Modularly Assembled Research Articles

Modular Assembly of Photoactive Lipid Nanoparticles on Red Blood Cells toward Enhanced Phototherapy Efficacy.

Photodynamic therapy has been developed as a promising treatment for malignant tumors, which inspires research into photosensitizers. However, the therapeutic efficacy of individual photosensitizers is often hampered by the physiological environment. The assembly of biological materials with synthetic molecules offers a strategy to enhance functionality while improving tolerance to varying physiological conditions. Herein, we present a biohybrid system for enhanced phototherapy efficacy through a simple two-step assembly process. Photoactive lipid nanoparticles were assembled based on synthesized conjugated molecules and lipophilic prodrugs, which were then modularly assembled with red blood cells (RBCs). Driven by hydrophobic and electrostatic interactions, hydrophobic conjugated molecules were efficiently incorporated into the RBCs, while lipophilic prodrugs were simultaneously inserted into the cell membranes. The engineered RBCs harnessed the natural oxygen transport capability, enabling the internal conjugated molecules to effectively produce reactive oxygen species (ROSs) even under oxygen-poor conditions. Meanwhile, the use of ROS-cleavable linkers in prodrugs enhanced drug release for chemotherapy, which is a perfect complement to photodynamic therapy. In vitro and in vivo experiments proved the improved phototherapy efficacy of the biohybrid system. Furthermore, the changes in aggregation directed Förster resonance energy transfer between conjugated molecules and fluorescent drugs provided a mechanism to track drug release from engineered RBCs. Therefore, the modular assembly of biohybrid systems can offer multiple functionalities required for phototherapy, on-demand drug release, and imaging.

Thiacalix[4]arene Etching of an Anisotropic Cu70H22 Intermediate for Accessing Robust Modularly Assembled Copper Nanoclusters.

Atom-precise metal nanoclusters (NCs) with large bulk (nuclearity >60) are important species for insight into the embryonic phase of metal nanoparticles and their top-down etching synthesis. Herein, we report a metastable rod-shaped 70-nuclei copper-hydride NC, [Cl@Cu70H22(PhC≡C)29(CF3COO)16]2+ (Cu70), with Cl- as the template, in which the Cl@Cu59 kernel adopts a distinctive metal packing mode along the bipolar direction, and the protective ligand shell exhibits corresponding site differentiation. In terms of metal nuclearity, Cu70 is the largest alkynyl-stabilized Cu-hydride cluster to date. As a typical highly active intermediate, Cu70 could undergo a transformation into a series of robust modularly assembled Cu clusters (B-type Cu8, A-A-type Cu22, A-B-type Cu23, and A-B-A-type Cu38) upon etching by p-tert-butylthiacalix[4]arene (H4TC4A), which could not be achieved by "one-pot" synthetic methods. Notably, the patterns of A and B blocks in the Cu NCs could be effectively modulated by employing appropriate counterions and blockers, and the modular assembly mechanism was illustrated through comprehensive solution chemistry analysis using HR-ESI-MS. Furthermore, catalytic investigations reveal that Cu38 could serve as a highly efficient catalyst for the cycloaddition of propargylic amines with CO2 under mild conditions. This work not only enriched the family of high-nuclear copper-hydride NCs but also provided new insights into the growth mechanism of metal NCs.

Integrated fatigue assessment method considering average stress effects of large-scale lattice wind turbine support structures

