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Indole was conjugated to deoxyuridine either directly or via ethynyl linkage to synthesize two new DNA monomers. Due to the high reactivity of indole toward electrophilic substitution reactions, unwanted iodination was observed during the oxidation step with iodine oxidizer in automated DNA synthesizer leading to an additional oligonucleotide having extra 126 mass unit beside the desired oligonucleotide. On the other hand, using CSO oxidizer ensured the formation of the wanted oligonucleotide. Oligonucleotide synthesis was confirmed by MALDI-TOF-MS analysis. Polypyrimidine strands containing non-iodinated indole nucleoside were able to form parallel triplexes, antiparallel RNA and DNA duplexes. The thermal denaturation experiments showed higher triplex stabilization for 5-(5-indolylethynyl)-2′-deoxyuridine over 5-(5-indolyl)-2′-deoxyuridine as the triple bond allows twisting to put the indole into a proper position within the triplex encouraging better π-π stacking.

In pancreatic Panc-1 cancer cells, an increase of oxidative stress enhances the level of 7,8-dihydro-8-oxoguanine (8OG) more in the KRAS promoter region containing G4 motifs than in non-G4 motif G-rich genomic regions. We found that H2O2 stimulates the recruitment to the KRAS promoter of poly [ADP-ribose] polymerase 1 (PARP-1), which efficiently binds to local G4 structures. Upon binding to G4 DNA, PARP-1 undergoes auto PARylation and thus becomes negatively charged. In our view this should favor the recruitment to the KRAS promoter of MAZ and hnRNP A1, as these two nuclear factors, because of their isoelectric points &gt, 7, are cationic in nature under physiological conditions. This is indeed supported by pulldown assays which showed that PARP-1, MAZ, and hnRNP A1 form a multiprotein complex with an oligonucleotide mimicking the KRAS G4 structure. Our data suggest that an increase of oxidative stress in Panc-1 cells activates a ROS-G4-PARP-1 axis that stimulates the transcription of KRAS. This mechanism is confirmed by the finding that when PARP-1 is silenced by siRNA or auto PARylation is inhibited by Veliparib, the expression of KRAS is downregulated. When Panc-1 cells are treated with H2O2 instead, a strong up-regulation of KRAS transcription is observed.

New compounds 5 and 9 using DNA bases e.g. Adenine 1 and Guanine 6 derivatives have been synthesized. The use of simple methods to synthesize compounds 5 and 9 were done using pyrimidine as an alternative DNA base ring. Another design to synthesize new simple pyrimidine rings utilizing thiourea and ethylcyano acetate to afford 6-amino-2-thiouracil was adopted. The reaction of thiouracil 10 with chloro cyano or chloro ester and ketone, resulted in the formation of adduct compounds 18-21 , rather than the formation of compound 17 . All the synthesized compounds were subjected to docking study, in order to gain insights into their binding modes against cyclin-dependent protein kinase 2 (CDK-2) that is involved heavily in cell cycle regulation and receptor protein B-cell lymphoma 2 (BCL-2) which is involved in cell apoptosis. These targets were selected based on their key roles in cancer progression via the regulation of the cell cycle and DNA replication. Molecular-docking analyses showed that compound 14e was the best docked ligand against both targets, as it displayed the lowest binding energy, critical hydrogen bonds and hydrophobic interactions with the targets.

KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.

The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.

Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.

Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now established a method, which conclusively demonstrates that an LNA ON can strand invade into duplex DNA. To make Zorros smaller in size and easier to design, we synthesized 3 0 –5 0 –5 0 –3 0 single-stranded Zorro-LNA (ssZorro) by using both 3 0 - and 5 0 -phosphoramidites. With ssZorro, a significantly greater extent and rate of double-strand invasion (DSI) was obtained than with conventional 2-ON Zorros. Introducing hydrophilic PEG-linkers connecting the two strands did not significantly change the rate or extent of DSI as compared to ssZorro with a nucleotide-based linker, while the longest alkyl-chain linker tested (36 carbons) resulted in a very slow DSI. The shortest alkyl-chain linker (3 carbons) did not reduce the extent of DSI of ssZorro, but significantly decreased the DSI rate. Collectively, ssZorro is smaller in size, easier to design and more efficient than conventional 2-ON Zorro in inducing DSI. Analysis of the chemical composition of the linker suggests that it could be of importance for future therapeutic considerations.

KRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription factor essential for KRAS transcription. It is based on the use of palmitoyl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with lipid-modified G4-decoy oligonucleotides and a lipid-modified cell penetrating TAT peptide. The potency of the strategy in pancreatic cancer cells is demonstrated by cell cytometry, confocal microscopy, clonogenic and qRT-PCR assays.

G-rich anti-parallel DNA triplexes were modified with LNA or α-l-LNA in their Watson-Crick and TFO strands. The triplexes were formed by targeting a pyrimidine strand to a putative hairpin formed by Hoogsteen base pairing in order to use the UV melting method to evaluate the stability of the triplexes. Their thermal stability was reduced when the TFO strand was modified with LNA or α-l-LNA. The same trend was observed when the TFO strand and the purine Watson-Crick strand both were modified with LNA. When all triad components were modified with α-l-LNA and LNA in the middle of the triplex, the thermal melting was increased. When the pyrimidine sequence was modified with a single insertion of LNA or α-l-LNA the ΔTm increased. Moreover, increasing the number of α-l-LNA in the pyrimidine target sequence to six insertions, leads to a high increase in the thermal stability. The conformational S-type structure of α-l-LNA in anti-parallel triplexes is preferable for triplex stability.

HRAS is regulated by two neighbouring quadruplex-forming GC-elements (hras-1 and hras-2), located upstream of the major transcription start sites (doi: 10.1093/nar/gku 5784). In this study we demonstrate that the C-rich strands of hras-1 and hras-2 fold into i-motif conformations (iMs) characterized under crowding conditions (PEG-300, 40% w/v) by semi-transitions at pH 6.3 and 6.7, respectively. Nondenaturing PAGE shows that the HRAS C-rich sequences migrate at both pH 5 and 7 as folded intramolecular structures. Chromatin immunoprecipitation shows that hnRNP A1 is associated under in vivo conditions to the GC-elements, while EMSA proves that hnRNP A1 binds tightly to the iMs. FRET and CD show that hnRNP A1 unfolds the iM structures upon binding. Furthermore, when hnRNP A1 is knocked out in T24 bladder cancer cells by a specific shRNA, the HRAS transcript level drops to 44 ± 5% of the control, suggesting that hnRNP A1 is necessary for gene activation. The sequestration by decoy oligonucleotides of the proteins (hnRNP A1 and others) binding to the HRAS iMs causes a significant inhibition of HRAS transcription. All these outcomes suggest that HRAS is regulated by a G-quadruplex/i-motif switch interacting with proteins that recognize non B-DNA conformations.

In a previous study we have demonstrated that two neighboring G-quadruplexes, hras-1 and hras-2, located immediately upstream of the major transcription start site of HRAS, bind MAZ, a nuclear factor that activates transcription (Cogoi, S.; et al. Nucl. Acid Res. 2014, 42, 8379). For the present study we have designed G4 oligonucleotides with anthraquinone insertions and locked nucleic acids (LNA) modifications mimicking quadruplex hras-1. Luciferase, qRT-PCR, and Western blot data demonstrate that these constructs efficiently down regulate HRAS in T24 bladder cancer cells. The inhibitory efficiency of the G4 oligonucleotides correlates with their nuclease resistance in the cell environment. By chromatin immunoprecipitation we show that the association of MAZ to the HRAS promoter is strongly attenuated by the designed G4 oligonucleotides, thus suggesting that these constructs behave with a decoy mechanism.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are promising drugs for the treatment of HIV when used in combination with other anti-HIV drugs such as nucleoside reverse transcriptase (RT) inhibitors and protease inhibitors. The first generation of NNRTIs have, however, suffered from the rapid development of resistance. This review discusses the properties of the FDA-approved NNRTI drugs and focuses on the recent efforts being made to produce second generation inhibitors that circumvent this resistance problem.

