Simon H. Friedman

Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups.

Diazo-based precursors of photolabile groups have been used extensively for modifying nucleic acids, with the intention of toggling biological processes with light. These processes include transcription, translation and RNA interference. In these cases, the photolabile groups have been typically depicted as modifying the phosphate backbone of RNA and DNA. In this work we find that these diazo-based reagents in fact react very poorly with backbone phosphates. Instead, they show a remarkable specificity for terminal phosphates and very modest modification of the nucleobases. Furthermore, the photo deprotection of these terminal modifications is shown to be much more facile than nucleobase modified sites. In this study we have characterized this regiospecificity using RNA duplexes and model nucleotides, analyzed using LC/MS/MS. We have also applied this understanding of the regio-specificity to our technique of light activated RNA interference (LARI). We examined 27-mer double-stranded precursors of siRNA (‘dsRNA’), and have modified them using the photo-cleavable di-methoxy nitro phenyl ethyl group (DMNPE) group. By incorporating terminal phosphates in the dsRNA, we are able to guide DMNPE to react at these terminal locations. These modified dsRNA duplexes show superior performance to our previously described DMNPE-modified siRNA, with the range of expression that can be toggled by light increasing by a factor of two.

Patterning of Gene Expression Using New Photolabile Groups Applied to Light Activated RNAi

The spacing, timing, and amount of gene expression are crucial for a range of biological processes, including development. For this reason, there have been many attempts to bring gene expression under the control of light. We have previously shown that RNA interference (RNAi) can be controlled with light through the use of siRNA and dsRNA that have their terminal phosphates modified with the dimethoxy nitro phenyl ethyl (DMNPE) group. Upon irradiation, these groups photolyze and release native RNA. The main problem with light activated RNA interference (LARI) to date is that the groups used only partially block RNA interference prior to irradiation, thus limiting the utility of the approach. Here, we describe a new photocleavable group, cyclo-dodecyl DMNPE (CD-DMNPE), designed to completely block the interaction of duplexes with the cellular machinery responsible for RNA interference prior to irradiation. This allowed us to switch from normal to a near complete reduction in gene expression using light, and to construct well-defined patterns of gene expression in cell monolayers. Because this approach is built on the RNA interference pathway, it benefits from the ability to quickly identify duplexes that are effective at low or subnanomolar concentrations. In addition, it allows for the targeting of endogenous genes without additional genetic manipulation. Finally, because of the regiospecificity of CD-DMNPE, it allows a standard duplex to be quickly modified in a single step. The combination of its efficacy and ease of application will allow for the facile control of the spacing, timing, and degree of gene expression in a range of biological systems.

