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Synthesis and in vitro Inhibition Properties of siRNA Conjugates Carrying Acridine and Quindoline Moieties
Abstract
The synthesis of RNA molecules carrying acridine or quindoline residues at their 3'- and 5'-termini is reported. These conjugates are fully characterized by MALDI-TOF mass spectrometry. Modified siRNA duplexes carrying acridine or quindoline moieties were evaluated for inhibition of the tumor necrosis factor. The conjugates showed inhibitory properties similar to those of unmodified RNA duplexes in HeLa cells transfected with oligofectamine. The fluorescent properties of acridine derivatives allow direct observation of the cytoplasmatic distribution of modified siRNA inside the cells.
... To expand the arsenal of available cationic lipids for siRNAs delivery, several lipids and lipoids libraries were screened, thereby generating lipids with high efficiency and less toxicity [58,59]. These studies led to the search for new hydrophobic molecules to enhance the cellular uptake of siRNAs [60][61][62][63]. Conjugation of amino-siRNAs with a small library of carboxyl-lipids including cholesterol, fatty-acids and bile acids resulted in hydrophobic siRNA derivatives that interact with lipoproteins [44]. ...
Antisense and small interfering RNA (siRNA) oligonucleotides have been recognized as powerful therapeutic compounds for targeting mRNAs and inducing their degradation. However, a major obstacle is that unmodified oligonucleotides are not readily taken up into tissues and are susceptible to degradation by nucleases. For these reasons, the design and preparation of modified DNA/RNA derivatives with better stability and an ability to be produced at large scale with enhanced uptake properties is of vital importance to improve current limitations. In the present study, we review the conjugation of oligonucleotides with lipids and peptides in order to produce oligonucleotide conjugates for therapeutics aiming to develop novel compounds with favorable pharmacokinetics.
... This interesting observation was confirmed by several studies indicating that the interaction of the 3'-end of the guide strand with the PAZ domain modulates the potency of siRNA derivatives. [43,44] Later, a small modification such as 1-ethynylribose introduced at the 3'-end of the guide strand was able to substantially reduce off-target effects without affecting-target knockdown potency by enhancing the binding of the 3'-end of the guide strand to the PAZ domain. [45] An interesting development to increase the stability of siRNAs against nuclease degradation was the replacement of the two 3'-terminal nucleotides by N-ethyl-N-coupled nucleosides. ...
The cover picture shows the three‐dimensional reconstruction of a DNA array described by the group of Dr. Seeman, obtained by A. Garibotti in Barcelona, and the crystal structure of the Argonaute protein from the Protein Data Bank (4Z4F). Both elements illustrate the research activity described in the Personal Account by R. Eritja and co‐workers (DOI:10.1002/tcr.202100270).
... This interesting observation was confirmed by several studies indicating that the interaction of the 3'-end of the guide strand with the PAZ domain modulates the potency of siRNA derivatives. [43,44] Later, a small modification such as 1-ethynylribose introduced at the 3'-end of the guide strand was able to substantially reduce off-target effects without affecting-target knockdown potency by enhancing the binding of the 3'-end of the guide strand to the PAZ domain. [45] An interesting development to increase the stability of siRNAs against nuclease degradation was the replacement of the two 3'-terminal nucleotides by N-ethyl-N-coupled nucleosides. ...
The last decade has witnessed the blooming of nucleic acids for therapeutic and diagnostic applications. In the present article, we describe the most important results from our group in this area covering the international context that surrounded this research. These include the study of modifications at the terminal and internal positions of siRNA duplexes to enhance nuclease resistance, increase loading of the antisense strand to RISC and avoid side effects such as activation of immune response and sense strand misloading. Then, we describe the design of novel lipid, carbohydrate and peptide conjugates to enhance cellular uptake. Finally, we describe the use of nanostructures for drug delivery and for the controlled deposition of matter on surfaces. We invite the readers to submerge into a highly interdisciplinary discipline that combines organic chemistry, biochemical assays, pharmacology issues as well as materials chemistry and structural studies in order to increase the applications of nucleic acids.
... 9,62 The study of bulky derivatives is also documented, but the compound synthesized in this work was never studied before in detail. 63 THR analogue has been explored in our lab recurrently due to its good silencing performance. 38,64 The unusual analogues MIR, GNA were newly incorporated at siRNA 3' overhangs. ...
