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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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: Despite a rise in automation, workers in the petroleum refining and petrochemical manufacturing industry are potentially exposed to various chemicals through inhalation while performing routine job duties. Many factors contribute to the degree of exposure experienced in this setting. The study objective was to characterize the impact of workplace conditions, anthropometric variability, and task orientation on exposure for a simulated routine operations task., Methods: A chemical exposure laboratory simulation study was designed to evaluate the dependent variable of chemical exposure level in the breathing zone for methane and sulfur hexafluoride. The independent variables were (i) posture of the worker, (ii) worker anthropometry, (iii) process configuration, and (iv) gas density., Results: Pipe height was a significant predictor of gas concentration measured in the breathing zone when located in a position that encouraged the gas to enter the breathing zone of the worker. Worker anthropometry had a major impact; tall worker's (male) chemical concentrations exceeded those of the short worker (female) for methane simulations but the opposite resulted for sulfur hexafluoride. Also, worker posture had a significant impact on gas exposure where nonneutral postures were found to have higher levels of chemical concentration., Conclusions: The study findings indicate that the breathing zone location is altered by posture and worker height, which changes the exposures relative to the emission source depending on the gas density of the chemicals that are present. As a result, qualitative risk assessment cannot be performed accurately without accounting for these factors. Practically, controls may need to account for worker size differences and posture adaptations., (Published by Oxford University Press on behalf of The British Occupational Hygiene Society 2022.)

