 6.
Figure 6: Melting temperature principle of analysis. A) Structure of target dsDNA in black and TFO B) principle of melting temperature analysis, rising the temperature a first melting point is recorded due to melting of triplex structure. A second melting point is registered at higher temperature due to dsDNA melting. Absorbance is directly proportional to melting temperature.
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Table 1: Target dsDNA sequences for each TFO
Name Sequence
Target Seq1 Fw 5’AGAAAGGAAGAAAAGGAAAGAGAAAGGAAGAAAAGGAAAG Target Seq1 Rw 5’CTTTCCTTTTCTTCCTTTCTCTTTCCTTTTCTTCCTTTCT Target Seq2 Fw 5’AAGGGAAAGGAAGGAAGAGAAGGGAAAGGAAGGAAGAG Target Seq2 Rw 5’CTCTTCCTTCCTTTCCCTTCTCTTCCTTCCTTTCCCTT
Target Seq3 Fw 5’GAGGGAGGGAAAAGGGAGGAAGGAGGGAGGGAAAAGGGAGGAAG Target Seq3 Rw 5’CTTCCTCCCTTTTCCCTCCCTCCTTCCTCCCTTTTCCCTCCCTC
ssDNA for dsDNA target were formed by incubating each complementary probe at a concentration of 100pmol in water solution. The sample was warmed up to 95 °C and slowly cooled down.
To assess the melting temperature experiment, target dsDNA was first synthesized.
Complementary ssDNA target oligonucleotides were ordered from Eurofins MWG Operon (Eiserberg Germany). Complementary probes were diluted in distilled water at equimolar concentration (100pmol/µL), warmed up to 95°C for 10 minutes, and slowly cooled down to RT in order to allow a dsDNA formation. Samples were stored at 4 or -20 °C.
Melting point was measured after incubation of 1µM dsDNA and 1µM TFO in 1x PBS pH 7 at 15°C and warmed up to 80°C. Measurements were made at a wavelength of 260 nm. Analyses were performed by University of Duisburg-Essen and Center for Nanointegration Duisburg-Essen (CENIDE).
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3.5 Triplex electrophoresis mobility shift assay (EMSA)
Electrophoresis mobility shift assay is a method used to validate probes triplex binding under electric field that shifts the localization of the free unbounded TFO from the hybridized ones (VASQUEZ et al., 2001, HELLMAN and FRIED, 2007).
To assess the EMSA, 1µg of male and female genomic DNA was isolated from blood and PstI digested (chapter 3.2, pp 30). gDNA was then incubated with 50 ng of DIG labeled TFO1, 2, and 3 in 1x PBS buffer pH 7 at different concentrations of MgCl2 (5, 10, and 20mM) at 19 °C for 1h. After incubation the samples were rapidly chilled on ice. A mobility electrophoresis in 0.8% agarose gel (TAE buffer) was performed at a RT of 5 °C. After electrophoresis, the gel was kept in 20x SSC for 10 minutes.
A blot was performed of the DNA/TFO sample after electrophoresis via upward capillary transfer of DNA from agarose gel onto a nylon membrane, using a 20x SSC buffer. DNA immobilization was achieved by UV irradiation (120x103 µJoule for 10 sec). As only TFOs were labeled to DIG, detection of DIG on the membrane was equivalent to detection of the triplex hybridization.
3.6 Sorted sperm nuclear matrix isolation
Bovine sperm were sorted as described by Rath et al. (RATH et al., 2009). More than 90% enrichment was achieved and purity validation of sorted samples was performed by reanalysis and by fluorescence in situ hybridization (FISH) (KAWARASAKI et al., 1998, RATH et al., 2009). Sperm were diluted in 10 mM Tris-
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HCl pH 7, 1mM EDTA. Sonication was performed at frequencies higher than 20 kHz to disrupt heads from tails and a density gradient Percoll (Sigma Percoll P-1644) centrifugation separated respective sub-fractions. A falcon tube was prepared with 3 phases of differently concentrated Percoll: 90% Percoll on the bottom of the tube, 67,5% Percoll gently charged on it and at last a 45% Percoll was charged on top. 1 mL of sonicated sperm sample was gently loaded over the 45% Percoll. Tube was centrifuged for 850 xg for 20 minutes. Percoll gradient centrifugation separated the sample with heads on the pellet and tails between phases of 65% and 90%. The procedure was repeated once.