The understanding of the dynamical and mechanistic aspects that lie behind siRNA-based gene regulation is a requisite to boost the performance of siRNA therapeutics. A systematic experimental and computational study on the 3’-overhang structural requirements for the design of more specific and potent siRNA molecules was carried out using nucleotide analogues differing in structural parameters such as sugar constraint, lack of nucleobase, distance between the phosphodiester backbone and nucleobase, enantioselectivity and steric hindrance. The results established a set of rules governing the siRNA-mediated silencing, indicating that the thermodynamic stability of the 5’-end is a crucial determinant for the antisense-mediated silencing but is not sufficient to avoid sense-mediated silencing. Both theoretical and experimental approaches consistently evidence the existence of a direct connection between PAZ/3’-overhang binding affinity and the siRNA’s potency and specificity. An overall description of the systems is thus achieved by atomistic simulations and free energy calculations that allow us to propose a robust and self-contained procedure for studying the factors implied in PAZ/3’-overhang siRNA interactions. A higher RNAi activity is associated with a moderate-to-strong PAZ/3’-overhang binding. Contrarily, lower binding energies compromise siRNA potency, increase specificity and favor siRNA downregulation by Ago2-independent mechanisms. This work provides in-depth details for the design of powerful and safe synthetic nucleotide analogues for substitution at 3’-overhang enabling to overcome some of the intrinsic siRNA disadvantages.
... Synthesis of galactose conjugates is a potential alternative to enhance siRNA uptake using sugar binding membrane receptors to mediate cell entry. Aviñó et al. [114] described the synthesis of galactose conjugate and its analogues conjugate of oligoribonucleotides (1-6) using phosphoramidite chemistry (Fig. 16). These carbohydrate compounds linked to siRNA duplexes entered the RNAi pathway to silence gene expression more efficiently than unmodified siRNA duplexes. ...
Statement of significance:
The delivery of siRNA into cells or tissues remains to be a challenge for its applications, an alternative strategy for siRNA delivery systems is direct conjugation of non-viral vectors with targeting moieties for cellular delivery. In comparison to macromolecules, small targeting molecules have attracted great attention due to their many potential advantages including significant simplicity and ease of production, good repeatability and biodegradability. This review will focus on the most recent advances in the delivery of siRNA using conjugates of small targeting molecules and non-viral delivery systems. Based the editor's suggestions, we hope the revised manuscript could provide more profound understanding to the conjugates of targeting molecules to vectors for mediation of siRNA.
... First introduced were phosphoramidite building blocks derived from L-threoninol (Asanuma et al., 2003), while a number of solid supports 124a to 124l derived from this scaffold were been reported several years later. The list of reporters used in 124a to 124l includes pyrene-1butyric acid, R 38 , anthraquinone-2-carboxylic acid, R 33 (Ben Gaied et al., 2009), 4-methyltrans-stilbazole, R 43 (Kashida et al., 2011), and a number of intercalating and/or fluorescent carboxylic acids, R 31 , R 45 to R 47 , R 50 , R 51 , and R 61 to R 63 (Aviñó et al., 2008(Aviñó et al., , 2012. At this point, it remains unclear whether the supports of general structure 124 offer any advantages over serinol-based solid supports aside from the ease of preparation. ...
This unit attempts to provide a reasonably complete inventory of over 280 solid supports available to oligonucleotide chemists for preparation of natural and 3'-modified oligonucleotides. Emphasis is placed on non-nucleosidic solid supports. The relationship between the structural features of linkers and their behavior in oligonucleotide synthesis and deprotection is discussed wherever the relevant observations are available. Curr. Protoc. Nucleic Acid Chem. 53:3.1.1-3.1.60. © 2013 by John Wiley & Sons, Inc.
... During RNAi, events occurring at the 39 end of siRNA involve binding of the PAZ domain with the nucleotide or compound at this position, followed by release and rebinding in a cyclic manner. In this context, several modifications of the nucleotides at the 3' end have been thoroughly investigated [10,[22][23][24][25]. However, it is not well understood whether compounds with stronger or weaker binding with PAZ domain could enhance or hinder the whole RNAi process. ...
RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.
Acridines are moderate binders of nucleic acids due to hydrophobic interactions leading to auto-insertion between two adjacent base pairs of nucleic acids (intercalation). Use of multiple acridines in one molecule can cause polyintercalation and enhanced binding to the nucleic acids. Intercalation is widely employed for targeting of cargos to proximity of nucleic acids. Moreover, when the nucleic acid processing enzymes are inhibited by intercalated acridines, the cure of various cancer or viral diseases can be achieved. Usually, acridines preferred site rich on GC moieties at dsDNA and G4 at quadruplexes. Structural aspects of interaction with nucleic acids can help to the reader in design of specific binders, especially for targeting of quadruplexes involved in cancer.
Oligonucleotide-based therapeutics may be one of the most promising approaches for the treatment of diseases. Although significant progress has been made in developing these agents as drugs, several hurdles remain to be overcome. One of the most promising approaches to overcome these difficulties is the preparation of modified oligonucleotides designed to increase cellular uptake and/or increase stability to nucleases. Herein, we report the developments done by our group in the synthesis of modified oligonucleotides directed to the generation of active compounds for gene inhibition. Specifically we will report the synthesis of novel nuclease-resistant oligonucleotides such as North bicyclo[3.1.0]hexane pseudosugars or N-coupled dinucleotide units. Also, the design of several siRNA conjugates carrying cell-penetrating peptides, lipids, intercalating agents, and carbohydrates will be described. Some of these novel derivatives show clear improvements in their biological and inhibitory properties.
RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3' end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.
Chemical modifications aimed at stabilizing the interaction between the 3'-end of siRNAs and the PAZ domain of RISC have been tested for their effect on RNAi activity. Such modifications contribute positively to the stability of siRNAs in human serum.
Synthetic oligonucleotides (ONs) are being investigated for various therapeutic and diagnostic applications. The interest in ONs arises because of their capability to cause selective inhibition of gene expression by binding to the target DNA/RNA sequences through mechanisms such as antigene, antisense, and RNA interference. ONs with catalytic activity (ribozymes and DNAzymes) against the target sequences, and ability to bind to the target molecules (aptamers), ranging from small molecules to proteins, are also known. Therefore ONs are considered potentially useful for the treatment of viral diseases and cancer. ONs also find use in the design of DNA microchips (a powerful bio-analytical tool) and novel materials in nanotechnology. However, the clinical success achieved so far with ONs has not been satisfactory, and the major impediments have been recognised as their instability against nucleases, lack of target specificity, and poor uptake and targeted delivery. Tremendous efforts have been made to improve the ON properties by either incorporating chemical modifications in the ON structure or covalently linking (conjugation) reporter groups, with biologically relevant properties, to ONs. Conjugation is of great interest because it can be used not only to improve the existing ON properties but also to impart entirely new properties. This tutorial review focuses on the recent developments in ON conjugation, and describes the key challenges in efficient ON conjugation and major synthetic approaches available for successful ON conjugate syntheses. In addition, an overview on major classes of ON conjugates along with their use in therapeutics, diagnostics and nanotechnology is provided.
A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells
was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is
negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered
that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr–PNA) could be effectively delivered to JAR cells (as
well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr–PNA conjugates,
one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 μM caused a reduction of MDM2
levels to ∼20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional
activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively
with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery
system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development
of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.
A carboxy derivative of the antimalarial cytotoxic drug cryptolepine was introduced into synthetic oligonucleotides by reaction of the carboxy derivative of cryptolepine with oligonucleotides carrying an amino group either at the 3'- or at the 5'-end. Oligonucleotides carrying the cryptolepine derivative bind their complementary sequences with greater affinity than unmodified ones. When cryptolepine is attached to a polypyrimidine oligonucleotide designed to form a parallel triplex, the triplex shows none or weak stabilization.
Synthetic small interfering RNA (siRNA) can suppress the expression of endogenous mRNA through RNA interference. It has been reported that siRNA can induce type I IFN production from plasmacytoid dendritic cells, leading to off-target effects. To separate immunostimulation from the desired gene-specific inhibitory activity, we designed RNA strands with chemical modifications at strategic positions of the ribose or nucleobase residues. Substitution of uridine residues by 2'-deoxyuridine or thymidine residues was found to decrease type I IFN production upon in vitro stimulation of human PBMC. Thymidine residues in both strands of a siRNA duplex further decreased immunostimulation. Fortunately, the thymidine residues did not affect gene-silencing activity. In contrast, 2'-O-methyl groups at adenosine and uridine residues reduced both IFN-alpha secretion and gene-silencing activity. Oligoribonucleotides with 2'-O-methyladenosine residues actively inhibited IFN-alpha secretion induced by other immunostimulatory RNAs, an effect not observed for strands with 2'-deoxynucleosides. Furthermore, neither 5-methylcytidine nor 7-deazaguanosine residues in the stimulatory strands affected IFN-alpha secretion, suggesting that recognition does not involve sites in the major groove of duplex regions. The activity data, together with structure prediction and exploratory UV-melting analyses, suggest that immunostimulatory sequences adopt folded structures. The results show that immunostimulation can be suppressed by suitable chemical modifications without losing siRNA potency by introducing seemingly minor structural changes.