1,8-Diazabicyclo[5.4.0]undec-7-ene salts of 2-methyl-4(5)-nitroimidazole or benzotriazole were obtained in crystalline form. Michael-type addition of these salts to (4S,5R)-(E)-4,6-di-O-acetyl-5-hydroxy-2-hexenal gave, after acetylation of the product, an isomeric mixture of acetylated 3-(azol-1-yl)-2,3-dideoxy-D-arabino-hexopyranosides and 3-(azol-1-yl)-2,3-dideoxy-D-ribo-hexofuranosides. Reaction of these peracetylated adducts with trimethylsilylated thymine in the presence of trimethylsilyl trifluoromethanesulfonate (TMS triflate) afforded the corresponding nucleosides which were deprotected by using methanolic ammonia. The nucleosides were found inactive against HIV-1 and HSV-1.

3'-Azido-2',3'-dideoxyuridines 6 and their corresponding alpha anomers 5 were synthesized by condensation of silylated 6-alkyl and 5,6-dialkyl substituted uracils 2 with methyl 3-azido-5-O-tert-butyldiphenylsilyl-2,3-dideoxy-alpha, beta-D-erythro-pentofuranoside (4). Compounds 5 and 6 were treated with tetrabutylammonium fluoride to obtain the deprotected nucleosides 7 and 8, respectively.

KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3'-end, whereas to enhance the folding and stability, two polycyclic aromatic hydrocarbon units (TINA or AMANY) were inserted internally, to cap the quadruplex. The most active G4-decoy (2998), which had two para-TINAs, strongly suppressed KRAS expression in Panc-1 cells. It also repressed their metabolic activity (IC50 = 520 nM), and it inhibited cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased the Kaplan-Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy.

Methyl 2-deoxy-3-O-[2-(formylamino)ethyl]-5-O-trityl-D-erythro- pentofuranoside (4) was obtained in a 3-O alkylation reaction by treatment with 2-chloroethylamine in DMF. Compound 4 afforded alpha nucleosides as the main products when condensed with uracils under the Vorbrüggen conditions. The nucleosides were deblocked by treatment with 80% acetic acid and subsequently with sodium methoxide in methanol.

5-Azido-2,5-dideoxy-beta-D-erythro-pentofuranosyl nucleosides 10 and their corresponding alpha-anomers 11 have been synthesized by condensation of methyl 3-O-acetyl-5-azido-2,5-dideoxy-beta-D-erythro-pentofuranoside (7) with silylated nucleobases followed by deprotection with methanolic ammonia. Reaction of silylated thymine (19) with methyl 2,3-di-O-benzoyl-5-deoxy-5-fluoro-D-arabino-pentofuranoside (15) and methyl 5-azido-2,3-di-O-benzoyl-5-deoxy-alpha-D-arabino-pentofuranoside (17 alpha) afforded a mixture of the alpha-nucleosides 20 and the acyclo nucleosides 5-fluoro- and 5-azido-2,3-O-dibenzoyl-5-deoxy-1-O-methyl-1-(thymin-1-yl)-D -arabinitol (22). Compounds 20 and 22 were deprotected with methanolic ammonia to give the acyclic nucleosides 21 and 23, respectively. The new nucleosides were inactive against HSV-1 and HIV-1.

Hydantoin nucleosides were synthesized from a protected methyl 2-deoxy-D-ribofuranoside in a Friedel-Crafts catalyzed silyl-Hilbert-Johnson reaction as modified by Vorbruggen. Atypical byproduts are accounted for by assuming the initial step being a ring opening of the sugar to give an acyclic glycos-1-yl cation

The condensation of 5'-O-protected 3'-O-(2-aminoethyl)thymidine with 1,2-dideoxy-1-thyminyl-beta-D-erythro-pentofuranuronic acid gives a T*T dimer with * representing a 3'-OCH2CH2NHC(O)-4' linkage connecting the two pentofuranosyl moieties. The incorporation of this dimer in oligonucleotide sequences show only moderately lowered Tm values when hybridized with a complementary DNA relative to the unmodified DNA duplex. Consistently, no looped-out or bubble-type structure could be detected in DNA duplexes with an internal T*T module. Moreover, the 5-atom carboxamide linker causes complete stop on DNA polymerization and on exonuclease III degradation.