Recovery of dexterous hand use is critical for functional outcome after stroke. Grip force recordings can inform on maximal motor output and modulatory and inhibitory cerebral functions, but how these actually contribute to recovery of dexterous hand use is unclear. This cohort study used serially assessed measures of hand kinetics to test the hypothesis that behavioural measures of motor modulation and inhibition explain dexterity recovery beyond that explained by measures of motor output alone. We also investigated the structural and functional connectivity correlates of grip force control recovery. Eighty-nine adults (median age = 54 years, 26% females) with first-ever ischaemic or haemorrhagic stroke and persistent arm and hand paresis were assessed longitudinally, at 3 weeks, and at 3 and 6 months after stroke. Kinetic measures included: maximal grip force, accuracy of precision and power grip force control, and ability to release force abruptly. Dexterous hand use was assessed clinically with the Box and Block Test and motor impairment with the upper extremity Fugl-Meyer Assessment. Structural and functional MRI was used to assess weighted corticospinal tract lesion load, voxel-based lesion symptom mapping and interhemispheric resting-state functional connectivity. Fifty-three per cent of patients had severe initial motor impairment and a majority still had residual force control impairments at 6 months. Force release at 3 weeks explained 11% additional variance of Box and Block Test outcome at 6 months, above that explained by initial scores (67%). Other kinetic measures did not explain additional variance of recovery. The predictive value of force release remained significant when controlling for corticospinal tract lesion load and clinical measures. Corticospinal tract lesion load correlated with recovery in grip force control measures. Lesions involving the parietal operculum, insular cortex, putamen and fronto-striatal tracts were also related to poorer force modulation and release. Lesions to fronto-striatal tracts explained an additional 5% of variance in force release beyond the 43% explained by corticospinal injury alone. Interhemispheric functional connectivity did not relate to force control recovery. We conclude that not only voluntary force generation but also force release (reflecting motor inhibition) are important for recovery of dexterous hand use after stroke. Although corticospinal injury is a main determinant of recovery, lesions to integrative somatosensory areas and fronto-parietal white matter (involved in motor inhibition) explain additional variance in post-stroke force release recovery. Our findings indicate that post-stroke upper limb motor impairment profiling, which is essential for targeted treatment, should consider both voluntary grasp generation and inhibition., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5' end and Sp diastereomers at the 3' end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5' end and Sp isomer at the 3' end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ability to infer from uncertain information is impaired in schizophrenia and is associated with hallucinations and false beliefs. The accumulation of information is a key process for generating a predictive internal model, which statistically estimates an outcome from a specific situation. This study examines if updating the predictive model by the accumulation of information in absence of feedback is impaired in schizophrenia. We explored the implicit adaptation to the probability of being instructed to perform a movement (33%-Go, 50%-Go, or 66%-Go) in a Go/NoGo task in terms of reaction times (RTs), electromyographic activity, and corticospinal excitability (CSE) of primary motor cortex (M1). CSE was assessed at two time points to evaluate prediction of the upcoming instruction based on previously accumulated information: at rest (preceding the warning signal) and at the Go/NoGo signal onset. Three groups were compared: patients with schizophrenia (n = 20), unaffected siblings (n = 16), and healthy controls (n = 20). Controls and siblings showed earlier movement onset and increased CSE with higher Go probability. CSE adaptation seemed long-lasting, because the two CSE measures, at least 1500 ms apart, strongly correlated. Patients with schizophrenia failed to show movement onset (RT) adaptation and modulation of CSE. In contrast, all groups decreased movement duration with increasing Go probability. Modulation of CSE in the anticipatory phase of the potential movement reflected the estimation of upcoming response probability in unaffected controls and siblings. Impaired modulation of CSE supports the hypothesis that implicit adaptation to probabilistic context is altered in schizophrenia., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Impairments in attentional, working memory and sensorimotor processing have been consistently reported in schizophrenia. However, the interaction between cognitive and sensorimotor impairments and the underlying neural mechanisms remains largely uncharted. We hypothesized that altered attentional processing in patients with schizophrenia, probed through saccadic inhibition, would partly explain impaired sensorimotor control and would be reflected as altered task-dependent modulation of cortical excitability and inhibition. Twenty-five stabilized patients with schizophrenia, 17 unaffected siblings and 25 healthy control subjects were recruited. Subjects performed visuomotor grip force-tracking alone (single-task condition) and with increased cognitive load (dual-task condition). In the dual-task condition, two types of trials were randomly presented: trials with visual distractors (requiring inhibition of saccades) or trials with addition of numbers (requiring saccades and addition). Both dual-task trial types required divided visual attention to the force-tracking target and to the distractor or number. Gaze was measured during force-tracking tasks, and task-dependent modulation of cortical excitability and inhibition were assessed using transcranial magnetic stimulation. In the single-task, patients with schizophrenia showed increased force-tracking error. In dual-task distraction trials, force-tracking error increased further in patients, but not in the other two groups. Patients inhibited fewer saccades to distractors, and the capacity to inhibit saccades explained group differences in force-tracking performance. Cortical excitability at rest was not different between groups and increased for all groups during single-task force-tracking, although, to a greater extent in patients (80%) compared to controls (40%). Compared to single-task force-tracking, the dual-task increased cortical excitability in control subjects, whereas patients showed decreased excitability. Again, the group differences in cortical excitability were no longer significant when failure to inhibit saccades was included as a covariate. Cortical inhibition was reduced in patients in all conditions, and only healthy controls increased inhibition in the dual-task. Siblings had similar force-tracking and gaze performance as controls but showed altered task-related modulation of cortical excitability and inhibition in dual-task conditions. In patients, neuropsychological scores of attention correlated with visuomotor performance and with task-dependant modulation of cortical excitability. Disorganization symptoms were greatest in patients with weakest task-dependent modulation of cortical excitability. This study provides insights into neurobiological mechanisms of impaired sensorimotor control in schizophrenia showing that deficient divided visual attention contributes to impaired visuomotor performance and is reflected in impaired modulation of cortical excitability and inhibition. In siblings, altered modulation of cortical excitability and inhibition is consistent with a genetic risk for cortical abnormality., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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'-Cβ-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and β-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'β-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'-Cβ-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cβ-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'β-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'-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During RNA-induced silencing complex (RISC) assembly the guide (or antisense) strand has to separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process was found to be facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without prior cleavage. Here we show that the potency of siRNA can be improved by modulating the internal thermodynamic stability profile with chemical modifications. Using a model siRNA targeting the firefly luciferase gene with subnanomolar IC50, we found that placement of thermally destabilizing modifications, such as non-canonical bases like 2,4-difluorotoluene or single base pair mismatches in the central region of the sense strand (9-12 nt), significantly improve the potency. For this particular siRNA, the strongest correlation between the decrease in thermal stability and the increase in potency was found at position 10. Controls with stabilized sugar-phosphate backbone indicate that enzymatic cleavage of the sense strand prior to strand dissociation is not required for silencing activity. Similar potency-enhancing effects were observed as this approach was applied to other functional siRNAs targeting a different site on the firefly luciferase transcript or endogenously expressed PTEN.

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA-protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176-183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 ('slicer' enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson-Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T(M)) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.

