Duplex Stability Research Articles

Effect of Internal and Bulge Loops on the Thermal Stability of Small DNA Duplexes.

The thermal stabilities of DNA duplexes analogous to the let-7 microRNA:lin-41 mRNA complex from Caenorhabditis elegans have been measured by free solution capillary electrophoresis. DNA duplexes with the same stems but different types of internal or bulge loops and a control with no loop have also been studied. The melting temperatures of the DNA derivatives increased linearly with the logarithm of the Na+ or K+ ion concentration in the solution. Peaks in the electropherograms corresponding to duplexes with internal or bulge loops exhibited extensive tailing at high temperatures, suggesting that denaturation occurred by slow exchange between the duplexes and their component single strands. The single strands did not separate completely from the duplexes in aqueous solutions; instead, they appeared as small subpeaks on the tails of the duplex peaks. However, complete separation of the duplexes from their component single strands was observed at 20 °C in solutions containing 300 mM tetrapropylammonium ions. In addition, counterion condensation appears to be significantly reduced in DNA duplexes containing internal or bulge loops.

Synthesis, Biophysical and Biological Evaluation of Splice-Switching Oligonucleotides with Multiple LNA-Phosphothiotriester Backbones.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2'-O-methyl phosphorothioate nucleotides for therapeutic applications. The nature of the triester alkyl group dictates the success of solid-phase synthesis; tertiary alkyl groups are lost during the P(III) oxidation step, whereas primary alkyl groups are partially cleaved during deprotection. In contrast, oligonucleotides containing secondary phosphothiotriester linkages are stable, and large numbers of triesters can be incorporated. The modified oligonucleotides have excellent duplex stability with complementary RNA and exhibit strong nuclease resistance. To expand synthetic flexibility, oligonucleotides containing multiple internal alkynyl phosphothiotriesters can be conjugated to lipids, carbohydrates, or small molecules through CuAAC click chemistry. Oligonucleotides containing LNA-THP phosphothiotriesters exhibit high levels of pre-mRNA splice switching in eukaryotic cells.

A Study Modeling Bridged Nucleic Acid-Based ASOs and Their Impact on the Structure and Stability of ASO/RNA Duplexes.

Antisense medications treat diseases that cannot be treated using traditional pharmacological technologies. Nucleotide monomers of bare and phosphorothioate (PS)-modified LNA, N-MeO-amino-BNA, 2',4'-BNANC[NH], 2',4'-BNANC[NMe], and N-Me-aminooxy-BNA antisense modifications were considered for a detailed DFT-based quantum chemical study to estimate their molecular-level structural and electronic properties. Oligomer hybrid duplex stability is described by performing an elaborate MD simulation study by incorporating the PS-LNA and PS-BNA antisense modifications onto 14-mer ASO/RNA hybrid gapmer type duplexes targeting protein PTEN mRNA nucleic acid sequence (5'-CTTAGCACTGGCCT-3'/3'-GAAUCGUGACCGGA-5'). Replica sets of MD simulations were performed accounting to two data sets, each set simulated for 1 μs simulation time. Bulk properties of oligomers are regulated by the chemical properties of their monomers. As such, the primary goal of this work focused on establishing an organized connection between the monomeric BNA nucleotide's electronic effects observed in DFT studies and the macroscopic behavior of the BNA antisense oligomers, as observed in MD simulations. The results from this study predicted that spatial orientation of MO-isosurfaces of the BNA nucleotides are concentrated in the nucleobase region. These BNA nucleotides may become less accessible for various electronic interactions when coupled as ASOs forming duplexes with target RNAs and when the ASO/RNA duplexes further bind with the RNase H. Understanding such electronic interactions is crucial to design superior antisense modifications with specific electronic properties. Also, for the particular nucleic acid sequence solvation of the duplexes although were higher compared to the natural oligonucleotides, their binding energies being relatively lower may lead to decreased antisense activity compared to existing analogs such as the LNAs and MOEs. Fine tuning these BNAs to obtain superior binding affinity is thus a necessity.

