Use of gamma-aminopropyl-coated glass surface for the patterning of oligonucleotides through oxime bond formation

The present work reports on the preparation of glass surfaces coated with NPPOC-protected aminooxy groups and their use for the patterning of oligonucleotides on glass slides and in capillary tubes. The method involves the use of surfaces coated with amino groups using (gamma-aminopropyl)triethoxy silane and subsequent grafting of the aminooxy groups by using the activated ester 1. The NPPOC-protected aminooxy groups on the surfaces can be cleaved upon irradiation. The free aminooxy groups so obtained are subsequently reacted with aldehyde-containing oligonucleotides to achieve efficient surface patterning.     

Efficient conjugation of oligonucleotides through aromatic oxime formation

The present work reports on the preparation of oligonucleotide conjugates via the formation of aromatic oxime linkage. The conjugation consists in the reaction between the oligonucleotide derivatized at 5′-extremity with a benzaldehyde moiety and an aminooxy reporter group. The conjugation was found highly efficient and was extended for the conjugation of phosphorothioate oligonucleotide. In addition, the stability of the so-formed oxime conjugate was investigated.     

Oxime bond formation for the covalent attachment of oligonucleotides on glass support

The chemical attachment of oligonucleotides on glass slides has been achieved using oxime bond formation. This method has been shown very efficient by comparison with the attachment of amino-oligonucleotides via reductive amination.     

Aldehydic oligonucleotide: a key intermediate for the preparation of oligonucleotide conjugates through oxime bond formation

Oligonucleotides functionalized with an aldehyde group are the key intermediates used for the preparation of peptide-oligonucleotide conjugates through the formation of an oxime linkage. Herein, we describe a brief overview of various synthetic protocols developed in our laboratory for the preparation of aldehyde containing oligonucleotides and their subsequent conjugation with peptides.     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

Efficient disulfide bond formation in virus-like particles

Virus-like particles (VLPs) consist of a virus's outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed.     

Submicron patterning of DNA oligonucleotides on silicon

The covalent attachment of DNA oligonucleotides onto crystalline silicon (100) surfaces, in patterns with submicron features, in a straightforward, two-step process is presented. UV light exposure of a hydrogen-terminated silicon (100) surface coated with alkenes functionalized with N-hydroxysuccinimide ester groups resulted in the covalent attachment of the alkene as a monolayer on the surface. Submicron-scale patterning of surfaces was achieved by illumination with an interference pattern obtained by the transmission of 248 nm excimer laser light through a phase mask. The N-hydroxysuccinimide ester surface acted as a template for the subsequent covalent attachment of aminohexyl-modified DNA oligonucleotides. Oligonucleotide patterns, with feature sizes of 500 nm, were reliably produced over large areas. The patterned surfaces were characterized with atomic force microscopy, scanning electron microscopy, epifluorescence microscopy and ellipsometry. Complementary oligonucleotides were hybridized to the surface-attached oligonucleotides with a density of 7 × 10¹² DNA oligonucleotides per square centimetre. The method will offer much potential for the creation of nano- and micro-scale DNA biosensor devices in silicon.     

Structural basis for cell surface patterning through NetrinG-NGL interactions

Brain wiring depends on cells making highly localized and selective connections through surface protein-protein interactions, including those between NetrinGs and NetrinG ligands (NGLs). The NetrinGs are members of the structurally uncharacterized netrin family. We present a comprehensive crystallographic analysis comprising NetrinG1-NGL1 and NetrinG2-NGL2 complexes, unliganded NetrinG2 and NGL3. Cognate NetrinG-NGL interactions depend on three specificity-conferring NetrinG loops, clasped tightly by matching NGL surfaces. We engineered these NGL surfaces to implant custom-made affinities for NetrinG1 and NetrinG2. In a cellular patterning assay, we demonstrate that NetrinG-binding selectivity can direct the sorting of a mixed population of NGLs into discrete cell surface subdomains. These results provide a molecular model for selectivity-based patterning in a neuronal recognition system, dysregulation of which is associated with severe neuropsychological disorders.     