The chemical processes involved in the preparation of oligonucleotide-intercalator conjugates are reviewed. Two strategies are used to covalently attach an intercalating agent to oligonucleotides; it can be either directly incorporated during the oligonucleotide chain assembly or linked to the unblocked oligonucleotide using a specific coupling reaction. The covalent attachment of different intercalating agents to various positions of an oligonucleotide chain: 5′-, 3′-, both 5′- and 3′-ends, internucleotidic phosphate, 1′- or 2′-position of a sugar residue or nucleic base is reported. Studies of the binding of these modified oligonucleotides to their targets are also presented together with some of the unique properties which are conferred upon the oligonuleotides by attachment of an intercalating agent.
Branched oligonucleotides with symmetric arms 6-15, which may contain biotin as a second recognition code, were prepared. These molecules are designed to be used for the directed assembly of nanomaterials. The branched structure of the desired oligonucleotides was confirmed by mass spectrometry on small branched oliognucleotides, by gel electrophoresis, and by hybridization with complementary oligonucleotide-nanoparticle conjugates, followed by visualization of the complexes by transmission electron microscopy.
Introduction:
There is great potential for antisense and siRNA oligonucleotides to become mainstream therapeutic entities thanks to their high specificity and wide therapeutic target space compared with small molecules. Despite this potential, the pharmacological targets within the cells are less accessible to oligonucleotides that are hydrophilic and often charged. Oligonucleotides access their intracellular targets mainly by means of endocytosis, but only a fraction of them reach their targets, as delivery requires functional synergy of cellular uptake and intracellular trafficking.
Areas covered:
This review provides an update on the progress of receptor-targeted delivery of oligonucleotides over the last 15 years and summarizes various targeting moieties for oligonucleotide delivery and coupling strategies. To inspire new strategies that can lead to oligonucleotides in the clinic, this review highlights how oligonucleotides successfully reach their intracellular targets by means of receptor-mediated endocytosis.
Expert opinion:
Understanding the mechanisms of oligonucleotide internalization has led to greater cellular uptake and superior endosomal release through the rational design of receptor-targeted delivery systems. Further improvements will again depend on a better understanding of the intracellular trafficking of oligonucleotides.
Oligonucleotide conjugates containing acridine and quindoline derivatives linked through a threoninol molecule were synthesized. We showed that these conjugates formed duplexes and quadruplexes with higher thermal stability than the corresponding unmodified oligonucleotides. When acridine is located in the middle of the sequence, DNA duplexes have a similar stability independently of the natural base present in front of acridine. Self-complementary oligonucleotides and thrombin binding aptamers (TBA) carrying the acridine and quindoline molecules are studied by NMR.
RNA interference (RNAi) is a collection of small RNA directed mechanisms that result in sequence specific inhibition of gene expression. The notion that RNAi could lead to a new class of therapeutics caught the attention of many investigators soon after its discovery. The field of applied RNAi therapeutics has moved very quickly from lab to bedside. The RNAi approach has been widely used for drug development and several phase I and II clinical trials are under way. However, there are still some concerns and challenges to overcome for therapeutic applications. These include the potential for off-target effects, triggering innate immune responses and most importantly obtaining specific delivery into the cytoplasm of target cells. This review focuses on the current status of RNAi-based therapeutics, the challenges it faces and how to overcome them.
Olignucleotide-based drugs show promise as a novel form of chemotherapy. Among the hurdles that have to be overcome on the way of applicable nucleic acid therapeutics, inefficient cellular uptake and subsequent release from endosomes to cytoplasm appear to be the most severe ones. Covalent conjugation of oligonucleotides to molecules that expectedly facilitate the internalization, targets the conjugate to a specific cell-type or improves the parmacokinetics offers a possible way to combat against these shortcomings. Since workable chemistry is a prerequisite for biological studies, development of efficient and reproducible methods for preparation of various types of oligonucleotide conjugates has become a subject of considerable importance. The present review summarizes the advances made in the solid-supported synthesis of oligonucleotide conjugates aimed at facilitating the delivery and targeting of nucleic acid drugs.