MicroLOCK: Highly stable microgel biosensor using locked nucleic acids as bioreceptors for sensitive and selective detection of let-7a

Chemically modified oligonucleotides can solve biosensing issues for the development of capture probes, antisense, CRISPR/Cas, and siRNA, by enhancing their duplex-forming ability, their stability against enzymatic degradation, and their specificity for targets with high sequence similarity as microRNA families. However, the use of modified oligonucleotides such as locked nucleic acids (LNA) for biosensors is still limited by hurdles in design and from performances on the material interface. Here we developed a fluorogenic biosensor for non-coding RNAs, represented by polymeric PEG microgels conjugated with molecular beacons (MB) modified with locked nucleic acids (MicroLOCK). By 3D modeling and computational analysis, we designed molecular beacons (MB) inserting spot-on LNAs for high specificity among targets with high sequence similarity (95%). MicroLOCK can reversibly detect microRNA targets in a tiny amount of biological sample (2μL) at 25°C with a higher sensitivity (LOD 1.3fM) without any reverse transcription or amplification. MicroLOCK can hybridize the target with fast kinetic (about 30min), high duplex stability without interferences from the polymer interface, showing high signal-to-noise ratio (up to S/N=7.3). MicroLOCK also demonstrated excellent resistance to highly nuclease-rich environments, in real samples. These findings represent a great breakthrough for using the LNA in developing low-cost biosensing approaches and can be applied not only for nucleic acids and protein detection but also for real-time imaging and quantitative assessment of gene targeting both in vitro and in vivo.

7-Deaza-2'-deoxyisoguanosine, a Noncanonical Nucleoside for Nucleic Acid Code Expansion and New DNA Constructs: Nucleobase Functionalization of Inverse Watson-Crick and Purine-Purine Base Pairs.

7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

2′-Deoxyribo-oligonucleotides duplexes and G-quadruplexes structures and thermal stability in 1‑butyl‑1-methylpiperidinium and 1-octyl-3-methylimidazolium bromide ionic liquid solutions

Conformation and thermal stability of duplexes made of various ratio of purine and pyrimidine, but also of two G-quadruplexes, were evaluated in two different ionic liquids, aliphatic 1‑butyl‑1-methylpiperidinium bromide and 1-octyl-3-methylimidazolium bromide with an aromatic moiety, by means of UV-spectroscopy and circular dichroism. It appeared that no conformational change occurred within all structures studied whatever the medium was, only thermal stabilities were affected.

Synthesis and Properties of RNA Modified with Thioamide Internucleoside Linkage.

Recent success of RNA therapeutics has reinvigorated interest in chemical modifications of RNA. As exemplified by the phosphorothioates, modifications of sugar-phosphate backbone have been remarkably impactful but relatively underexplored in therapeutic RNAs. The present study reports synthesis, thermal stability, and RNA interference activity of RNAs modified with thioamide linkages. Compared to the previously studied amide-modified RNA, thioamide linkages strongly destabilized a short self-complementary RNA model duplex. However, in short interfering RNAs amides and thioamides had a similar effect on duplex stability and target RNA cleavage activity and specificity. Hence, the thioamide may be added to the toolbox of chemical biologist as a useful backbone modification well tolerated by the RNA interference machinery.

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 – 78.2 °C for 10–100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1–3 °C, and denaturation power increases in the order MeOH<EtOH<MeCN. Next, we investigated the RNA duplex melting using selected chromatographic techniques and within the 10–90 °C column temperature range. Melting temperature obtained with size exclusion chromatography in 25 mM sodium phosphate buffer was ∼ 70 °C, about 5 °C lower compared to DSC values. The apparent melting temperature for a reversed-phase chromatography experiment was < 10 °C for 10 mM ammonium acetate mobile phase with acetonitrile eluent. Ion-pair reversed-phase with 10 mM triethylamine, 100 mM hexafluoroisopropanol and MeCN as eluent yielded Tm 49.3–54.9 °C. Hydrophilic interaction chromatography experiments with 10 mM ammonium acetate and ∼ 50 % acetonitrile mobile phase showed high duplex stability (Tm ∼ 80 °C). The observed chromatographic Tm values suggest that the formation of an organic/aqueous solvent layer on the chromatographic sorbent surface affects the duplex stability more significantly than the composition of the mobile phase alone.

7-Deazapurine and Pyrimidine Nucleoside and Oligonucleotide Cycloadducts Formed by Inverse Diels-Alder Reactions with 3,6-Di(pyrid-2-yl)-1,2,4,5-tetrazine: Ethynylated and Vinylated Nucleobases for Functionalization and Impact of Pyridazine Adducts on DNA Base Pair Stability and Mismatch Discrimination.

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

DNA-PAINT Probe Modifications Support High-Resolution Imaging with Shorter Binding Domains.

