Per T. Jørgensen

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

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.

Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes

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.

Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule

The thrombin‐binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G‐quadruplex‐forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three‐carbon spacer (spacer‐C3), unlocked nucleic acid (UNA) and 3′‐amino‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C3 introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.

Synthesis of annelated analogues of 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442) using 1,3-oxazine-2,4(3H)-diones as key intermediates

Condensation of ethyl 3-phenyl-2-oxocyclopentanecarboxylate 5 with 2-(S-methylthio)isourea followed by hydrolysis with HCl gave 6,7-dihydro-7-phenylcyclopenta[e][1,3]oxazine-2,4(3H,5H)-dione (10a). 7,8-Dihydro-8-phenyl-6H-cyclohexa[e][1,3]oxazine-2,4(3H,5H)-dione (10b) was synthesised by reacting 2-phenylcyclohexanone (9b) with N-(chlorocarbonyl) isocyanate. The oxazines 10a,b were reacted with ammonia to obtain the corresponding uracil derivatives 12a,b which after silylation were alkylated with diethoxymethane using trimethylsilyl triflate (TMS-triflate) as the catalyst or alkylated with chloromethyl ethyl ether to give annelated MKC-442 analogues 2,3 which are locked in a conformation close to the one of MKC-442. In spite of this, only moderate activities were found against HIV-1 for the annelated analogues of MKC-442.

LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures

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.

Synthesis of locked pyranosyl nucleic acid (LpNA)

A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation.

Unlocked nucleic acid modified primer-based enzymatic polymerization assay: towards allele-specific genotype detection of human platelet antigens

Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.

Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole intercalator to a triplex oligonucleotide and to a three-way junction

A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.

Unexpected isolation of 4-isothiocyanatomethylene-4H-pyridine-1-carboxylic acid ethyl ester as potential template in organic synthesis

Abstract The synthesis of 4‐isothiocyanatomethyl‐pyridine 4 in 36% yield by Hasegawa and Kotani (Japanese patent 49088878, 1974) has spurred us to investigate this preparation in detail. In addition to this compound, 4‐isothiocyanatomethylene‐4H‐pyridine‐1‐carboxylic acid ethyl ester 3 can be isolated. The synthesis of both compounds 3 and 4 were optimized to 75% and 50% yield respectively. Reaction of compound 3 with methylamine gave thiourea derivatives 5, the same product obtained on reacting 4‐isothiocyanatomethyl‐pyridine 4 with methylamine. We succeed in adjusting the reaction conditions to obtain high yield either from compound 3 or isothiocyanate derivatives 4.