An octathymidylate covalently linked via Its 3′-end to an acridine derivative inhibited the cytopathic effect of Simian Virus
SV40 on CV-1 cells in culture. Control experiments revealed that this effect was virus-specific and did not arise as a result
of ollgonucleotide degradation by nucleases. A photoactive probe was covalently attached to the 5′-end of the ligonucleotideacridine
conjugate. Upon UV-irradiation, photocrosslinking was shown to occur at the A.T-rich region within the viral origin of replication.
A local triple helix can form at moderate salt concentrations with two octathymldylate-acridine conjugates bound to the octaadenylate
sequence. Alternatively the octathymidylate-acridine conjugate can bind to the major groove of duplex DNA forming a local
triple helix. Different mechanisms are discussed to explain the inhibition of viral DNA replication.
This review summarizes the effect of conjugating small molecules and large biomacromolecules to antisense oligonucleotides to improve their therapeutic potential. In many cases, favorable changes in pharmacokinetic and pharmacodynamic properties were observed. Opportunities exist to change the terminating mechanism of antisense action or to enhance the RNase H mode of action via conjugate formation.
Methyl Red H aggregate of predetermined size is successfully synthesized from the DNA conjugate involving multiple Methyl Red moieties in sequence. In the single stranded state, hypsochromicity monotonically increases with the number of incorporated dyes: the peak maximum of the conjugate involving six Methyl Reds appears at 415 nm, and the shift is as great as 69 nm (3435 cm(-)(1)) with respect to the monomeric transition. This large hypsochromicity accompanied by the narrowing of the band clearly demonstrates that H aggregate is formed in the single strand. H aggregation is further promoted at higher ionic strength. Upon addition of complementary DNA below the T(m), however, this H band disappears and a new peak appears at 448 nm, indicating that aggregated structure is changed by the duplex formation. This spectral change is completely reversible so that the H band at 415 nm appears again above T(m). Thus, aggregated structure can be reversibly controlled by the formation and dissociation of the DNA duplex.
In mammalian cells, RNA duplexes of 21-23 nucleotides, known as small interfering RNAs (siRNAs) specifically inhibit gene expression in vitro. Here, we show that systemic delivery of siRNAs can inhibited exogenous and endogenous gene expression in adult mice. Cationic liposome-based intravenous injection in mice of plasmid encoding the green fluorescent protein (GFP) with its cognate siRNA, inhibited GFP gene expression in various organs. Furthermore, intraperitoneal injection of anti-TNF-alpha siRNA inhibited lipopolysaccharide-induced TNF-alpha gene expression, whereas secretion of IL1-alpha was not inhibited. Importantly, the development of sepsis in mice following a lethal dose of lipopolysaccharide injection, was significantly inhibited by pre-treatment of the animals with anti-TNF-alpha siRNAs. Collectively, these results demonstrate that synthetic siRNAs can function in vivo as pharmaceutical drugs.
RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.
RNA interference (RNAi) is triggered by double-stranded RNA helices that have been introduced exogenously into cells as small interfering (si)RNAs or that have been produced endogenously from small non-coding RNAs known as microRNAs (miRNAs). RNAi has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNAs and miRNAs, that trigger RNAi has shed light on the RNAi machinery. In particular, it has highlighted the assembly and function of the RNA-induced silencing complex (RISC), and has provided guidelines to efficiently silence genes for biological research and therapeutic applications of RNAi.
A method for the preparation of oligomers by linking chromophore units is described. Specifically, the synthesis of chromophore units having a protected-hydroxyl group and a phosphoramidite function is described, along with a method to link several units using solid-phase phosphite-triester protocols.
Oligonucleotide sequences were prepared using solid-phase methodology. The syntheses were carried out on an Applied Biosystems Model 3400 DNA synthesizer using 1 mmol scale. Oligoribonucleotides were purified by DMT-on-based protocols and by reversed-phase HPLC
- General
General. Oligonucleotide sequences were prepared using solid-phase methodology. The syntheses
were carried out on an Applied Biosystems Model 3400 DNA synthesizer using 1 mmol scale.
Oligoribonucleotides were purified by DMT-on-based protocols and by reversed-phase HPLC.
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