RESUMO
Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.
Assuntos
Acetilgalactosamina , RNA Interferente Pequeno , Animais , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Administração Oral , Camundongos , Ratos , Interferência de RNA , Masculino , Disponibilidade Biológica , Humanos , Ratos Sprague-Dawley , Macaca fascicularis , Fígado/metabolismo , Macaca mulattaRESUMO
Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.
Assuntos
Ácidos Nucleicos , Animais , Camundongos , Ratos , RNA Interferente Pequeno , Nucleotídeos , Interferência de RNA , AcetilgalactosaminaRESUMO
In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using 'click' chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5'-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.
Assuntos
Inativação Gênica , Interferência de RNA , RNA Circular , RNA Interferente Pequeno , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.
Assuntos
Organofosfonatos , Nucleotídeos de Pirimidina , Animais , Lipossomos , Camundongos , Modelos Moleculares , Nanopartículas , Conformação de Ácido Nucleico , Nucleosídeos , Nucleotídeos , Oligonucleotídeos , Fosfatos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.
Assuntos
Acetilgalactosamina , Porfirias Hepáticas , Acetilgalactosamina/farmacocinética , Receptor de Asialoglicoproteína/metabolismo , Hepatócitos/metabolismo , Humanos , Porfirias Hepáticas/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification.
Assuntos
Acetilgalactosamina/química , Carboidratos/química , Interferência de RNA , RNA Interferente Pequeno/química , Álcoois Açúcares/química , Animais , Células COS , Chlorocebus aethiops , Exorribonucleases , Hepatócitos/metabolismo , Camundongos , Conformação de Ácido Nucleico , Pré-Albumina/genética , Ribonucleotídeos/químicaRESUMO
Development of molecules capable of binding to specific sequences of double-stranded (ds) DNA continues to attract considerable interest, as this may yield useful tools for applications in life science, biotechnology, and medicine. We have previously demonstrated sequence-unrestricted of dsDNA using Invader probes, i.e., DNA duplexes that are energetically activated through incorporation of +1 interstrand zipper arrangements of O2'-intercalator-functionalized RNA monomers. Nonetheless, recognition of extended dsDNA target regions remains challenging due to the high stability of the corresponding probes. To address this, we introduce toehold Invader probes, i.e., Invader probes with 5'-single-stranded overhangs. This design provides access to probes with shortened double-stranded segments, which facilitates probe denaturation. The single-stranded overhangs can, furthermore, be modified with affinity-enhancing modifications like LNA (locked nucleic acid) monomers to additionally increase target affinity. Herein, we report the biophysical and dsDNA-targeting properties of different toehold Invader designs and compare them to conventional Invader probes. LNA-modified toehold Invader probes display promising recognition characteristics, including greatly improved affinity to dsDNA, excellent binding specificity, and fast recognition kinetics, which enabled recognition of chromosomal DNA targets that have proven refractory to recognition by conventional Invader probes. Thus, toehold Invader probes represent another step toward a robust, oligonucleotide-based approach for sequence-unrestricted dsDNA-recognition.
Assuntos
OligonucleotídeosRESUMO
Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, are energetically activated for sequence-unrestricted recognition of double-stranded DNA (dsDNA) as they are engineered to violate the neighbor exclusion principle, while displaying high affinity towards complementary DNA sequences. The impact on Invader-mediated dsDNA-recognition upon additional modification with different non-nucleotidic bulges is studied herein, based on the hypothesis that bulge-containing Invader probes will display additionally disrupted base-stacking, more extensive denaturation, and improved dsDNA-recognition efficiency. Indeed, Invader probes featuring a single central large bulge - e.g., a nonyl (C9) monomer - display improved recognition of model DNA hairpin targets vis-à-vis conventional Invader probes (C50 values â¼1.5 µM vs. â¼3.9 µM). In contrast, probes with two opposing central bulges display less favorable binding characteristics. Remarkably, C9-modified Invader probes display perfect discrimination between fully complementary dsDNA and dsDNA differing in only one of eighteen base-pairs, underscoring the high binding specificity of double-stranded probes. Cy3-labeled bulge-containing Invader probes are demonstrated to signal the presence of gender-specific DNA sequences in fluorescent in situ hybridization assays (FISH) performed under non-denaturing conditions, highlighting one potential application of dsDNA-targeting Invader probes.
Assuntos
DNA/química , Sondas Moleculares/química , Pirenos/química , Animais , Bovinos , Linhagem Celular , Hibridização in Situ Fluorescente , Masculino , Desnaturação de Ácido Nucleico , Termodinâmica , Cromossomo YRESUMO
Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cß-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and ß-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'ß-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cß-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cß-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'ß-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cß-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.
Assuntos
Oligonucleotídeos/química , Estabilidade de RNA/genética , RNA Interferente Pequeno/química , Termodinâmica , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Fosfatos/química , Interferência de RNA , Ribonucleases/química , Ribose/química , Uridina/química , Uridina/genéticaRESUMO
Over the past three decades, a wide range of pyrene-functionalized oligonucleotides have been developed and explored for potential applications in material science and nucleic acid diagnostics. Our efforts have focused on their possible use as components of Invader probes, i.e., DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. We have previously demonstrated that Invader probes based on 2'-O-(pyren-1-yl)methyl-RNA monomers are energetically activated for sequence-unrestricted recognition of chromosomal DNA targets under non-denaturing conditions. As part of ongoing efforts towards delineating structure-property relationships and optimizing Invader probes, we report the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2'-O-(7-neo-pentylpyren-1-yl)methyl-uridine monomer V and 2'-O-(7-tert-butyl-1-methoxypyren-5-yl)methyl-uridine monomer Y. ONs modified with monomer V display increased DNA affinity (ΔTm up to +10.5 °C), while Y-modified ONs display lower DNA affinity and up to 22-fold increases in fluorescence emission upon RNA binding. Although these monomers display limited potential as building blocks for Invader probes, their photophysical properties render them of interest for diagnostic RNA-targeting applications.
