Hexaethylene Glycol Spacer Research Articles

An Improved PEG-Linked Solid Support for Minimizing Process-Related Impurities During Solid-Phase Synthesis of DNA and RNA Sequences.

The preparation of controlled pore glass (CPG) supports, functionalized with several hexaethylene glycol spacers, to alleviate the problems associated with the porosity of commercial CPG supports is described in this article. The pore size of CPG restricts the diffusion of reagents to the leader nucleoside embedded in porous supports; this inhibits efficient solid-phase syntheses of DNA and RNA sequences and, by default, the purity of those sequences through formation of a shorter than full-length oligonucleotide. Functionalization of a CPG support with five hexaethylene glycol spacers led to a 42% reduction in process-related impurities contaminating oligonucleotide sequences, compared to that obtained using the commercial long-chain alkylamine (LCAA) CPG support. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Preparation of the hydroxylated CPG support 3 Basic Protocol 2: Automated preparation of the CPG support 6 Basic Protocol 3: Automated preparation of the poly(hexaethylene glycol)-derived CPG 7 Basic Protocol 4: Automated functionalization of the poly(hexaethylene glycol)-derived CPG support 7 with leader deoxyribo- and ribonucleosides to provide the CPG support 9 Basic Protocol 5: Automated syntheses of DNA and RNA sequences on poly(hexaethylene glycol)-derived CPG support 9 and on a commercial long-chain alkylamine (LCAA) CPG support Support Protocol: Release and deprotection of the DNA and RNA sequences linked to the poly(hexaethylene glycol)-derived CPG support 10 and commercial LCAA-CPG support Basic Protocol 6: Comparative RP-HPLC analyses of crude, fully deprotected DNA or RNA sequences released from the poly(hexaethylene glycol)-derived CPG support 10 and from a commercial LCAA-CPG support.

An expedient process for reducing the formation of process-related impurities during solid-phase synthesis of potential nucleic acid-based drugs

With the intent of mitigating the formation of process-related impurities during solid-phase synthesis of DNA or RNA sequences, a hydroxylated controlled-pore glass support conjugated to three, five or seven hexaethylene glycol spacers was prepared and demonstrated to provide a more efficient and robust synthesis process. Indeed, the use of a support conjugated to five hexaethylene glycol spacers led to a 19% up to 42% reduction of process-related impurities contaminating synthetic nucleic acid sequences, when compared to that obtained from the same DNA/RNA sequences synthesized using a commercial long-chain alkylamine controlled-pore glass support under highly similar conditions.

Controlled Pore Glass-based oligonucleotide affinity support: towards High Throughput Screening methods for the identification of conformation-selective G-quadruplex ligands

Target selectivity is one of the main challenges in the search for small molecules able to act as effective and non-toxic anticancer and/or antiviral drugs. To achieve this goal, handy, rapid and reliable High Throughput Screening methodologies are needed. We here describe a novel functionalization for the solid phase synthesis of oligonucleotides on Controlled Pore Glass, including a flexible hexaethylene glycol spacer linking the first nucleoside through the nucleobase via a covalent bond stable to the final deprotection step. This allowed us preparing fully deprotected oligonucleotides still covalently attached to their supports. In detail, on this support we performed both the on-line synthesis of different secondary structure-forming oligonucleotides and the affinity chromatography-based screenings of conformation-selective G-quadruplex ligands. By using a fluorescent core-extended naphthalene diimide with different emitting response upon binding to sequences folding into G-quadruplexes of different topologies, we have been able to discriminate not only G-quadruplex vs. duplex DNA structures, but also different G-quadruplex conformations on the glass beads by confocal microscopy.

Gas-Phase Fragmentation Studies of Biotinylated, Hexaethylene Glycol–Spacered Oligosaccharides—Molecular Probes—Using Electrospray Mass Spectrometry on a Hybrid High-Resolution Mass Spectrometer

The electrospray ionization high-resolution mass spectra of biotinylated hexaethylene glycol–spacered molecular probes bearing biologically relevant carbohydrate moieties in positive and negative modes were recorded and interpreted. Collisionally induced decay mass spectra (positive mode) revealed different patterns depending on the charge of the parent ion, attached cations (or ions), the composition, and the sequence of carbohydrate fragments. The most intense peaks (two series) originated from the sequential cleavage of glycoside bonds resulting in charge location on the reducing end (Y series observed for all of the test compounds) or nonreducing end (B series). Hexaethylene glycol chain fragmentation giving rise to the cleavage of the C–O bond remote from the biotin moiety was observed. Other fragment ions lighter than the above by a difference of (C2H4O) n were absent or much smaller. Similar fragmentation was found for all of the nonsulfated biotinylated glycosides with the hexaethylene glycol spacer thus demonstrating that this type of fragmentation was characteristic of such molecular probes. Similar cleavages along with biotin moiety decay via the elimination of H2S and H2CS were observed for negative ions in the collisionally induced decay mass spectra of sulfated and neutral molecular probes.