Modular assembly techniques for ultra-high lattice wind turbine support structures are gaining popularity as a viable solution for wind energy development in low wind speed areas. However, efficient fatigue assessment of the overall structure is crucial for the widespread promotion and application of these structures. Existing engineering methods for fatigue assessment suffer from limitations in calculation efficiency and often fail to account for the effects of average stress. To address these issues, a novel integrated fatigue assessment method is proposed, which is both fast and efficient while considering the impact of average stress. The method enables the computation of cumulative fatigue damage and fatigue life for each component, as well as the identification of the location of the greatest cumulative fatigue damage in the structure. An onshore ultra-high lattice wind turbine support structure, modularly assembled to a height of 180 m, is utilized as an application example to illustrate the specific implementation process of the proposed method. The effect of different fatigue load conditions and transfer functions on cumulative fatigue damage is analyzed, and the relationship between transfer function and fatigue accumulation damage is examined. The findings indicate that average stress plays a crucial role in the cumulative fatigue damage of ultra-high lattice wind turbine support structures, and the proposed integrated fatigue assessment method simplifies the calculation process for fatigue-resistant designs substantially.

Modular coupling MOF nanozyme with natural enzyme on hollow fiber membrane for rapid and reusable detection of H2O2 and glucose.

A novel strategy based on gradient porous hollow fiber membrane (GPF) is proposed for the modular assembly of enzyme-nanozyme cascade systems. The porous structure of GPF provided sufficient specific surface area, while the gradient structure effectively minimized the leaching of enzymes and nanozymes. To enhance stability, we prepared and immobilized metal-organic framework (MOF) nanozymes, resulting in the fabrication of GPF-MOF with excellent stability and reusability for colorimetric H2O2 detection. To improve specificity and expand the detection range, micro-crosslinked natural enzymes were modularly assembled, using glucose oxidase as the model enzyme. The assembled system, GPF-mGOx@MOF, achieved a low detection limit of 0.009mM and a linear range of 0.2 to 11mM. The sensor retained 87.2% and 80.7% of initial activity after being stored for 49 days and 9 recycles, respectively. Additionally, the reliability of the biosensor was validated through glucose determination of human blood and urine samples, yielding comparable results to a commercial glucose meter.

PH-Dependent Release of Vancomycin from Modularly Assembled Collagen Laminates

To prevent surgical site infections, antibiotics can be released from carriers made of biomaterials, such as collagen, that support the healing process and are slowly degraded in the body. In our labs we have developed collagen laminates that can be easily assembled and bonded on-site, according to medical needs. As shown previously, the asymmetric assembly leads to different release rates at the major faces of the laminate. Since the pH changes during the wound healing and infection, we further examined the effect of an acidic and alkaline pH, in comparison to pH 7.4 on the release of vancomycin from different collagen samples. For this purpose, we used an additively manufactured sample holder and quantified the release by HPLC. Our results show that the pH value does not have any influence on the total amount of released vancomycin (atelocollagen sponge pH 5.5: 71 ± 2%, pH 7.4: 68 ± 8%, pH 8.5: 74 ± 3%, bilayer laminate pH 5.5: 61 ± 6%, pH 7.4: 69 ± 4% and pH 8.5: 67 ± 3%) but on the time for half-maximal release. At an acidic pH of 5.5, the swelling of the atelocollagen sponge is largely increased, leading to a 2-3 h retarded release, compared to the physiological pH. No changes in swelling were observed at the basic pH and the compound release was 1-2 h delayed. These effects need to be considered when choosing the materials for the laminate assembly.

Modular Assembly of MXene Frameworks for Noninvasive Disease Diagnosis via Urinary Volatiles.

Volatile organic compounds (VOCs) in urine are valuable biomarkers for noninvasive disease diagnosis. Herein, a facile coordination-driven modular assembly strategy is used for developing a library of gas-sensing materials based on porous MXene frameworks (MFs). Taking advantage of modules with diverse composition and tunable structure, our MFs-based library can provide more choices to satisfy gas-sensing demands. Meanwhile, the laser-induced graphene interdigital electrodes array and microchamber are laser-engraved for the assembly of a microchamber-hosted MF (MHMF) e-nose. Our MHMF e-nose possesses high-discriminative pattern recognition for simultaneous sensing and distinguishing of complex VOCs. Furthermore, with the MHMF e-nose being a plug-and-play module, a point-of-care testing (POCT) platform is modularly assembled for wireless and real-time monitoring of urinary volatiles from clinical samples. By virtue of machine learning, our POCT platform achieves noninvasive diagnosis of multiple diseases with a high accuracy of 91.7%, providing a favorable opportunity for early disease diagnosis, disease course monitoring, and relevant research.