DNA-based Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is an effective super resolution microscopy technique, and its optimization is key to improve nanoscale detection. The state-of-the-art improvements that are at the base of this optimization have been first routinely validated on DNA nanostructure devices before being tested on biological samples. This allows researchers to finely tune DNA-PAINT imaging features in a more controllable in vitro environment. Dye-labeled oligonucleotide probes with short hybridization domains can expand DNA-PAINT's detection by targeting short nucleotide sequences and improving resolution, speed, and multiplexing. However, developing these probes is challenging as their brief bound state makes them difficult to capture under routine imaging conditions. To extend dwell binding times and promote duplex stability, we introduced structural and chemical modifications to our imager probes. The modifications included mini-hairpins and/or Bridged Nucleic Acids (BNA); both of which increase the thermomechanical stability of a DNA duplex. Using this approach we demonstrate DNA-PAINT imaging with approximately 5 nm resolution using a 4-nucleotide hybridization domain that is 43% shorter than previously reported probes. Imager probes with such short hybridization domains are key for improving detection on DNA nanostructure devices because they have the capability to target a larger number of binding domains per localization unit. This is essential for metrology applications such as Nucleic Acid Memory (NAM) where the information density is dependent on the binding site length. The selected imager probes reported here present imaging resolution equivalent to current state-of-the-art DNA-PAINT probes, creating a strategy to image shorter DNA domains for nanoscience and nanotechnology alike.

Micro Target: MicroRNA Target Prediction and Validation with Experimentally Positive and Negative Examples

MicroRNAs (miRNAs) usually controls the gene by binding to complementary sites of 3’ untranslated region of its target genes. Numerous criteria-based and machine learning approaches are available in the literature to predict miRNA–mRNA interactions, but most of them struggle with either high false positive or false negative rates and also don’t show good validation with experimentally validated positive and negative examples. Here we present microTarget, a new computational approach for identifying miRNA target genes which are based on complementarity score, thermodynamic duplex stability and also independent of conservation of target sites in related genomes. In this article, we validated our algorithm using positive and negative data from the literature in various human tissues, and our method outperformed existing computational methods such as miRanda, RNA22, and PITA. Receiver operating characteristic curves (ROC) and Matthew's correlation coefficient (MCC) were calculated using experimentally validated data, and they reveal that microTarget greatly improves miRNA target prediction compared to the three algorithms employed individually. Additionally, an F-score analysis demonstrated that microTarget greatly enhances the relevance of the other techniques. Thus, microTarget is a useful tool for biologists looking for miRNA targets and integrating them into biological contexts.

Synthesis and characterization of oligonucleotides containing 2′-O,4′-C-methyleneoxy-bridged 5-methyluridine with an unsubstituted bridge

In this study, a 2′-O,4′-C-methyleneoxy-bridged 5-methyluridine with three consecutive acetal groups (TaNA with a thymine base) was synthesized. The synthesized TaNA was incorporated into oligonucleotides (ONs), and their properties were compared with those of ONs containing the 7′-methylated analog (Me-TaNA) modifications previously reported by us. The duplex stability of TaNA with single-stranded RNA was comparable to that of Me-TaNA. However, the triplex stability of TaNA-modified ONs with double-stranded DNA was slightly lower than that of Me-TaNA-modified ONs. Additionally, the enzymatic stability of TaNA-modified ONs was lower than that of Me-TaNA-modified ONs. These results suggest that the 7′-methyl substitution on the TaNA structure is crucial for enhancing the properties of modified ONs.

Accelerated Screening of Alternative DNA Base‐Organic Molecule‐Base Architectures via Integrated Theory and Experiment

AbstractOrganic molecule‐mediated noncanonical DNA self‐assembly expands the standard DNA base‐pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base‐pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub‐stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule‐incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA‐molecule architectures beyond Watson–Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA‐based delivery system and probe design for sensitive detection of certain molecules.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we have focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation (RMSD) values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability plays a crucial role.

Impact of Single Acyclic Phosphonate Nucleotide (ZNA) Modifications on DNA Duplex Stability.

An acyclic phosphonate-linked nucleic acid backbone (ZNA) demonstrated the capability to support duplex formation and propagate genetic information in vivo, unveiling its potential for evolution into a synthetic genetic system (XNA). To determine the structural impact of such modification, modified Dickerson Drew DNA dodecamers (DDDs) were prepared by solid phase synthesis, each containing either an (R) or (S) isomeric form of a cytosine ZNA nucleotide. While the DDD is known to adopt a stable duplex, both duplex and hairpin forms were simultaneously observed for both modified oligonucleotides by NMR spectroscopy over a broad temperature range (5-65 °C). Diffusion-ordered spectroscopy (DOSY) experiments allowed to separate duplex and hairpin signals based on the different diffusion constants of both conformational states. For the oligomer containing (R)-ZNA, only the duplex form occurred at 5 °C, while it was not possible to determine by NMR a single hairpin conformation at higher temperatures. In the case of the (S)-ZNA nucleoside modified oligomer, both hairpin and duplex forms were observable at 0 °C, while a single hairpin conformation was detected at 37 °C, suggesting a higher destabilizing effect on dsDNA.