Chemoselective oxime and thiazolidine bond formation: a versatile and efficient route to the preparation of 3'-peptide-oligonucleotide conjugates

Oligonucleotides carrying an aldehyde moiety at the 3'-end were synthesized by the oxidation of a 1,2-diol precursor. These were coupled to peptides bearing a cysteine residue for thiazolidine formation and an aminooxy group for oxime formation. The conjugation reaction proved very efficient and selective, thereby allowing the preparation of 3'-peptide-oligonucleotide conjugates in good yield. The conjugation was achieved in aqueous solution without using any protection strategy. Moreover, the present approach neither requires the use of peptide in excess nor changes the hybridization properties of the conjugates.     

Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides

A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self-quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluorophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies.     

Glutathionylation of lens proteins through the formation of thioether bond

Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.     

The oxime bond formation as an efficient chemical tool for the preparation of 3',5'-bifunctionalised oligodeoxyribonucleotides

The simultaneous conjugation of peptides or carbohydrates at the 3'- and 5'-end of oligodeoxyribonucleotides was achieved very efficiently through chemoselective oxime bond formation. The method employs bifunctionalised oligonucleotides in single step without the need of protection strategy, under mild acidic conditions. The conjugates were obtained in high yields by reacting an oxyamine containing reporter groups (peptide, mono- and disaccharide) with an oligonucleotide carrying an aldehyde at each extremity.     

RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.     

Geminin is required for left-right patterning through regulating Kupffer's vesicle formation and ciliogenesis in zebrafish

Geminin plays an important role in coordinating the cell cycle with anterior–posterior patterning during embryonic development. However, whether it is involved in the regulation of left–right (LR) patterning remains unknown. Here, we reported that geminin is required for setting up heart and visceral laterality during zebrafish development. Defective heart and visceral laterality was observed in geminin morphants. Further study demonstrated that the left-sided nodal/spaw in the lateral plate mesoderm (LPM) as well as the sideness of its downstream targets lefty2 and lefty1 was perturbed in geminin morphants. Upstream of the left-sided Nodal signal along the regulatory cascade of LR asymmetry, knock down of geminin resulted in defective Kupffer’s vesicle (KV) formation and ciliogenesis rather than middle line defects. Predominant distribution of an antisense morpholino against geminin in dorsal forerunner cells (DFCs) led to defective KV morphogenesis and perturbed LR asymmetry, similar to those of geminin morphants, indicating a cell-autonomous role of geminin in regulating KV formation and ciliogenesis. Our results demonstrate that geminin is required for proper KV formation and ciliogenesis, thus playing an important part in setting up LR asymmetry.     

Abiotic formation of oligonucleotides on basalt surfaces

The complication and further evolution of abiotic syntheses products occurred under environmental influences at the prebiological stage. From this point of view, the influence of some types of irradiation on the organic molecules adsorbed on the surfaces of volcanic rocks, appeared to be of great importance. In this connection, the effect of gamma rays on the AMP molecules adsorbed on mineral surfaces such as cinders and ashes has been studied. It has been shown that they can polymerize with the formation of oligonucleotides. The treatment of oligomers obtained by venom phosphodiesterase has shown that a polymeric product has mainly 3–5 and 2–5 bonds between nucleotides. The results obtained have been discussed from the evolutionary aspect.     

Sequence specificity of Z-DNA formation in oligonucleotides

The sequence specific requirement for B----Z transition in solution was examined in d(CGTGCGCACG), d(CGTACGTACG), d(ACGTACGT) in presence of various Z-inducing factors. Conformational studies show that inspite of the alternating nature of purines and pyrimidines, the aforementioned sequences do not undergo B----Z transition under the influence of NaCl, hexamine cobalt chloride and ethanol. A comparison with the crystal structures of an assorted array of purine and pyrimidine sequences show that the sequence requirement for B----Z transition is much more stringent in solution as compared to the solid state. The disruptive influence of AT base pairs in B to Z transition is discussed.