Modular assembly of enzyme loaded nanoparticles in 3D hollow fiber electrode for electrochemical sensing

We have developed a three-dimensional (3D) enzyme loading strategy by modular assembly of enzyme loaded nanoparticles with porous conductive membranes. A 3D conductive structure was fabricated as electrode via the poly-(3,4-ethylenedioxythiophene) (PEDOT) in-situ synthesized on hollow fiber membranes (HFMs) framework, retaining the original gradient porous structures and capillary effect. Using physical entrapment pattern, enzyme-carrying nanoparticles were modularly assembled in the 3D conductive support and their spatial distribution can be regulated by particle size-tuning. The obtained enzyme entrapped 3D composites can be applied as a promising electrode in electrochemical sensing and catalysis. HFMs provide large surface area and gradient porous structure for entrapping the enzyme-carrying nanoparticles in the confined micro-space. The physical entrapment improves the enzymatic stability and promotes full contact between the enzymes and the conductive/catalytic substrates. The conductive support creates an interconnected network to increase the electrode area and the response current, thus enhancing the sensitivity of biosensors. It shows fast current response (~3 s), wide linear range (glucose, 2–24 mM; lactic acid, 0.1–6 mM), high sensitivity, and low detection limit (glucose, 100 µM; lactic acid, 10 µM) towards cancer cell metabolism monitoring. This electrode structure can be further applied in multi-target array detection towards metabolic markers by modulating different enzymes on the conductive HFMs.

Modularly Assembled Upconversion Nanoparticles for Orthogonally Controlled Cell Imaging and Drug Delivery.

Upconversion nanoparticles (UCNPs) have been used effectively as light transducers to convert near-infrared irradiation to short-wavelength emissions for photoactivation in deep tissues. UCNPs with single/multiple emissions under excitation at a single wavelength can be used for simultaneous activation of single or multiple photosensitive molecules only; an ideal multifunctional UCNP nanoplatform should not only have the ability to load multiple molecules but also should activate them at the right time with the right dose when necessary, depending upon the application for which it is used. The control of many biological processes requires complex (simultaneous or subsequent) photoactivation at different time points. Subsequent photoactivation requires UCNPs with orthogonal fluorescence emissions, which can be controlled independently. So far, there are only a few reports about UCNPs with orthogonal emissions. Synthesis of these orthogonal emission nanoparticles is complicated and tedious because nanoparticles with multiple shells need to be synthesized, and different lanthanide ions need to be doped into different shells. Also, there is no flexibility for changing the doped ions and emission profile after the nanoparticles are produced. Here, we have demonstrated a versatile method to modularly assemble individual UCNPs into UCNP clusters (UCNPs-C) with adjustable emissions. The synthesis is much easier, and there is a lot of flexibility in changing the particle size, shape, doped ions, and emission profile. We have demonstrated the use of such UCNPs-C for color encoding at the nanoscale. We further designed orthogonal photoactivatable UCNPs-C (OP-UCNPs-C), which can be independently activated under 980 nm excitation for red emission and 808 nm excitation for UV/blue emission. These OP-UCNPs-C were used for independent activation of processes for cell imaging (980 nm) and drug delivery (808 nm). In comparison to the traditional nonprogrammed activation, a programmed controlled imaging and drug delivery process could guarantee highly targeted and enhanced cell death of cancerous cells.

Hypoxia-Inducible Factor Drives Vascularization of Modularly Assembled Engineered Tissue.

Using two inhibitory methods, we demonstrated that hypoxia-inducible factor (HIF) plays an important role in vascularizing and oxygenating modularly-assembled engineered tissues. Each inhibitory technique elucidated a different mechanism by which this occurred. Whereas systemic inhibition negatively impacted early recruitment of host-derived cells, genetic inhibition in grafted endothelial cells was detrimental to their survival. Taken together, our study suggests that methods of HIF-mediated mechanisms could be harnessed to tune the extent and rate of vascularization in engineered tissue constructs.

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes

BackgroundGuayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome.ResultsThe 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N50 = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus.ConclusionThe sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Modular assembly of superstructures from polyphenol-functionalized building blocks.

The organized assembly of particles into superstructures is typically governed by specific molecular interactions or external directing factors associated with the particle building blocks, both of which are particle-dependent. These superstructures are of interest to a variety of fields because of their distinct mechanical, electronic, magnetic and optical properties. Here, we establish a facile route to a diverse range of superstructures based on the polyphenol surface-functionalization of micro- and nanoparticles, nanowires, nanosheets, nanocubes and even cells. This strategy can be used to access a large number of modularly assembled superstructures, including core-satellite, hollow and hierarchically organized supraparticles. Colloidal-probe atomic force microscopy and molecular dynamics simulations provide detailed insights into the role of surface functionalization and how this facilitates superstructure construction. Our work provides a platform for the rapid generation of superstructured assemblies across a wide range of length scales, from nanometres to centimetres.

Targeting the r(CGG) Repeats That Cause FXTAS with Modularly Assembled Small Molecules and Oligonucleotides

We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)exp) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)exp toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)expin vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)exp’s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2′-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

Features of Modularly Assembled Compounds That Impart Bioactivity Against an RNA Target

Transcriptomes provide a myriad of potential RNAs that could be the targets of therapeutics or chemical genetic probes of function. Cell-permeable small molecules, however, generally do not exploit these targets, owing to the difficulty in the design of high affinity, specific small molecules targeting RNA. As part of a general program to study RNA function using small molecules, we designed bioactive, modularly assembled small molecules that target the noncoding expanded RNA repeat that causes myotonic dystrophy type 1 (DM1), r(CUG)(exp). Herein, we present a rigorous study to elucidate features in modularly assembled compounds that afford bioactivity. Different modular assembly scaffolds were investigated, including polyamines, α-peptides, β-peptides, and peptide tertiary amides (PTAs). On the basis of activity as assessed by improvement of DM1-associated defects, stability against proteases, cellular permeability, and toxicity, we discovered that constrained backbones, namely, PTAs, are optimal. Notably, we determined that r(CUG)(exp) is the target of the optimal PTA in cellular models and that the optimal PTA improves DM1-associated defects in a mouse model. Biophysical analyses were employed to investigate potential sources of bioactivity. These investigations show that modularly assembled compounds have increased residence times on their targets and faster on rates than the RNA-binding modules from which they were derived. Moreover, they have faster on rates than the protein that binds r(CUG)(exp), the inactivation of which gives rise to DM1-associated defects. These studies provide information about features of small molecules that are programmable for targeting RNA, allowing for the facile optimization of therapeutics or chemical probes against other cellular RNA targets.

Rational Design of Bioactive, Modularly Assembled Aminoglycosides Targeting the RNA that Causes Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)(exp)) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6'-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)(exp). However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a d-Arg(9) molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r(CUG)(exp); pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA.

Using modularly assembled ligands to bind RNA internal loops separated by different distances.

Looped in: Modular assembly provides a unique opportunity to tune the affinity and specificity of ligands for a target biomolecule. Herein, we describe our investigations into how the distance between bis-benzimidazole ligand modules (see graphic) affects affinity and specificity for RNA internal loops.

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a ‘modular assembly’ method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

The role of flexibility in the rational design of modularly assembled ligands targeting the RNAs that cause the myotonic dystrophies.