Gas-phase fragmentation studies of biotinylated oligomannuronopyranosides under conditions of collisionally activated dissociation

High-resolution electrospray ionization mass spectra (MS and MS/MS) of a series of glycoconjugates containing biotin and oligomannuronopyranosyl residues linked via a hexaethylene glycol spacer were recorded in the positive and negative ion modes and interpret. In collision activation mass spectra of the positively charged ions (CID MS2), two series of the most abundant peaks were attributed to the fragments resulting from a glycosidic bond cleavage with localization of a charge at either reducing (Y series) or non-reducing end (B series). Fragmentation of hexaethylene glycol chain observed at relatively high activation energies (30 eV or higher) occurred predominantly at the C—O bond most distant from the biotin moiety. In the mass spectra of the negatively charged ions of the studied compounds, the peaks of B, C, and Z series (a systematic nomenclature for carbohydrate fragmentations was used) were present along with the peak analogous to the aforementioned one, which arose from the cleavage of the same C—O bond. The distinctive feature of the fragmentation of the negative ions is the elimination of H2S and H2CS from the biotin moiety.

Hairpin ODN-based ligands as potential inhibitors of HMGB1 cytokine activity

We here report the design, synthesis, and biological activity of a kinked hairpin-loop DNA acting at low nM concentrations as a strong inhibitor of HMGB1 (High-Mobility Group Box-1), a nuclear protein with cytokine activity in a number of inflammatory diseases. Lead compound optimization has been realized by inserting different oligo-ethylene glycol spacers at the 5′-end and loop positions of the natural hairpin DNA, in order to improve its enzymatic stability and structuring capability, as well as its overall pharmacokinetic properties. Thermal stability data as well as activity assays proved that the ODN which contained two hexa-ethylene glycol spacers, one at the 5′-end and the other in the loop, was the best candidate to inhibit HMGB1. Plasma stability assays and hydrodynamic volume measurements afforded further encouraging results in view of future in vivo evaluation of the optimized ligand.

Conjugation of PEG and gold nanoparticles to increase the accessibility and valency of tethered RNA splicing enhancers

Since alternative splicing patterns are exhibited by most mammalian genes, the functional properties of a gene may be changed for therapeutic purposes by nudging splicing from one pattern to another. Splicing to a refractory exon can be enhanced using bifunctional targeted oligonucleotide enhancers of splicing (TOES), which anneal to a target exon and carry additional sequence motifs to recruit activator proteins. We have tested here the effectiveness of increasing the availability of the activating domain by using click chemistry to insert spacers of hexaethylene glycol (HEG) or abasic DNA between the activating and annealing domains of the oligonucleotide. We show that the presence of one or two HEG spacers can increase the effectiveness of an oligonucleotide, but the inclusion of 11 or 20 reduced the activity. To increase the valency of the oligonucleotides further, the annealing and activating domains were conjugated to gold nanoparticles (GNPs). We show that activity of these GNP–RNA conjugates in splicing assays depended on the size of the GNP conjugates, the RNA sequence and the directionality (i.e. attachment to GNP at either the 5′ or 3′-ends) of the RNA strands conjugated to the GNP surface. Strikingly, only the activating domains were required.

First-in-Human Trial of a STAT3 Decoy Oligonucleotide in Head and Neck Tumors: Implications for Cancer Therapy

Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as "undruggable." We developed a decoy targeting STAT3 and conducted a phase 0 trial. Expression levels of STAT3 target genes were decreased in head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control. Decoys have not been amenable to systemic administration due to instability. To overcome this barrier, we linked the oligonucleotide strands using hexaethylene glycol spacers. This cyclic STAT3 decoy bound with high affinity to STAT3 protein, reduced cellular viability, and suppressed STAT3 target gene expression in cancer cells. Intravenous injection of the cyclic STAT3 decoy inhibited xenograft growth and downregulated STAT3 target genes in the tumors. These results provide the first demonstration of a successful strategy to inhibit tumor STAT3 signaling via systemic administration of a selective STAT3 inhibitor, thereby paving the way for broad clinical development. This is the fi rst study of a STAT3-selective inhibitor in humans and the fi rst evidence that a transcription factor decoy can be modifi ed to enable systemic delivery. These findings have therapeutic implications beyond STAT3 to other “undruggable” targets in human cancers.