Sperm heads were treated for de-membranization with 10% TritonX-100 in a buffer made of 10 mM Tris-HCl pH 7, 1 mM EDTA and 250 mM sucrose (KURODA and PORTER, 1987). Washing steps were performed by centrifugating the samples at 20000 x g for 5 minutes and resuspending it in the 10 mM Tris-HCl pH 7, 1 mM EDTA and 250 mM sucrose . At last sperm nuclear matrix were diluted in distilled water.
To confirm that membranes were mostly dissolved without a loss of nuclear structure, a Neil red staining for phospholipids was assessed as follows: A Nile red stock solution was prepared (500 µg/mL) in acetone and diluted with 75% aqueous glycerol to a concentration of 2.5 µg/mL. One drop of Neil red was used on sperm and sperm nuclear matrix was fixed on a slide. Neil red staining has an excitation wavelength between 515-530 nm and an emission wavelength between 526 and 605 nm (DIANE L. PECHT and STEPHEN K. DURHAM, 2007).
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A control group of normal sperm not subjected to demembranization was also stained with the same procedure. Images of demembranized sperm nuclear matrix and normal spermatozoa were evaluated under a confocal microscope (Figure 7).
Light microscopical visualization was performed using an Axioplan 200 and a confocal imaging system LSM510 (Carl Zeiss MicroImaging GmbH, Jena, Germany) within the spectrum of visible light. A (Helium-Neon-Red) Helium-Neon-green laser of 543 nm was used to excite the Nile Red staining with emission detection from 560 nm.
Figure 7: Confocal microscopy images of bovine sperm nuclear matrix isolation. A) Normal bovine sperm stained with Neil red; B) Same sample of bovine sperm demembranized and stained also with Neil red.
3.7 Triplex fluorescence in situ hybridization (T-FISH)
Microscope slides of sorted bovine sperm nuclear matrix were prepared by letting a drop of sample suspension fall from a height of about 20 cm on the center of the slide. Once dried, the slides were incubated at 19 °C for 2 hours in triplex hybridization buffer (1x PBS pH 7 and 10 mM MgCl2 and 1 mM of fluorescent labeled
38
TFO probes). Chemical hybridization is based on Hoogsteen binding between the TFOs and the sperm nuclear matrix.
A single washing step was assessed using hybridization buffer (1x PBS pH 7 and 10 mM MgCl2) at 19 °C. Finally slides were dehydrated with ascending ethanol series and mounted with Vectashield ® mounting media. Slides were stored at 4°C or immediately analyzed under a fluorescence microscope (Olympus BX60). In total 200 observations were made.
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 had to have the same location as a hybridization duplex.
First, TFO1, 2 and 3 were used separately, on a second step, all three probes were used together to analyze a possible better outcome. 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. Since the result of the assay was a YES/NO hybridization, a Chi square analysis of 200 observations was performed.
Probes used for triplex fluorescent in situ hybridization were:
TFO1: 5'-YXYTYXXYTXYTYTXXYTYX-Alexa488 TFO2: 5'-TYXXXYTYXXYTXXYTXYX-Alexa488 TFO3: 5'-XYXXXYXXXYTTYXXXYXXYTX-Alexa488
39 3.8 Fluorescence in situ hybridization (FISH)
Fluorescent in situ hybridization is a common method used to identify specific gene sequences on the sperm heads based on a Watson and Crick chemical hybridization (RENS et al., 2001).
Slides with bovine sorted sperm were prepared by letting a drop fall from about 10 cm above on microscopes slides. Samples were dried for about 30 minutes at 37 °C.
Slides were treated for de-condensation with 3M NaOH for 3 minutes, rapidly washed twice in H2O and dehydrated with ascending ethanol series (70%, 90%, and 100%).
Slides were incubated at 37°C for 2 hours in hybridization solution buffer previously prepared (2x SSC, 30% formamide and 1mM of fluorescent labeled probe) (SILAHTAROGLU et al., 2007).
First washing step was made using 0.1x SSC tween 1% at 41°C for 5 minutes and in 0.1x SSC for 5 minutes at RT. Finally, slides were dehydrated with ascending ethanol series. Vectashield ® mounting media was used, and slides were stored at 4°C or immediately analyzed under a fluorescence microscope (Olympus BX60).
Probes for duplex FISH were:
TFO1: 5'- XYTYXXTYTYXTYXXYTYXY-Alexa488 TFO2: 5'- XTXTYXXTYXXYTYXXXYT-Alexa488 TFO3: 5'- XTYXXYXXXTTYYXXXYXXXYX-Alexa488
3.9 Gold nanoparticle synthesis and conjugation to TFOs
AuNPs were obtained by pulsed laser-ablation in liquid (Walter, Petersen et al. 2010, Barcikowski and Compagnini 2013, Rath, Barcikowski et al. 2013, Rehbock, Merk et
40
al. 2013, Taylor, Barchanski et al. 2014, Tiedemann, Taylor et al. 2014). Laser generation of AuNPs was performed utilizing a Spitfire Pro femtosecond laser system (Spectra-Physics) providing 120 fs laser pulses at a wavelength of 800 nm. Ablation was performed while moving a 5×5 mm gold foils at a constant speed of 60 mm×min- 1 in a spiral. Conjugation to TFOs was performed ex situ in water solution. The TFO1 probe was chosen as candidate to be labeled to AuNPs. It was synthesized with a 10 thyminie spacer and a thiol group on the 5’ prime end. 5'-(THIOL)-TTTTTTTTTT- YXYTYXXYTXYTYTXXYTYX. Thiol group was added because it binds to gold surfaces with high affinity and is used as stabilizing agents which bind to the surface of the AuNPs by formation of Au-sulfur bonds (TEMPLETON et al., 2000).
3.10 Gold nanoparticle hybridization and SPR shift analysis
AuNP/TFO conjugates (16.9 µg/mL and 22.5 µg/mL) were incubated with 26 ng/mL of male and female bovine genomic DNA extracted from lymphocytes Hybridization conditions were provided in 1x PBS pH 7 buffer and at atemperature of 19 °C. SPR shift analyses were performed by photometric analysis from a wavelength of 200 up to 700 nm. SPR analyses were made by Center for Nanointegration Duisburg-Essen (CENIDE).
Hybridization of AuNP/TFO conjugates were tested on sorted sperm nuclear matrix by incubation of 22.6 µg/mL AuNPs conjugate with 1x106 of sorted sperm nuclear matrix. SPR shift analyses were performed by UV-Vis analysis from a wavelength of 200 up to 700 nm. SPR analyses were performed by Center for Nanointegration Duisburg-Essen (CENIDE).
41 3.11 Statistical analysis
DNA and sperm samples were taken from two Holstein bulls and cows respectively, and analysed together to avoid individual variability. The software R i386 313 was used for computational statistical analysis. Since data are based on a yes/no hybridization, chi-square contingency test of independence and a Pairwise comparison of proportions was performed to compare the different TFOs and their hybridization with XY and XX genome. A percentage of the hybridization values was calculated and a one way ANOVA with t-test was performed with 95% of confidential interval.
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
association
Hits on Y- chromosome AGAAAGGAAGAAAAGGAAAG AC_000159.1;
NC_007304.5;
NC_007304.5
110 AAGGGAAAGGAAGGAAGAG NC_016145.1;
NC_007307.5;
NC_007299.5
18 GAGGGAGGGAAAAGGGAGGAAG NC_016145.1;
NW_001502062.2 17 AAAGGAGGAGAAGGAAAAGGAGGGG NW_001505400.1;
NC_016145.1;
NW_003100714.1
142 AGGGGAAGGAGAGAGAAGAGGGGGA NC_016145.1;
AC_000174.1 70 GGGGAAGGAGAGAGAAGAGGGGGAA AC_000174.1;
NC_016145.1 70 AAGGGGAAGGAGAGAGAAGAGGGGG NC_016145.1;
NW_001502173.1;
AC_000174.1
75 AAAGGGGAAGGAGAGAGAAGAGGGG NC_016145.1;
NW_001502173.1;
AC_000174.1
75 AAGGGGAAGGAGAGAGAAGAGGGGA NC_016145.1;
AC_000174.1 75 GGGGAAGGAGAGAGAAGAGGGGGAG NC_016145.1;
AC_000174.1 70
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
53
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).