Modularly assembled ligands were designed to target the RNAs that cause two currently untreatable neuromuscular disorders, myotonic dystrophy types 1 (DM1) and 2 (DM2). DM1 is caused by an expanded repeating sequence of CUG, and DM2 is caused by expanded CCUG repeats. Both are present in noncoding regions and fold into hairpins with either repeating 1x1 nucleotide UU (DM1) or 2x2 nucleotide 5'-CU/3'-UC (DM2) internal loops separated by two GC pairs. The repeats are toxic because they sequester the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Rational design of ligands targeting these RNAs was enabled by a database of RNA motif-ligand partners compiled by using two-dimensional combinatorial screening (2DCS). One 2DCS study found that the 6''-azido-kanamycin A module binds internal loops similar to those found in DM1 and DM2. In order to further enhance affinity and specificity, the ligand was assembled on a peptoid backbone to precisely control valency and the distance between ligand modules. Designed compounds are more potent and specific binders to the toxic RNAs than MBNL1 and inhibit the formation of the RNA-protein complexes with nanomolar IC(50) values. This study shows that three important factors govern potent inhibition: 1) the surface area sequestered by the assembled ligands; 2) the spacing between ligand modules since a longer distance is required to target DM2 RNAs than DM1 RNAs; and 3) flexibility in the modular assembly scaffold used to display the RNA-binding module. These results have impacts on the general design of assembled ligands targeting RNAs present in genomic sequence.

Reply to “Genome editing with modularly assembled zinc-finger nucleases”

To the Editor: The publications cited by Kim et al. describing successful construction of ZFNs by modular assembly only further support our original conclusion that this method has a high failure rate for engineering functional zinc finger arrays.1 Two of the three reports cited provide data that enable calculation of failure rates for modular assembly.2, 3 Although it is true that modular assembly yielded ZFNs for ~25% of the DNA sites targeted, failure rates measured instead by the number of zinc finger proteins tested are remarkably consistent with those reported in our original Correspondence.1 For example, at the human CCR5 gene, Kim et al. screened 315 pairs of ZFNs for activity;3 this large-scale effort yielded only a small number of functional ZFN pairs (93.3% failure rate for ZFN pairs tested). Similarly, for the tobacco SuRB gene,2 we tested 32 zinc finger arrays in vitro but identified only three with functional activity (91.6% failure rate for zinc finger arrays tested). These data are consistent with our original predicted failure rates of ~94% and ~76% for modularly assembled ZFN pairs and zinc finger arrays, respectively.1 We believe that failure rates measured by numbers of zinc finger arrays or ZFN pairs tested rather than by numbers of DNA sites targeted are more relevant statistics for potential ZFN users because these influence how many proteins must be modularly assembled and tested for each potential site. We also respectfully disagree with various statements Kim et al. make regarding Oligomerized Pool ENgineering (OPEN), a selection-based method for engineering zinc finger arrays.4 Kim et al. contend that our recent report2 shows that “modularly assembled ZFNs…outperformed OPEN ZFNs in terms of mutation frequencies” and that “each method [modular assembly and OPEN] gave rise to successful genome modification at one out of four target sites.” However, the modularly assembled and OPEN ZFNs in our study were designed to recognize different DNA target sites and therefore Kim et al.’s conclusion is based on an indirect comparison. For the single site where direct comparison was possible, only the OPEN approach yielded functional ZFNs. In addition, we found in a different direct comparison of OPEN and modular assembly at five different target sites in the EGFP gene that OPEN ZFNs were active at four sites whereas modularly assembled ZFNs showed activity at only one site.4 Furthermore, the OPEN ZFNs outperformed the modularly assembled ZFNs at this one site. We believe that the higher activity and success rate of OPEN ZFNs is most likely due to the method’s explicit consideration of the well-established context-dependent behavior of zinc fingers,1, 4 a parameter that is largely ignored by modular assembly. Kim et al assert that “OPEN ZFNs thus far are largely limited to target GNN repeat sequences (i.e. 5’-GNN GNN GNN-3’)”. However, ~40% (11 out of 28) of the half-sites successfully targeted in endogenous genes by OPEN ZFNs to date actually contain one or more non-GNN subsites (Supplementary Table 1). Furthermore, potential users of the ZFN technology should be aware that with both modular assembly and OPEN it is easiest to target sequences composed entirely of GNN subsites. Kim et al. further suggest “that careful choice and use of reliable modules could improve success rates [of modular assembly].” However, given that our original Correspondence showed that modular assembly has considerably higher failure rates for target sites harboring one or more non-GNN subsites than for those composed solely of GNN subsites,1 taking this approach could also substantially restrict the targeting range of the method. In summary, we advise potential users to carefully weigh the effort required both to engineer and to validate ZFNs. Although modular assembly is simpler to perform than the selection-based OPEN method, our direct comparisons suggest that OPEN is more efficient than modular assembly for engineering functional ZFNs. As researchers who have practiced both methods,2, 4–6 we have concluded that modular assembly requires as much (if not more) time and effort to use than OPEN when one considers the requirement to screen hundreds of largely non-functional modularly assembled ZFNs for cellular activity. Nonetheless, the Zinc Finger Consortium continues to make reagents and software for both modular assembly and OPEN available to academic scientists (http://www.addgene.org/zfc; www.zincfingers.org/software-tools.htm). We believe that rather than attempting to improve the success rate of modular assembly, future efforts should instead focus on further simplification of selection-based techniques or development of more effective design-based methods that account for the context-dependent behavior of zinc finger domains.