Construction of a Photoactivated Insulin Depot

Insulin is a primary tool for the treatment of type I diabetes. However, multiple challenges accompany its administration. Because of low oral bioavailability, it is typically injected multiple times per day, a significant lifetime burden on patients. Moreover, because of fluctuating blood sugar levels, the required amounts of insulin continuously vary over the course of a day. A current solution to the challenge of multiple daily injections is the use of an insulin pump, which delivers insulin transdermally through a cannula, an awkward and limiting solution. We have conceived of a less invasive approach, in which light is used to activate the release of insulin from a covalently linked depot (Figure 1). The elements of the system are a polymer, a photocleavable linker, and the therapeutic, insulin. Ideally, the polymer should be insoluble, so that it remains at the site of injection, and biodegradable, so that after the majority of the insulin has been released, the polymer can be cleared from the system. Insulin has features that make it particularly amenable to the photoactivated depot (PAD) approach. Because blood sugar levels vary greatly, the required concentrations of insulin in the blood also vary greatly from hour to hour. Moreover, the total volume of insulin required in a day is small, on the order of 1 mg, the equivalent of 1 mL. Even with a 10:1 ratio of carriers to insulin in a PAD, a typical 250 mL injection of insulin would contain the equivalent of 25 days insulin. The principle challenge is to engineer a material in which insulin maintains its integrity during the synthetic process, and is retained effectively until light releases it in a precisely metered fashion and in its native form. We have successfully made this material by using a new azide derivative of the 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) group that reacts with insulin. The final link to the insoluble resin is made through a “click”-type reaction with a strained cyclooctyne (DBCO) that is resin-bound. We demonstrate that insulin modified with both one and two DMNPE–azide moieties is photoreleased in a similar fashion, with the dimodified species being photolyzed in two sequential reactions, each of which has identical kinetics. This suggests chemically similar sites of modification. Furthermore, we demonstrate that the insulin modified with DMNPE–azide efficiently reacts with resin-bound DBCO, and that this final material shows efficient and metered photorelease of native insulin when using a 365 nm lightemitting diode (LED). We are pursuing multiple ways of making the photocleavable link between insulin and an insoluble resin. One of the most direct ways of accomplishing this is using a “click” approach to make the final link between resin and insulin (Figure 2). We started from the known DMNPE derivative 2, which contains both carboxy and ketone groups. This was modified with an amino azide 1 to make the corresponding amide 3 in good yield. The ketone group was then transformed into the hydrazone 4 by using hydrazine, and finally converted to the diazo derivative 5 by using MnO2, a process based on previously developed DMNPE chemistry in our group. We found that a parallel approach, in which propargyl amine was condensed with 2, was not effective. The diazo–DMNPE–azide 5 was reacted with insulin directly in DMSO, using a 2:1 mole ratio. HPLC analysis of the reaction mixture shows two main, well-resolved peaks as well as a minor one (Figure 3a). The compounds in these peaks were isolated and identified by using ESI–MS as unreacted insulin (58%), monomodified insulin (“insulin monoazide” 6, 32%), and dimodified insulin (“insulin diazide” 7, 9%). A less highly modified sample was prepared for the resin modification experiments using a 1:1 ratio of insulin to 5. This reaction product showed 75% unreacted insulin, 23% insulin monoazide 6, and 2% insulin diazide 7. For the resin linking, this mixture was used directly, because Figure 1. Overall approach to the photoactivated depot (PAD). A drug, insulin in this case, is linked to an insoluble but biodegradable resin, through a photocleavable linker. The conjugate is injected in a shallow depot cutaneously or subcutaneously. Irradiation breaks the link of insulin from the resin, thereby allowing it to diffuse away from the resin and be absorbed by the systemic circulation. Ultimately the resin is biodegraded.

An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications

RNA interference (RNAi) has become a powerful tool for investigating gene function, and, in addition, shows potential for the development of therapeutic agents. RNAi can be triggered in a variety of eukaryotic cells using small interfering RNA (siRNA), their double-stranded precursors (double-stranded RNA) and short hairpin precursors (shRNA). Here, we describe a protocol for analyzing these RNAs and their modifications using electrospray ionization mass spectrometry (ESI-MS). This protocol involves the desalting of nucleic acids using ammonium acetate precipitation, followed by characterization using ESI-MS. This protocol has been chiefly used for analyzing siRNAs and their chemical modifications, but it has also been used and can be applied to the analysis of a wide range of native and modified oligonucleotides. This protocol provides accurate information on molecular weight for a range of nucleic acids and can be completed in less than a day.

Small molecule/Nucleic acid affinity chromatography: application for the identification of telomerase inhibitors which target its key RNA/DNA heteroduplex

The purpose of this work is to develop methods for identifying high-affinity nucleic acid binding species from soluble mixtures of compounds. We have developed and applied an affinity chromatography method for identifying small molecules with high affinity for the telomerase RNA/DNA duplex. An affinity resin was derivatized with an RNA/DNA duplex which represents the key structure that forms during telomerases catalytic cycle. A soluble mixture of compounds was applied to this resin and the compounds which bound to the highest extent were also confirmed to be the best inhibitors of the enzyme. This correlation of affinity for the RNA/DNA duplex with telomerase inhibition both supports the duplex as the target of these compounds, and suggests that the affinity method may be applied for the identification of higher affinity inhibitors from soluble mixtures of compounds.

Enhanced Light-Activated RNA Interference Using Phosphorothioate-Based dsRNA Precursors of siRNA

ABSTRACTPurposeTo improve light-activated RNA interference by incorporating phosphorothioate linkages into the dsRNA used. The rationale behind this approach is that the groups have the potential to improve nuclease stability and therefore prevent cleavage of photolabile groups from the RNA termini prior to photolysis.MethodsPhotolabile groups (di-methoxy nitro phenyl ethyl or DMNPE) were incorporated into multiple double-stranded precursors of siRNA (dsRNA) that had six, two or no phosphorothioate linkages at the 3′ and 5′ ends of the strands. They were analyzed for their ability to toggle light-activated RNA interference with light and for serum stability.ResultsIncorporation of phosphorothioate linkages increased serum stability of all dsRNA examined. Presence of DMNPE groups reduced overall stability of the modified RNA relative to the analogous species without DMNPE modification. DMNPE-modified dsRNA with two phosphorothioate linkages in each strand significantly improved the window of expression toggled by light.ConclusionsIncorporating phosphorothioate groups into dsRNA both stabilizes them towards degradation by serum enzymes, as well as improves them as the basis for light-activated RNA interference.

The synthesis of tetra-modified RNA for the multidimensional control of gene expression via light-activated RNA interference

Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process are siRNA or dsRNA that have been modified with four photocleavable groups of dimethoxy nitro phenyl ethyl (DMNPE), located on the four terminal phosphate groups of the duplex RNA. These mediators can be easily synthesized and purified using two readily available products: synthetic RNA oligonucleotides and DMNPE-hydrazone. The synthesis of the tetra-DMNPE–modified duplex RNA is made possible by a remarkable regiospecificity of DMNPE for terminal phosphates (over internal phosphates or nucleobases) that we have previously identified. The four installed DMNPE groups effectively limit RNAi until irradiation cleaves them, releasing native, active siRNA. By using the described protocol, any process that is mediated by RNAi can be controlled with light. Although other methods exist to control gene expression with light by using specialized reagents, this method requires only two commercially available products. The protocol takes ∼3 d in total for the preparation of modified RNA.

Inhibition of therapeutically important polymerases with high affinity bis-intercalators.

We have previously demonstrated that polymerases such as telomerase can be inhibited by molecules (e.g., intercalators) that target the key RNA/DNA duplex substrate. In this work we show that this also holds true for reverse transcriptase, and show that the lead intercalators can be modified to increase inhibition efficacy. Specifically, we use the strategy of multiple simultaneous intercalation, by linking two intercalators with a variable linker. The rationale behind this design is that a specific linker has the potential to increase affinity and specificity for the target duplex. We have synthesized a library of 45 ethidium bis-intercalators in which the distance between intercalators is systematically varied. We observe that members of the dimer library have improved telomerase and reverse transcriptase inhibition, relative to the monomeric leads. We show that this improvement in inhibition over mono-intercalators is most prominent when non-productive sites of inhibitor binding are limited in the assay mix. When this is done, a 400-fold increase in inhibition efficacy is observed.

A Tetra-Orthogonal Strategy for the Efficient Synthesis of Scaffolds Based on Cyclic Peptides

We have developed a straightforward and robust strategy for synthesizing a family of cyclic peptide scaffolds for the presentation of defined moieties in a wide range of orientations. Specifically we are exploring quinoxaline as the moiety, as a potential nucleic acid binding motif. The method requires the use of four degrees of orthogonality, which in turn allow the extension of the main chain, incorporation of the target side chains, on-resin cyclization, and the revelation of an amino group upon cleavage to increase solubility. We show that related approaches fail for a range of reasons, including the failure of cyclization. Following the optimization of the approach with a single cyclic peptide, we synthesized a family of all possible bis and tris quinoxaline adducts showing by ESI–MS that the desired full length cyclic product is produced in a majority of cases.

The ULTIMATE Reagent: A Universal Photo‐Cleavable & Clickable Reagent for the Regiospecific and Reversible End Labeling of Any Nucleic Acid

There is a need for methods to chemically incorporate photocleavable labels into synthetic and biologically sourced nucleic acids in a chemically defined and reversible manner. We have previously demonstrated that the light‐cleaved diazo di‐methoxy nitro phenyl ethyl (diazo‐DMNPE) group has a remarkable regiospecificity for modifying terminally phosphorylated siRNA. Building on this observation, we have identified conditions under which a diazo‐DMNPE reagent that we designed (diazo‐DMNPE‐azide or DDA) is able to singly modify any nucleic acid (RNA, DNA, single‐stranded, double‐stranded, 3′ or 5′ phosphate). It can then be modified with any clickable reagent to incorporate arbitrary labels such as fluorophores into the nucleic acid. Finally, native nucleic acid can be regenerated directly through photolysis of the reagent. Use of the described approach should allow for the tagging of any nucleic acid, from any source—natural or unnatural—while allowing for the light‐induced regeneration of native nucleic acid.