Synthesis and Evaluation of New Spacers for Use as dsDNA End-Caps

A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl-protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. End-caps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. End-caps containing either a terthiophene or a naphthalene tetracarboxylic acid diimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A*T base pair. Spacers containing only methylene (-CH(2)-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

Abstract 4139: Development of a transcription factor decoy for efficient systemic administration in head and neck cancer

Abstract This study was aimed to develop a transcription factor decoy for efficient systemic administration in head and neck cancer. Signal Transducer and Activator of Transcription-3 (STAT3) is upregulated in numerous human cancers including head and neck squamous cell carcinoma (HNSCC). Small molecules that target STAT3 are limited by lack of specificity. We developed a transcription factor decoy targeting STAT3 consisting of a 15-mer double-stranded oligonucleotide with three phosphorothioate modifications (PTO) at the 3′ and 5′ end of the double-stranded structure. This decoy exhibits antitumor efficacy in preclinical cancer models and is now being tested in a phase 0 human trial with intratumoral inoculation. To enable systemic administration, the STAT3 decoy formulation has been modified to enhance stability and uptake into tumors. A series of chemically modified STAT3 decoys have been designed to increase resistance to nucleases and enhance stability when administered in vivo. These include tetra-nucleotide single stranded DNA sequences (DN4), 18-atom hexa-ethyleneglycol spacers (DS18), and locked nucleic acid (LNA-1) modifications to enhance stability in serum. The modified STAT3 decoys were tested for serum nuclease degradation, melting temperature, binding to pSTAT3 protein, uptake and biologic activity. LNA-1 showed increased half-life of up to 8 h in serum, whereas DN4 is stable up to 4 h, DS18 up to 3 h compared with the parent STAT3 decoy, which is resistant only up to 2 h. The melting temperature demonstrated increased thermal stability for all the modified decoys compared to the parent STAT3 decoy. The DN4, DS18 and LNA-1 modified decoy bound to recombinant STAT3 protein as well as the parent decoy and inhibited head and neck cancer cell proliferation in vitro at nanomolar concentrations. The modified STAT3 decoys (DN4, DS18 and LNA-1) showed increased uptake into head and neck cancer cells similar compared with the parent STAT3 decoy. These modified decoys will be tested for their effect on STAT3 target gene expression and in vivo antitumor efficacy when administered systemically with anticipation of implementing a phase I clinical trial if tumor growth inhibition is observed upon intravenous injection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4139.

Surface Plasmon Resonance Study of Protein−Carbohydrate Interactions Using Biotinylated Sialosides

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

Electrooptical Properties of Main-Chain Mesogenic Polymers in Nematic State and Dilute Solutions

Electrooptical and dielectric properties of two aromatic polyesters with hexaethyleneglycol spacers (as well as their low molecular weight structural analogues) were studied in dilute solutions and in nematic state using the methods of flow birefringence, equilibrium electric birefringence, and orientational elastic deformations in electric fields. It was shown that the macromolecules under investigation orientated in the electric field according to the small-scale mechanism, and the Kerr constant K values are in good agreement with the dipole structure of their molecules. The sign of dielectric anisotropy Δϵ for the polymer nematics under investigation coincides with the sign of K in dilute solutions. The values of Δϵ for polymer nematics are close to the same characteristics for their low molecular weight structural analogues. The comparative analysis of the data obtained for these polymers in dilute solutions and in the nematic state is carried out.

A novel carboxy-functionalized photocleavable dinucleotide analog for the selection of RNA catalysts

A new multifunctional dinucleotide analog is synthesized for the application in in vitro selection experiments with linker-coupled reactants. For this purpose it contains a 5′-pCC ligation site, three flexible hexaethylene glycol spacers, a photocleavable o-nitrobenzyl unit and a 3′-terminal carboxy-function which can be derivatized with potential reactants as demonstrated with three model compounds.

Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.

A novel linker for the synthesis of oligonucleotides on a glass support is described. Oligonucleotides synthesised on the support remain tethered to the support after ammonia treatment and are shown to take part in sequence specific hybridisation reactions. These hybridizations were carried out with oligonucleotides synthesised on 'ballotini' solid sphere glass beads and microscope slides. The linker has a hexaethylene glycol spacer, bound to the glass via a glycidoxypropyl silane, terminating in a primary hydroxyl group that serves as starting point for automated or manual oligonucleotide synthesis.