Two G-quadruplex forming sequences, 5′-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5′-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge–charge repulsion. Contrary to that, we found that attachment of the 5′-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation—forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed.

The 3'-iodonucleosides 4 have been synthesized by condensation of silylated 5-alkyluracils 2 with methyl 5-O-tert-butyldiphenylsilyl-2,3-dideoxy-3-iodo-D-threo-pentofur anoside (3). 4 was treated with sodium azide and the deprotected nucleoside 5 was subsequently obtained by treatment with tetrabutylammonium fluoride. The nucleoside 4 produced the corresponding 2',3'-didehydro-2',3'-dideoxy nucleoside 6 and 3',4'-didehydro-2',3'-dideoxy nucleoside 7 in elimination reactions on treatment with sodium methoxide.

Methyl 2-C-allyl-2,6-anhydro-3-deoxy-4,5:7,8-di-O-isopropylidene-D-glycero- D-talo-octonate (9a), the protected alpha-allyl C-glycoside of 3-deoxy-D-manno-2-octulosonic acid (KDO), has been synthesized using a photochemically initiated radical reaction. A phenyl thioglycoside (8) was used as the substrate and allyltributyltin as the acceptor. The stereoselectivity of the reaction was 90:10 in favour of the talo-isomer (alpha-).

Fetal neocortical tissue was grafted into neocortical lesion cavities made in newborn rats. After two weeks survival,in vitrobinding of[H3]- estradiol to cytosolic preparations provided evidence of estrogen receptors within the transplants. The observed high levels correspond to previous work demonstrating elevated estrogen receptor levels during the first postnatal week in the rat cerebral cortex.

We report the first synthesis of the L isomer of 3'-azido-3'-deoxythymidine (L-AZT). L-Arabinose was used as starting material for preparation of appropriately protected α,β-unsaturated aldehyde 5-Michael-type addition of azide to 5 gave 3-azido-2,3-dideoxypentofuranose suitable for nucleoside coupling with silylated thymine to afford after deprotection L-AZT, 1-(3-azido,-2,3-dideoxy-α-L-erythro-pentofuranosyl)thymine, 1-(3-azido-2,3-dideoxy-β-L-threo-pentofuranosyl)thymine and 1-(3-azido-2,3-dideoxy-α-L-threo-pentofuranosyl)thymine. Anti-HIV activity of L-AZT is discussed

The 3'-iodonucleoside 4 and the 3'-O-methylsulfonylthymidine 9 have been synthesized by condensation of silylated uracils 2 with methyl 5-O-tert-butyldiphensilyl-2,3-dideoxy-3-iodo-D-threo-pentofuran oside (3) and methyl 5-O-tert-butyldiphenylsilyl-2-deoxy-3-methylsulfonyl-D-erythro- pentofuranoside (8), respectively. The nucleoside 4 and 9 produced the corresponding 2',3'-didehydro-2',3'-dideoxynucleosides 5 in an elimination reaction on treatment with sodium methoxide. The compounds 5b showed no antiviral activity against HIV-1.

Mercuric-catalyzed hydrolysis of acetylated L-rhamnal 1 gives an alpha,beta-unsaturated aldehyde 2. 1,4-Addition of DBU-phthalimide salt with concomitant acetyl shift resulted in L-ribo and L-arabino isomers of 5-O-acetyl-2,3,6-trideoxy-3-phthalimido-hexofuranose 3 and 4. After acetylation at the anomeric center, coupling with silylated thymine resulted in three new nucleosides, with L-acosamine and L-ristosamine of furanose configuration as the carbohydrate moiety. The target compounds have been evaluated for their antiviral activity against HIV and HSV-1.

Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA–TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA–TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA–TFOs were found to abrogate the formation of a DNA–protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA–TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome.