Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene ...expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA’s oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3′-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3′-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.
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Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently ...been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.
We compare the duplex stabilizing properties of 2′-fluorinated nucleic acid analogues with furanose and non-furanose ring systems and dissect the relative contributions of hydration, sugar ...conformation, and fluorine configuration toward the overall T m value. We find that the stabilization imparted by fluorine substitution is additive over that obtained by restricting the conformation of the sugar ring itself. Our studies support further evaluation of fluorinated nucleic acid analogues with non-furanose sugar rings as surrogates of 2′-F RNA for therapeutic antisense applications.
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A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5′-end of antisense oligonucleotide using ...phosphoramidite chemistry. Conjugation of the 5′-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5′-triantennary GalNAc cluster conjugated ASOs and helps understand the structure–activity relationship for targeting hepatocytes with oligonucleotide therapeutics.
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The anticancer nucleoside 5-fluoro 2′-deoxyuridine-5′-phosphate (5-FdU-P) was attached via an amide chain linker to a triantennary GalNAc cluster as a means to deliver the drug to ...hepatic cells that recognize the amino sugar units.
We report the design and synthesis of 2′-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified ...oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-d-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2′-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3′-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2′-fluoro RNA (FRNA), 2′-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the 3H2 and 2H3 conformations, similar to the C3′-endo/C2′-endo conformation equilibrium seen in natural furanose nucleosides. In the 2H3 conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3′-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the 3H2 (C3′-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex d(GCGTA)-T*-d(ACGC)2. In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.
Abstract
We recently showed that site-specific incorporation of 2′-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance ...therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5′-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5′-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2′-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5′-Me and R-5′-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5′-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.
Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. ...Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2′-N-(pyren-1-yl)carbonyl-2′-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (ΦF = 0.45−0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.
The multi-biomarker disease activity (MBDA) test measures 12 serum protein biomarkers to quantify disease activity in RA patients. A newer version of the MBDA score, adjusted for age, sex, and ...adiposity, has been validated in two cohorts (OPERA and BRASS) for predicting risk for radiographic progression. We now extend these findings with additional cohorts to further validate the adjusted MBDA score as a predictor of radiographic progression risk and compare its performance with that of other risk factors.
Four cohorts were analyzed: the BRASS and Leiden registries and the OPERA and SWEFOT studies (total N = 953). Treatments included conventional DMARDs and anti-TNFs. Associations of radiographic progression (ΔTSS) per year with the adjusted MBDA score, seropositivity, and clinical measures were evaluated using linear and logistic regression. The adjusted MBDA score was (1) validated in Leiden and SWEFOT, (2) compared with other measures in all four cohorts, and (3) used to generate curves for predicting risk of radiographic progression.
Univariable and bivariable analyses validated the adjusted MBDA score and found it to be the strongest, independent predicator of radiographic progression (ΔTSS > 5) compared with seropositivity (rheumatoid factor and/or anti-CCP), baseline TSS, DAS28-CRP, CRP SJC, or CDAI. Neither DAS28-CRP, CDAI, SJC, nor CRP added significant information to the adjusted MBDA score as a predictor, and the frequency of radiographic progression agreed with the adjusted MBDA score when it was discordant with these measures. The rate of progression (ΔTSS > 5) increased from < 2% in the low (1-29) adjusted MBDA category to 16% in the high (45-100) category. A modeled risk curve indicated that risk increased continuously, exceeding 40% for the highest adjusted MBDA scores.
The adjusted MBDA score was validated as an RA disease activity measure that is prognostic for radiographic progression. The adjusted MBDA score was a stronger predictor of radiographic progression than conventional risk factors, including seropositivity, and its prognostic ability was not significantly improved by the addition of DAS28-CRP, CRP, SJC, or CDAI.
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