Binding properties of ruthenium(II) polypyridyl complexes [Ru(bpy)2(L)]2+(L=7-F-dppz and 7OCH3–-dppz) with poly(A)-poly(U) under dilute and molecular crowding conditions

Two ruthenium(II) polypyridyl complexes, [Ru(bpy)2(7-F-dppz)]2+ (Ru1, bpy = 2,2′-bipyridine, 7-F-dppz = 7-fluorodipyrido[3,2-a:2′,3′-c]phenazine) and [Ru(bpy)2(7OCH3–-dppz)]2+ (Ru2, 7OCH3–-dppz = 7‑methoxy-dipyrido-[3,2-a,2′,3′-c]-phenazine), were synthesized. The binding properties of Ru1 and Ru2 to duplex RNA poly(A)-poly(U) have been explored through spectroscopic techniques and viscometry. Spectral titration and viscosity experiments show that the binding mode of Ru1 and Ru2 to double-stranded RNA(dsRNA) in dilute solution and molecular crowded environment are intercalation, and that the binding affinity of Ru1 for poly(A)-poly(U) is greater than that of Ru2. Thermal denaturation experiments showed that two complexes have good thermal stability for dsRNA in two environments. Fluorescence titrations showed that Ru1 and Ru2 could act as molecular "light switch" for duplex poly(A)-poly(U) in both environments. Surprisingly, the binding effect of ligands and poly(A)-poly(U) was significantly reduced under crowded condition, and the thermal stability of RNA duplex was less significant than that of dilute solution. The reason of this result is due to the water activity in molecular crowded environment, the increased viscosity of the medium leading to a decrease in the affinity of the ligand for the RNA duplex, and also the electronic effect of the ligand on the binding affinity of the poly(A)-poly(U). Results of the present work facilitate our understanding of the two environments as well as the interaction between ruthenium(II) polypyridyl complexes and dsRNA is influenced by the electronegativity of the substituents.

Enhanced Sequence-Specific DNA Recognition Using Oligodeoxynucleotide-Benzimidazole Conjugates.

Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties. In the present study, five Hoechst 33258 derived benzimidazoles were conjugated to T rich ODNs and their hybridization effectiveness was tested. Thermal denaturation studies revealed significant stabilization of complementary duplexes by ODN-benzimidazole conjugates, with the extent of stabilization being highly dependent on the length of the linker between DNA and benzimidazole. The increases in thermal stability were determined to be due to the binding of the benzimidazole moiety to the duplex. Circular dichroism and molecular modeling studies provided insights toward the influence of conjugation on duplex structure and how linker length impacts placement of the benzimidazole moiety in the minor groove. Furthermore, thermal denaturation studies with the complementary strand containing a single base mismatch or being RNA revealed that covalent conjugation of benzimidazoles to an ODN also enhances the sequence specificity. The fundamental studies reported herein provide a strategy to improve the stability and specificity properties of the ODN probes, which can be of use for targeting and diagnostics applications.

Selective Duplex Formation in Mixed Sequence Libraries of Synthetic Polymers.

Recognition-encoded melamine oligomers (REMO) are synthetic polymers that feature an alternating 1,3,5-triazine-piperazine backbone and side-chains equipped with either a phenol or phosphine oxide recognition unit. An automated method for the solid-phase synthesis (SPS) of REMO of any specified sequence has been developed starting from dichlorotriazine monomer building blocks. Complementary homo-oligomers with either six phenols or six phosphine oxides were synthesized and shown to form a stable duplex in nonpolar solvents by NMR denaturation experiments. The duplex was covalently trapped by equipping the ends of the oligomers with an azide and an alkyne group and using a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. The SPS methodology was adapted to synthesize mixed sequence libraries by using a mixture of two different dichlorotriazine building blocks in each coupling cycle of an oligomer synthesis. The resulting libraries contain statistical mixtures of all possible sequences. The self-assembly properties of these libraries were screened by using the CuAAC reaction to trap any duplexes present. In mixed sequence libraries of 6-mers, the trapping experiments showed that only sequence-complementary oligomers formed duplexes at micromolar concentrations in dichloromethane. The automated synthesis approach developed here provides access to large libraries of mixed sequence synthetic polymers, and the covalent trapping experiment provides a convenient tool for screening functional properties of mixtures. The results suggest high-fidelity sequence-selective duplex formation in mixtures of 6-mer sequences of the REMO architecture.

Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans.

Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on catalytic residues for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.