Triplex-hybridizing bioconjugated gold nanoparticles for specific Y-chromosome sequence targeting of bull spermatozoa

Abstract

Gold nanoparticles (AuNPs) are widely used in biomedical applications for drug targeting and bioimaging. This often neccesitates their functionalization with biomolecules carrying a defined biological function, yielding gold nanoparticle bioconjugates. The utilization of triplex-forming oligonucleotides (TFOs) as ligands gives access to nanoconjugates as tools for specific DNA-related nanotargeting via triplex hybridization. Since triplex hybridization with nanobioconjugates has to date not been shown on biologically relevant samples, sex-specific sperm marking may be an appropriate model system to demonstrate the opportunities of this targeting method in vitro. In this study, we focused on specific labeling of repetitive target sites enriched on the bovine Y-chromosome using triplex forming oligonucleotides. First, the functionality of a specific locked nucleic acid (LNA) sequence was confirmed on bovine free DNA and on demembranated sperm heads. Thereafter, the influence of AuNPs on triplex hybridization was spectrophotometrically analyzed employing synthetic dsDNA, genomic DNA and demembranated sperm heads. Results from the SPR-peak shift indicate that TFO-AuNP hybridize to bovine gDNA in a qualitative and significant manner. These results confirm successful triplex hybridization on biologically relevant target sites as well as the establishment of a method to use gold nanoparticles as a suitable tool for sex-selective hybridization.

Perspective of Surfactant‐Free Colloidal Nanoparticles in Heterogeneous Catalysis

“…The LAL technique grants both options of either obtaining pure or modified nanoparticle surfaces after subsequent functionalization. In the latter case, starting from the natural (surfactant‐free) surface, functionalization and sub‐monolayer surfactant coverages of various organic molecules are accessible . Hence, one of the major advantages of laser‐based catalyst preparation is the free accessibility of the nanoparticle surface providing model materials in various compositions without necessary procedural adaptions.…”

“…Hence, surfactant‐free nanoparticle systems provide an ideal reference system for mechanistic studies to develop or verify kinetics or structure‐activity correlations. A subsequently targeted functionalization either with surfactants or by deposition on support materials enables parametric studies where only a single parameter is varied while the others are maintained. Thus, surfactant‐free NPs may also be used as reference material for other independent investigations of nanoparticle properties in current research.…”

“…Additionally, the surfactant‐free nanoparticle surfaces provide an ideal prerequisite for a systematic study of the influence of surfactants in comparison with bare NPs . Apart from that, intended NP functionalization can be performed independently of other additives to modify the particle properties even at sub‐monolayer concentrations . Hence, regarding scientific studies as well as potential industrial applications, the laser‐based catalyst preparation method offers the possibility of precisely designed nanomaterials with potentially augmented catalytic activity and selectivity.…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Additionally, nanoparticles have been tested as non‐photobleaching alternative labels for flow cytometry‐based sex‐sorting procedures which has limitations, such as reduction in sperm fertility. Gold nanoparticles (AuNPs), which are widely used in biomedical applications, conjugated with locked nucleic acids (LNAs) and cell‐penetrating peptides could be a suitable tool for sperm sex sorting (Barchanski et al, ; Gamrad et al, ). Functionalization of AuNPs with bull Y‐chromosome‐specific triplex target sequences were successfully hybridized with genomic DNA and the condensed sperm nuclear matrix (Gamrad et al, ).…”

“…Gold nanoparticles (AuNPs), which are widely used in biomedical applications, conjugated with locked nucleic acids (LNAs) and cell‐penetrating peptides could be a suitable tool for sperm sex sorting (Barchanski et al, ; Gamrad et al, ). Functionalization of AuNPs with bull Y‐chromosome‐specific triplex target sequences were successfully hybridized with genomic DNA and the condensed sperm nuclear matrix (Gamrad et al, ). Future improvements regarding the use of nanoparticles and molecular probes may represent a promising strategy for sex sorting of pig sperm.…”