Natural Oligonucleotides Research Articles

Fluorine‐18‐ and iodine‐125‐labelling of spiegelmers

AbstractSpiegelmers are high‐affinity l‐enantiomeric oligonucleotide ligands (aptamers) that display high resistance to enzymatic degradation compared to d‐oligonucleotides. Spiegelmers belong to the third generation of aptamers, and are currently extensively investigated as potential therapeutic agents. We have previously developed an original method to label natural oligonucleotides with radiohalogens and particularly with fluorine‐18, the most widely used positron‐emitter, t1/2: 109.8 min. Using the same strategy, we herein report the labelling of Spiegelmers, both with fluorine‐18 for positron emission tomography imaging and iodine‐125 for high resolution autoradiography. Three 25‐mer l‐oligonucleotides have been used, differing (a) by the position of the terminal phosphorothioate monoester group (3′‐ or 5′‐end, and therefore differing by the position of the labelling on the macromolecule) and (b) by the nature of the backbone sugar moiety (2′‐OH or 2′‐H, therefore covering the RNA and DNA series, respectively). N‐(4‐[18F]fluorobenzyl)‐2‐bromoacetamide was synthesized in three radiochemical steps from 4‐cyano‐N,N,N‐trimethylanilinium trifluoromethanesulfonate and HPLC‐purified in 90 min (typical production: 2.2–2.4 GBq starting from a batch of 22–24 GBq of [18F]fluoride). N‐(4‐[125I]iodobenzyl)‐2‐bromoacetamide was synthesized from the corresponding trimethylsilyl derivative (one pot, two radiochemical steps) and HPLC‐purified in 60 min (typical production: 24 MBq starting from 37 MBq of Na[125I]I). Coupling of the Spiegelmers with the appropriate HPLC‐purified [radiolabelled]‐halobenzyl‐2‐bromoacetamide (MeOH/PBS (0.1 M, pH 8), 10 min, 120°C) gave the corresponding labelled conjugated Spiegelmers after RP‐HPLC purification. For fluorine‐18, the whole synthetic procedure yields up to 1.1 GBq of pure labelled Spiegelmers in 160 min with a specific radioactivity of 37–74 GBq/μmol at the end of synthesis starting from 22–24 GBq of [18F]fluoride. For iodine‐125, the whole synthetic procedure allows producing up to 7.4 MBq of pure labelled Spiegelmers in 100 min with a specific radioactivity of 11–37 GBq/μmol starting from 37 MBq of Na[125I]I. Copyright © 2003 John Wiley & Sons, Ltd.

Synthesis and Properties of Oligodeoxynucleotide Analogs with Bis(methylene) Sulfone Bridges

AbstractA convergent, solution‐phase synthesis was developed for the bis(methylene) sulfone‐bridged oligodeoxynucleotide analogs (SNA) 5′‐d(HOCH2‐Tso2Tso2Tso2Cso2Tso2Tso2Tso2T‐CH2SO$\rm{_{3}^{-}}$)‐3′ (35b) and 5′‐d(HOCH2‐Tso2Tso2Tso2Tso2Tso2Tso2Tso2T‐CH2SO$\rm{_{3}^{-}}$)‐3′ (34c) (SO2 corresponds to CH2SO2CH2 instead of OP(O)(O−)(O). In these, the phosphodiester linkages are replaced by non‐ionic bis(methylene) sulfone linkers. The general strategy involved convergent coupling of 3′,5′‐bishomo‐β‐D‐deoxyribonucleotide analogs functionalized at the 6′‐end (CH2C(5′)) as bromides or mesylates and at the CH2C(3′) position as thiols, with the resulting thioether being oxidized to the corresponding sulfone. A single charge was introduced at the terminal CH2C(3′) position of the octamers to increase their solubility in water. During the synthesis, it became apparent that the key intermediates generated secondary structures through either folding or aggregation in a variety of solvents. This generated unusual reactivity and was unique for very similar structures. For example, although the dimeric thiol d(BzOCH2‐Tso2C‐CH2SH) (14b) was a well‐behaved synthetic intermediate, the tetrameric thiol d(TrOCH2‐Tso2Tso2Tso2toC‐CH2SH) derived from the corresponding thioacetate was rapidly converted to a disulfide by very small amounts of oxidant (28→29, Scheme 6), while the analogous tetrameric thiol d(BzOCH2‐Tso2TsTso2T‐CH2SH) (26), differing only by a single heterocycle, was oxidized much more slowly (Bz=PhCO, Tr=Ph3C, to=2‐MeC6H4CO (at N4 of dc)). The sequence‐dependent reactivity, well known in many classes of natural products (including polypeptides), is not prominent in natural oligonucleotides. These results are discussed in light of the proposal that the repeating negative charge in nucleic acids is key to their ability to serve as genetic molecules, in particular, their capability to support Darwinian evolution. The ability of 5′‐d(HOCH2‐Tso2Tso2Tso2Cso2Tso2Tso2Tso2T‐CH2SO$\rm{_{3}^{-}}$)‐3′ (35b) to bind as a third strand to duplex DNA was also examined. No triple‐helix‐forming propensity was detected in this molecule.

Structural modifications of antisense oligonucleotides

Antisense oligonucleotides are efficient tools for the inhibition of gene expression in a sequence specific way. Natural oligonucleotides are decomposed rapidly in biological systems, which strongly restrict their application. In contrast, artificial oligonucleotides are designed to be more stable against degradation than the target mRNA, which results in a catalytic effect of the drug. Modification of the phosphate linkage has been the first successful strategy for antisense drug developments and Fomivirsene the first antisense drug in therapy. The launch of Fomivirsene has resulted in a revolutionary spin off to antisense research leading to a second generation of antisense oligonucleotides, which are stable against oligonucleotide cleaving enzymes. Among these, oligonucleotides bearing an alkoxy substituent in position 2′ were the most successful ones. The third generation of antisense oligonucleotides contains structure elements, which enhance the antisense action. Zwitterionic oligonucleotides show remarkable results, first, because the stability against ribozymes is largely increased, and secondly, because the electrostatic repulsion between the anionic sense and the zwitterionic antisense cords is minimized. Promising new target molecules in antisense reseach are oligonucleotide chimäres, which enhance the antisense action (chimäres with intercalators, chelators or polyamines) or enable an application as sequence specific detectors (chimäres with biotin, fluorescein or radioligands).

NMR Study of A Heterochiral DNA Hairpin: Impact of L-Enantiomery in the Loop

We carried out a structural study of the DNA heterochiral strand d (AGCTTATCAT(L)CGATAAGCT), -AT(L)C-, where T(L) (L thymine) replaces T (natural D-thymine). -AT(L)C- is a structural analog of -ATC- that belongs to a strong topoisomerase II DNA cleavage site and which has been shown to resolve into a hairpin structure with a stem formed by eight Waston-Crick base-pairs and a single residue loop closed by an A.C sheared base-pair. Although -AT(L)C-, like its parent -ATC-, folds into a hairpin structure at low and high DNA concentrations it displays a lower stability (Tm of 56 °C versus 58.5 °C). Several NMR features in—AT(L)C- account for the disruption of the A.C pairing in the loop and a weakening of the C.G base-pair stability at the stem-loop junction. For instance, the exchange of the loop imino protons with solvent is accelerated compared with the natural oligonucleotide -ATC-. The higher flexibility of the heterochiral loop is confirmed by the results of NMR restrained molecular dynamics. In the calculated final structures of—AT(L)C-, the T10(L) residue moves the A9 and C11 residues away, thus preventing the loop closure through a C.A sheared base-pair and the achievement of a good base-base or sugarbase stacking. Actually, most of the stabilizing interactions present in -ATC- are lost in the heterochiral -AT(L)C- explaining its weaker stability.

Neutral liposome-mediated delivery process of fluorescein-modified oligonucleotides in cultured human keratinocytes

We propose a model of the intracellular delivery process in which fluorescein-labeled natural oligonucleotides (F-DNA) are transferred into the nuclei of cultured human keratinocytes. By encapsulation in neutral multilamellar lecithin liposomes, the F-DNA appeared to be protected against intracellular interactions with cellular materials and nuclease attacks in the cytoplasm during the process. The intracellular behavior of F-DNA and fluorescent phospholipid-labeled liposomes was observed by means of fluorescence analysis. Results showed that: F-DNA encapsulated in neutral multilamellar liposomes reached the cellular nuclei more efficiently than either free F-DNA, or F-DNA in unilamellar liposomes; the liposomal membranes appeared to be left in the cytoplasm. The reaction of F-DNA with complementary DNA was suggested by a rapid quenching of the fluorescence in the nucleus. In addition, the fluorescence decrease was evidently suppressed in the cytoplasm, indicating a protective effect of the neutral multilamellar liposomes against the interaction of F-DNA with cytoplasmic materials. The application of these findings to ‘photo’-antisense studies has been discussed, where suppression of a gene expression is attempted by using oligonucleotide-attached fluorescein with the aid of a photo-induced covalent binding property.

Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers.

The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated. These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present. It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli. The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers. Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers. Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H. As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.

2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA): synthesis and triplex-forming properties

For development of ideal antisense and antigene molecules, various chemical modifications of oligonucleotides have been studied. However, despite their importance, there is only limited information available on the triplex-forming ability of the conformationally restricted or locked oligonucleotides. We report herein that 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA) modification of triplex-forming oligonucleotide (TFO) significantly enhances the binding affinity towards target dsDNA. On Tm measurements, the triplex with the 2′,4′-BNA oligonucleotides were found to be stabilized with ΔTm/modification of +4.3 to +5°C at pH 6.6 compared to the triplexes with the unmodified oligonucleotide. By means of gel-retardation assay, the binding constant of the 2′,4′-BNA oligonucleotide at pH 7.0 was at least 300-fold higher than that of the natural oligonucleotide. In addition, the 2′,4′-BNA oligonucleotide clearly showed the inhibition of the NF-κB transcription factor (p50)-target dsDNA binding by forming a stable triplex at pH 7.0.

PNA-RELATED OLIGONUCLEOTIDE MIMICS AND THEIR EVALUATION FOR NUCLEIC ACID HYBRIDIZATION STUDIES AND ANALYSIS

DNA mimics containing phosphonate analogues of PNAs (pPNAs), particularly PNA-pPNA hybrids as well as hetero-oligomers consisted of pPNA units and PNA-like molecules on the base of trans-4-hydroxy-L-proline (HypNA) have been synthesized. The evaluation of their effectiveness in assays based on the hybridization technique in the comparison with natural oligonucleotides and classical PNAs has shown a high potential of these mimics as sensor molecules for nucleic acid based diagnostics and as molecular probes for mRNA isolation.

New Boron Analogues of Pyrophosphates and Deoxynucleoside Boranophosphates

Tetraethyldicyanoborane pyrophosphate (2) and 3'-(diethylphosphite-cyanoborano)-5'-dimethoxytrityl.N4-benzoyl-deoxycytidine (3) have been synthesized in 70% and 76% yields, respectively. The compatibility of the substituted boranophosphates with common protecting groups is hereby demonstrated. Boron containing biologically active compounds, such as nucleosides and nucleotides 1-6 and amino acids 7-9 are important due to their potential therapeutic activity, research and diagnostic applications. Many boron containing compounds have shown promising activity as anticancer, 10. 11. 12 antiinflammatory,13 and antiosteoporotic 13agents. Oligonucleotdes in which a non-bridging oxygen atom is replaced by a borane(BH3) group are a very important class of modified nucleic acids. 1. 3. 14-16 The BH3 group is isoelectronic with oxygen in natural oligonucleotides and isoelectronic and isostructural with the oligonucleotide methyl phosphonates, which are nuclease resistant. On the other hand, the α-borano triphosphates are good substrates for DNA polymerases and incorporation of boranophosphates into DNA causes an increase in the resistance to exo- and endonucleases 2. 17a as compared to non-modified DNA. There are also notable applications of the α-borano triphosphates in PCR sequencing 17a and nucleic acid detection 17b.

Synthesis of oligodeoxyribonucleotides containing oleylamine moieties

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3'- or 5'-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of the snake venom phosphodiesterase hydrolysis of oligonucleotides containing oleylamine residues in the 3'-terminal units was shown to be markedly lower than that of natural oligonucleotides.

A two B-Z junction containing DNA resolves into an all right-handed double-helix

Natural and artificial oligonucleotides are capable of assuming many different conformations and functions. Here we present results of an NMR restrained molecular modelling study on the conformational preferences of the modified decanucleotide d((m)C1G2(m)C3G4C5(L)G6(L)(m)C7G8(m)C9G10) .d((m)C11G12(m)C13G14C15(L)G (L)16(m)C17-G18(m)C19G20 ) which contains L deoxynucleotides in its centre. This chimeric DNA was expected to form a right-left-right-handed B-type double-helix (BB*B) at low salt concentration. Actually, it matured into a fully right-handed double helix with its central C(L)pG(L) core forming a right-handed Z-DNA helix embedded in a B-DNA matrix (BZ*B). The interplay between base-base and base-sugar stackings within the core and its immediately adjacent residues was found to be critical in ensuring the stabilisation of the right-handed helix. The structure could serve as a model for the design of antisense oligonucleotides resistant to nucleases and capable of hybridising to natural DNAs and RNAs.

Invasion of Strongly Binding Oligonucleotides into tRNA Structure

Interaction of yeast tRNAPhe with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2′-deoxyuridine was investigated. The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides. The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65–76 of the tRNAphe.

Synthesis and biophysical studies of oligonucleotides containing hydroxamate linkages

Novel oligonucleotide dimers containing hydroxamate linkages ( 15 and 16) were synthesized, incorporated into oligonucleotide sequences and studied for their hybridization properties with complementary DNA and RNA targets. The modified oligonucleotides showed similar binding properties and enhanced resistance to exonucleases compared to natural oligonucleotides.

Syntheses and binding studies of oligonucleotides containing N-hydroxycarbamate linkages: potential DNA cleaving antisense oligomers

An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides.

Site-selective electron transfer from purines to electrocatalysts: voltammetric detection of a biologically relevant deletion in hybridized DNA duplexes

The one-electron oxidation of guanine nucleobases is of interest for understanding the mechanisms of mutagenesis, probing electron-transfer reactions in DNA, and developing sensing schemes for nucleic acids. The electron-transfer rates for oxidation of guanine by exogenous redox catalysts depend on the base paired to the guanine. An important goal in developing the mismatch sensitivity is to identify a means for monitoring the current resulting from electron transfer at a single base in the presence of native oligonucleotides that contain all four bases. The nucleobase 8-oxo-guanine (8G) is selectively oxidized by the redox catalyst Os(bpy)(3)(3+/2+) (bpy = 2,2'-bipyridine) in the presence of native guanine. Cyclic voltammograms of Os(bpy)(3)(2+) show current enhancements indicative of nucleobase oxidation upon addition of oligonucleotides that contain 8G, but not in the presence of native guanine. As expected, similar experiments with Ru(bpy)(3)(2+) show enhancement with both guanine and 8G. The current enhancements for the 8G/Os(III) reaction increase in the order 8G-C approximately 8G.T < 8G.G < 8G.A < 8G, the same order as that observed for guanine/Ru(III). This site-selective mismatch sensitivity can be applied to detection of a TTT deletion, which is important in cystic fibrosis. The base 8G can be effectively used in conjunction with a low-potential redox catalyst as a probe for selective electron transfer at a single site. Because of the high selectivity for 8G, rate constants can be obtained that reflect the oxidation of only one base. The mismatch sensitivity can be used to detect biologically relevant abnormalities in DNA.

Synthesis and hybridization property of sugar and phosphate linkage modified oligonucleotides

1-[3-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-3-C-hydroxymethyl-2-O-(2-methoxyethoxymethyl)-β-d-erythro-pentofuranosyl]thymine (13) was synthesized from 1,2-isopropylidene-d-xylose (1) as a building block of modified oligonucleotides. Three types of novel oligonucleotides were synthesized from 13 and their Tms were compared with those of the corresponding natural oligonucleotides. It was found that our synthesized oligomers had lower affinity to DNA and RNA than the natural oligomers.

Stability of Phosphorothioate Oligonucleotides in Aqueous Ammonia in Presence of Stainless Steel

Antisense oligonucleotides as modulators of gene expression represent an exciting new drug technology. Oligodeoxyribonucleotide phosphorothioates are now among the most intensively investigated nuclease-resistant antisense analogs, as evidenced by a number of ongoing clinical trials against several disease targets. Structurally, these differ from natural oligonucleotides by the replacement of one of two nonbridging oxygen atoms by a sulfur atom at each internucleotide linkage. Among factors in the successful development of these complex molecules to support broad clinical trials have been advances made in automation, analysis and purification. The large scale synthesis of oligonucleotide phosphorothioates is presently carried out by initial formation of the internucleotide phosphite linkage using solid-phase phosphoramidite chemistry followed by sulfurization. Efficient synthesis of 20-mer oligophosphorothioates has been achieved on 0.15 mole scale with only 1.75-fold excess of amidite synthons. However, as the scale of synthesis increases to meet future market demands, issues related to fast and efficient synthesis, automation, scalability and product purification are also being investigated. One issue has been the protocol for final product deprotection. Since deprotection involves large quantities of saturated aqueous ammonium hydroxide, one might consider use of stainless steel reactors to withstand resulting vapor pressure at 55°C. A recent report,1 however, discusses the instability of dimer phosphorothioates in aqueous ammonia in the presence of metal ions. As this is potentially an important issue for phosphorothioate oligonucleotide synthesis, we describe herein our findings regarding deprotection of a 20-mer oligodeoxyribonucleotide phosphorothioate with aqueous ammonia during process development studies.