Rotation of a DNA or RNA nucleotide out of the double helix and into a protein pocket ('base flipping') is a mechanistic feature common to some DNA/RNA-binding proteins. Here, we report the structure of HhaI methyltransferase in complex with DNA containing a south-constrained abasic carbocyclic sugar at the target site in the presence of the methyl donor byproduct AdoHcy. Unexpectedly, the locked south pseudosugar appears to be trapped in the middle of the flipping pathway via the DNA major groove, held in place primarily through Van der Waals contacts with a set of invariant amino acids. Molecular dynamics simulations indicate that the structural stabilization observed with the south-constrained pseudosugar will not occur with a north-constrained pseudosugar, which explains its lowered binding affinity. Moreover, comparison of structural transitions of the sugar and phosphodiester backbone observed during computational studies of base flipping in the M.HhaI-DNA-AdoHcy ternary complex indicate that the south-constrained pseudosugar induces a conformation on the phosphodiester backbone that corresponds to that of a discrete intermediate of the base-flipping pathway. As previous crystal structures of M.HhaI ternary complex with DNA displayed the flipped sugar moiety in the antipodal north conformation, we suggest that conversion of the sugar pucker from south to north beyond the middle of the pathway is an essential part of the mechanism through which flipping must proceed to reach its final destination. We also discuss the possibility of the south-constrained pseudosugar mimicking a transition state in the phosphodiester and sugar moieties that occurs during DNA base flipping in the presence of M.HhaI.

Oligodeoxyribonucleotides containing pseudorotationally locked sites derived from bicyclo[3.1.0]hexane pseudosugars have been synthesized using adenosine, thymidine and abasic versions of North- and South-methanocarba nucleosides. The reaction conditions for coupling and oxidation steps of oligonucleotide synthesis have been investigated and optimized to allow efficient and facile solid-phase synthesis using phosphoramidite chemistry. Our studies demonstrate that the use of iodine for P(III) to P(V) oxidation leads to strand cleavage at the sites where the pseudosugar is North. In contrast, the same cleavage reaction was not observed in the case of South pseudosugars. Iodine oxidation generates a 5'-phosphate oligonucleotide fragment on the resin and releases the North pseudosugar into the solution. This side reaction, which is responsible for the extremely low yields observed for the incorporation of the North pseudosugar analogs, has been studied in detail and can be easily overcome by replacing iodine with t-butylhydroperoxide as oxidant.

Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.

To further our understanding of the functional roles of different motor cortical areas, we made a quantitative comparison of the density of corticospinal projections from primary motor cortex (M1) and supplementary motor area (SMA) to spinal motor nuclei supplying hand and finger muscles in four macaque monkeys. We also compared the action of corticospinal outputs excited by electrical stimulation of these two areas on upper limb motoneurons recorded in three anaesthetized macaques. The hand representations of SMA and M1 were first identified using structural magnetic resonance imaging scans and intracortical microstimulation. In the anatomical study we then made focal injections of wheatgerm agglutinin- horseradish peroxidase into these representations, which were subsequently confirmed by analysis of retrograde cortical labelling. Densitometric analysis showed that corticospinal projections from M1 were denser and occupied a greater proportion of the hand muscle motor nuclei than did projections from SMA. In caudal Th1 the densest projections from M1 occupied 81% of this motoneuronal area, compared with only 6% from SMA. In the electrophysiological study, bipolar intracortical stimulation of the hand representation of M1 and SMA evoked direct (D) and indirect (I) corticospinal volleys. Volleys elicited by M1 stimulation had larger amplitudes and faster conduction velocities than those evoked from the SMA. Intracellular recordings were made from 84 contralateral upper limb motoneurons. M1 and SMA stimulation evoked markedly different responses in tested motoneurons: EPSPs were larger and more common from M1 (88% of motoneurons) than from SMA (48%). Some motoneurons (16/84) showed evidence of excitatory postsynaptic potentials mediated by monosynaptic action of the D-wave evoked from M1; these early effects were not observed from the SMA. In most motoneurons (74/84) EPSPs had segmental latencies indicating that they were due to monosynaptic action of the I-wave. The results are consistent with cortico-motoneuronal (CM) connections originating from both SMA and M1 converging upon single motoneurons, but those from M1 are far more numerous and exert stronger excitatory effects than from the SMA. Thus although they may function in parallel, the two CM projections probably make different contributions to upper limb motor control.

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na(+.)O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na(+) by K(+), Rb(+) or Cs(+) and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb(+) or Cs(+) also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 A.