Thioctic acid derivatives as building blocks to incorporate DNA oligonucleotides onto gold nanoparticles

Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid-gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid-L- Threoninol derivatives containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.This work is supported by the European Commission (Grants FP7-NMP-213382-2, FUNMOL and NMP4-LA-2011-262943, MULTIFUN), by the Spanish Ministry of Education (Grant CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208) and the Instituto de Salud Carlos III (CB06_01_0019). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Modulation of the stability of i-motif structures using an acyclic threoninol cytidine derivative

The effect of aTNA (acyclic threoninol nucleic acids) units on the stability of intramolecular i-motifs was investigated by spectroscopic techniques. The replacement of selected positions of the C-core can modulate the stability at different pH ranges.This work was supported by Spanish Ministry of Economy and Competitiveness (grants CTQ2014-52588-R and CTQ2012-38616-C02-02), the Generalitat de Catalunya (grant 2014 SGR 1106; 2014 SGR 187) and the Communities MULTIFUN (contract NMP4-LA-2011-262943). CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Peer reviewe

Synthesis, Cell-Surface Binding, and Cellular Uptake of Fluorescently Labeled Glucose−DNA Conjugates with Different Carbohydrate Presentation

8 páginas, 5 figurasOligonucleotide conjugates carrying carbohydrates at the 5′-end have been prepared. Glucose, fucose, and saccharides containing glucose at the nonreducing end were attached to DNA strands using the classical phosphoramidite chemistry. Two types of spacers and a dendron scaffold helped to obtain a diversity of sugar presentations in the DNA conjugates. Cellular surface adsorption and cellular uptake of carbohydrate oligonucleotide antisense sequences were measured using flow cytometric analysis. Conjugates with the glucose moiety linked through long spacers (15 to 18 atom distances) were internalized better than those with short linkers (4 atom distance) and than DNA control strands without sugar modification. Conjugates with tetravalent presentation of glucose did not improve cell uptake.This work was supported by Consejo Superior de Investigaciones Científicas (PIF06-045); Spanish Ministry of Science (grants BFU2007-63287, BFU2007-62062, CTQ2006-01123,CTQ2009-13705), Generalitat de Catalunya (2009/SGR/208),and Instituto de Salud Carlos III (CIBER-BNN, CB06_01_0019). RL thanks CSIC for a JAE contract.Peer reviewe

Synthesis of Oligonucleotides Carrying Thiol Groups Using a Simple Reagent Derived from Threoninol

Oligonucleotides carrying thiol groups are useful intermediates for a remarkable number of applications involving nucleic acids. In this study, DNA oligonucleotides carrying tert-butylsulfanyl (t-BuS) protected thiol groups have been prepared. A building block derived from threoninol has been developed to introduce a thiol group at any predetemined position of an oligonucleotide. The resulting thiolated oligonucleotides have been used for the preparation of oligonucleotide conjugates and for the functionalization of gold nanoparticles using the reactivity of the thiol groups

Solid-phase synthesis of oligodeoxynucleotides containing N4-[2-(t-butyldisulfanyl)ethyl]-5-methylcytosine moieties

An efficient route for the synthesis of the phosphoramidite derivative of 5- methylcytosine bearing a tert-butylsulfanyl group protected thiol is described. This building block is used for the preparation of oligonucleotides carrying a thiol group at the nucleobase at the internal position of a DNA sequence. The resulting thiolated oligonucleotides are useful intermediates to generate oligonucleotide conjugates carrying molecules of interest at internal positions of a DNA sequence.This research was supported by the EECC (grants DYNAMO, NEST-ADV028669, FUNMOL, FP7-NMP-213382-2), by the Spanish Ministry of Education (BFU2007-63287) and the Generalitat de Catalunya (2009/SGR/208). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Peer reviewe

Synthesis of oligonucleotides carrying thiol groups using a simple reagent derived from threoninol