Controlling the Specificity of Modularly Assembled Small Molecules for RNA via Ligand Module Spacing: Targeting the RNAs That Cause Myotonic Muscular Dystrophy

Myotonic muscular dystrophy types 1 and 2 (DM1 and DM2, respectively) are caused by expansions of repeating nucleotides in noncoding regions of RNA. In DM1, the expansion is an rCUG triplet repeat, whereas the DM2 expansion is an rCCUG quadruplet repeat. Both RNAs fold into hairpin structures with periodically repeating internal loops separated by two 5'GC/3'CG base pairs. The sizes of the loops, however, are different: the DM1 repeat forms 1 x 1 nucleotide UU loops while the DM2 repeat forms 2 x 2 nucleotide 5'CU/3'UC loops. DM is caused when the expanded repeats bind the RNA splicing regulator Muscleblind-like 1 protein (MBNL1), thus compromising its function. Therefore, one potential therapeutic strategy for these diseases is to prevent MBNL1 from binding the toxic RNA repeats. Previously, we designed nanomolar inhibitors of the DM2-MBNL1 interaction by modularly assembling 6'-N-5-hexyonate kanamycin A (K) onto a peptoid backbone. The K ligand binds the 2 x 2 pyrimidine-rich internal loops found in the DM2 RNA with high affinity. The best compound identified from that study contains three K modules separated by four propylamine spacing modules and is 20-fold selective for the DM2 RNA over the DM1 RNA. Because the modularly assembled K-containing compounds also bound the DM1 RNA, albeit with lower affinity, and because the loop size is different, we hypothesized that the optimal DM1 RNA binder may display K modules separated by a shorter distance. Indeed, here the ideal DM1 RNA binder has only two propylamine spacing modules separating the K ligands. Peptoids displaying three and four K modules on a peptoid scaffold bind the DM1 RNA with K(d)'s of 20 nM (3-fold selective for DM1 over DM2) and 4 nM (6-fold selective) and inhibit the RNA-protein interaction with IC(50)'s of 40 and 7 nM, respectively. Importantly, by coupling the two studies together, we have determined that appropriate spacing can affect binding selectivity by 60-fold (20- x 3-fold). The trimer and tetramer also bind approximately 13- and approximately 63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and nontoxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and, (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.

An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins.

Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this ‘modular assembly’ approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 ‘three-finger’ ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, Kd values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here.