Novel Hoogsteen-like bases for configurational recognition of the T-A base pair by DNA triplex formation.

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides binding duplex A-T or G-C base-pair DNA sequences specifically at homopurine sites in the major groove as T-A-T and C+.G-C triplets. Here we report the successful modeling of novel unnatural nucleosides that recognize the T-A DNA base pair by Hoogsteen interaction. Since the DNA triplex can be considered to assume an A-type or B-type conformation, these novel Hoogsteen nucleotides are tested within model A-type and B-type conformation triplex structures. A triplet consisting of the T-A base pair and one of the novel Hoogsteen nucleotides replaces the central T.A-T triplet in the triplex using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration. The entire triplex is energy minimized and the presence of any structural or energetic perturbations due to the central triplet is assessed with respect to the unmodified energy-minimized (T.A-T)11 proposed starting structures. Incorporation of these novel triplets into both A-type and B-type natural tiplex structures provokes minimal change in the configuration of the central and adjacent triplets. The plan is to produce a series of Hoogsteen-like bases that preferentially bind the T-A major groove in either an A-type or B-type conformation. Selective recognition of the T-A major groove with respect to the G-C major groove, which presents similar keto and amine placement, is also assessed with configurational preference. Evaluation of the triplex solution structure by using these unnatural bases as binding conformational probes is a prerequisite to the further design of triplet forming bases.

Novel Hoogsteen-like bases for configurational recognition of the T-A base pair by DNA triplex formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides binding duplex A-T or G-C base-pair DNA sequences specifically at homopurine sites in the major groove as T-A-T and C+.G-C triplets. Here we report the successful modeling of novel unnatural nucleosides that recognize the T-A DNA base pair by Hoogsteen interaction. Since the DNA triplex can be considered to assume an A-type or B-type conformation, these novel Hoogsteen nucleotides are tested within model A-type and B-type conformation triplex structures. A triplet consisting of the T-A base pair and one of the novel Hoogsteen nucleotides replaces the central T.A-T triplet in the triplex using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration. The entire triplex is energy minimized and the presence of any structural or energetic perturbations due to the central triplet is assessed with respect to the unmodified energy-minimized (T.A-T)11 proposed starting structures. Incorporation of these novel triplets into both A-type and B-type natural tiplex structures provokes minimal change in the configuration of the central and adjacent triplets. The plan is to produce a series of Hoogsteen-like bases that preferentially bind the T-A major groove in either an A-type or B-type conformation. Selective recognition of the T-A major groove with respect to the G-C major groove, which presents similar keto and amine placement, is also assessed with configurational preference. Evaluation of the triplex solution structure by using these unnatural bases as binding conformational probes is a prerequisite to the further design of triplet forming bases.

Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs (review).

Polymeric, nucleic acid drugs must be protected from endogenous nucleases and delivered to target cell nuclei in order to maximize their activity. Constructs expressing therapeutic genes, antisense oligonucleotides and ribozymes can be delivered into cells by viral vectors, but concerns over safety and clinical utility have led to research into the development of alternative, non-viral delivery systems. Antisense and ribozyme drug development has focused upon modifications to the natural oligonucleotide chemistry which make the molecules resistant to nuclease degradation. These novel oligonucleotides cannot be generated by transgenes and must be administered in similar fashion to conventional drugs. However, oligonucleotides cannot cross membranes by passive diffusion and intracellular delivery for these drugs is very inefficient. Here we review the recent advances in forming lipid-DNA particles designed to mimic viral delivery of DNA. Most evidence now supports the hypothesis that lipid-DNA drugs enter target cells by endocytosis and disrupt the endosomal membrane, releasing nucleic acid into the cytoplasm. The mechanisms of particle formation and endosome disruption are not well understood. Cationic lipids are employed to provide an electrostatic interaction between the lipid carrier and polyanionic nucleic acids, and they are critical for efficient packaging of the drugs into a form suitable for systemic administration. However, their role in endosome disruption and other aspects of successful delivery leading to gene expression or inhibition of mRNA translation are less clear. We discuss the propensity of lipid-nucleic acid particles to undergo lipid mixing and fusion with adjacent membranes, and how phosphatidylethanolamine and other lipids may act as factors capable of disrupting bilayer structure and the endosomal pathway. Finally, we consider the challenges that remain in bringing nucleic acid based drugs into the realm of clinical reality.