Assuntos
Pirenos/química , RNA/química , Alquilação , Relação Dose-Resposta a Droga , Estrutura Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Four probe chemistries are characterized and compared with respect to thermal denaturation temperatures (Tms), thermodynamic parameters associated with duplex formation, and recognition of mixed-sequence double-stranded (ds) DNA targets: (i) oligodeoxyribonucleotides (ONs) modified with Locked Nucleic Acid (LNA) monomers, (ii) MPγPNAs, i.e., single-stranded peptide nucleic acid (PNA) probes that are functionalized at the γ-position with (R)-diethylene glycol (mini-PEG, MP) moieties, (iii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers, and (iv) intercalating nucleic acids (INAs), i.e., DNA duplexes with opposing insertions of 1-O-(1-pyrenylmethyl)glycerol bulges. Invader and INA probes, which are designed to violate the nearest-neighbor exclusion principle, denature readily, whereas the individual probe strands display exceptionally high affinity towards complementary DNA (cDNA) as indicated by increases in Tms of up to 8 °C per modification. Optimized Invader and INA probes enable efficient and highly specific recognition of mixed-sequence dsDNA targets with self-complementary regions (C50 = 30-50 nM), whereas recognition is less efficient with LNA-modified ONs and fully modified MPγPNAs due to lower cDNA affinity (LNA) and a proclivity for dimerization (LNA and MPγPNA). A Cy3-labeled Invader probe is shown to stain telomeric DNA of individual chromosomes in metaphasic spreads under non-denaturing conditions with excellent specificity.
Assuntos
DNA/química , Sondas Moleculares/química , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Animais , Bovinos , Linhagem Celular , Núcleo Celular/química , Sondas Moleculares/síntese química , Estrutura MolecularRESUMO
We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'ß) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and ß epimers could be obtained by a nonstereoselective approach starting from 2'-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the ß-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.
Assuntos
Inativação Gênica , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Ribonucleotídeos/genética , Termodinâmica , Uridina/química , Uridina/metabolismoRESUMO
Development of hybridization-based probes that enable recognition of specific mixed-sequence double-stranded DNA (dsDNA) regions is of considerable interest due to their potential applications in molecular biology, biotechnology, and medicine. We have recently demonstrated that nucleic acid duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl RNA monomers are inherently activated for recognition of mixed-sequence dsDNA targets, including chromosomal DNA. In the present work, we follow up on our previous structure-activity relationship studies and explore if the dsDNA-recognition efficiency of these so-called Invader probes can be improved by using larger intercalators than pyrene. Oligodeoxyribonucleotides modified with 2'-O-(triphenylen-2-yl)methyl-uridine monomer X and 2'-O-(coronen-1-yl)methyl-uridine monomer Z form extraordinarily stabilized duplexes with complementary DNA (ΔTm's per modification of up to 13 °C and 20 °C, respectively). Invader probes based on X- and Z-monomers are shown to recognize model dsDNA targets with exceptional binding specificity, but are less efficient than reference probes modified with 2'-O-(pyren-1-yl)methyl-uridine monomer Y. The insight from this study will inform further optimization of Invader probes.
Assuntos
Crisenos/química , DNA/química , Compostos Policíclicos/química , RNA/química , Sequência de Bases , DNA/genética , Sequências Repetidas Invertidas , Desnaturação de Ácido Nucleico , Relação Estrutura-Atividade , Temperatura , Uridina/químicaRESUMO
Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers.
Assuntos
DNA/química , Ácidos Nucleicos/química , Oligonucleotídeos/síntese química , Uridina/química , Estrutura Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
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.
Assuntos
Ácidos Nucleicos/química , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos/química , Compostos Organofosforados/química , Compostos Organofosforados/síntese química , RNA/química , Uridina/análogos & derivados , Conformação Molecular , Conformação de Ácido Nucleico , Uridina/síntese química , Uridina/químicaRESUMO
Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and--more recently--engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔT(m)/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.
Assuntos
DNA/química , Pirenos/química , RNA/química , Alquilação , Sequências Repetidas Invertidas , Modelos Moleculares , Conformação de Ácido Nucleico , Compostos Organofosforados/química , Estabilidade de RNA , TermodinâmicaRESUMO
The invasion has begun: Invaders are shown to recognize DNA hairpins in cell-free assays and chromosomal DNA during non-denaturing fluorescence in situ hybridization (nd-FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.
RESUMO
The development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable because of their potential as tools for detection, regulation, and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl-RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2'-O-(pyren-1-yl)methyl-RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2'-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprising C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular biology, nucleic acid diagnostics, and biotechnology.
Assuntos
DNA/análise , Nucleotídeos/síntese química , RNA/síntese química , Conformação Molecular , Nucleotídeos/química , RNA/químicaRESUMO
The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with "+1 interstrand zippers" of 2'-N-(pyren-1-yl)methyl-2'-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2'-O-(pyren-1-yl)methyl-RNA or 2'-N-methyl-2'-N-(pyren-1-yl)methyl-2'-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics.
Assuntos
DNA/química , Pirenos/química , Timidina Monofosfato/análogos & derivados , Espectroscopia de Ressonância Magnética , Modelos Químicos , Conformação de Ácido Nucleico , Oligonucleotídeos , Espectrometria de Fluorescência , Timidina Monofosfato/químicaRESUMO
Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.