Oligonucleotides carrying thiol groups are useful intermediates for a remarkable number of applications involving nucleic acids. In this study, DNA oligonucleotides carrying tert-butylsulfanyl (t-BuS) protected thiol groups have been prepared. A building block derived from threoninol has been developed to introduce a thiol group at any predetemined position of an oligonucleotide. The resulting thiolated oligonucleotides have been used for the preparation of oligonucleotide conjugates and for the functionalization of gold nanoparticles using the reactivity of the thiol groups.This work was supported by the Spanish Ministry of Education (CTQ2010-20541-C03-01), the EECC (FUNMOL, FP7-NMP-213382-2), the AECID (A1/038984/11) and the Generalitat de Catalunya (2009/SGR/208). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. RF is recipient of a FPI contract from the Spanish Ministry of Science. We are thankful to Alejandra V. Garibotti for the analysis and purification of oligonucleotide 12 and Marta Vilaseca for the measurement of the mass of oligonucleotide 12 by electrospray.Peer reviewe

Synthesis and Properties of Oligonucleotides Carrying Isoquinoline Imidazo[1,2-a]azine Fluorescent Units

Oligonucleotides carrying novel fluorescent compounds with a dipolar isoquinoline imidazo[1,2-a]azine core were prepared. Analysis of the melting curves demonstrates that DNA duplexes carrying these fluorescent labels at their ends have a slight increase in DNA duplex stability. The UV absorption and fluorescent properties of the oligonucleotide conjugates were analyzed. The fluorescent label is sensitive to duplex formation, as cooperative melting curves are also observed at 366 nm and fluorescence has a large increase upon denaturation. Cell uptake studies allow observation of these fluorescently labeled oligonucleotides internalized into HeLa cells.This study was supported by the “Dirección General de Investigación Científica y Técnica” (grant BFU2007-63287, CTQ2010-20541 and CTQ2009-07758), and the Generalitat de Catalunya (2009/SGR/208). N. K. thanks the DGICYT for Ph.D. fellowship.Peer reviewe

Article Synthesis of Oligonucleotides Carrying Thiol Groups Using a Simple Reagent Derived from Threoninol

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Biophysical and RNA interference inhibitory properties of oligonucleotides carrying tetrathiafulvalene groups at terminal positions

Oligonucleotide conjugates carrying a single functionalized tetrathiafulvalene (TTF) unit linked through a threoninol molecule to the 3′ or 5′ ends were synthesized together with their complementary oligonucleotides carrying a TTF, pyrene, or pentafluorophenyl group. TTF-oligonucleotide conjugates formed duplexes with higher thermal stability than the corresponding unmodified oligonucleotides and pyrene- and pentafluorophenyl-modified oligonucleotides. TTF-modified oligonucleotides are able to bind to citrate-stabilized gold nanoparticles (AuNPs) and produce stable gold AuNPs functionalized with oligonucleotides. Finally, TTF- oligoribonucleotides have been synthesized to produce siRNA duplexes carrying TTF units. The presence of the TTF molecule is compatible with the RNA interference mechanism for gene inhibition.The authors thank Dr. Anna Avi˜n´o for providing the nonmodified oligonucleotides 11–14.This researchwas supported by the European Commission (Grants FP7-FUNMOL 213382 and NMP4-LA-2011-262943, MULTIFUN), by the Spanish Ministry of Education (grant CTQ2010-20541, SAF2010-15440), the Generalitat de Catalunya (2009/SGR/208), the Czech Science Foundation (P207/10/2214), the Ministry of Education, Youth and Sports of the Czech Republic (7E09054), and the Institute of Organic Chemistry and Biochemistry AS CR (RVO: 61388963). The authors declare no conflict of interests.Peer reviewe

Glucose conjugation of anti-HIV-1 oligonucleotides containing unmethylated CpG motifs reduces their immunostimulatory activity

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5′-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.Peer Reviewe