In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells.

I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Challenges of Assessing Exon 53 Skipping of the Human DMD Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2'-O-methyl (2'OMe), locked nucleic acid (LNA)-2'OMe, 2'-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2'OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2'OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.

Telomeric i-motifs and C-strands inhibit parallel G-quadruplex extension by telomerase.

Telomeric C-rich repeated DNA sequences fold into tetrahelical i-motif structures in vitro at acidic pH. While studies have suggested that i-motifs may form in cells, little is known about their potential role in human telomere biology. In this study, we explore the effect of telomeric C-strands and i-motifs on the ability of human telomerase to extend G-rich substrates. To promote i-motif formation at neutral pH, we use telomeric sequences where the cytidines have been substituted with 2'-fluoroarabinocytidine. Using FRET-based studies, we show that the stabilized i-motifs resist hybridization to concomitant parallel G-quadruplexes, implying that both structures could exist simultaneously at telomeric termini. Moreover, through telomerase activity assays, we show that both unstructured telomeric C-strands and telomeric i-motifs can inhibit the activity and processivity of telomerase extension of parallel G-quadruplexes and linear telomeric DNA. The data suggest at least three modes of inhibition by C-strands and i-motifs: direct hybridization to the substrate DNA, hybridization to nascent product DNA resulting in early telomerase dissociation, and interference with the unique mechanism of telomerase unwinding and extension of a G-quadruplex. Overall, this study highlights a potential inhibitory role for the telomeric C-strand in telomere maintenance.

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of the first viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectroscopy. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

Sequence-Controlled Spherical Nucleic Acids: Gene Silencing, Encapsulation, and Cellular Uptake.

Antisense oligonucleotides (ASOs) can predictably alter RNA processing and control protein expression; however, challenges in the delivery of these therapeutics to specific tissues, poor cellular uptake, and endosomal escape have impeded progress in translating these agents into the clinic. Spherical nucleic acids (SNAs) are nanoparticles with a DNA external shell and a hydrophobic core that arise from the self-assembly of ASO strands conjugated to hydrophobic polymers. SNAs have recently shown significant promise as vehicles for improving the efficacy of ASO cellular uptake and gene silencing. However, to date, no studies have investigated the effect of the hydrophobic polymer sequence on the biological properties of SNAs. In this study, we created a library of ASO conjugates by covalently attaching polymers with linear or branched [dodecanediol phosphate] units and systematically varying polymer sequence and composition. We show that these parameters can significantly impact encapsulation efficiency, gene silencing activity, SNA stability, and cellular uptake, thus outlining optimized polymer architectures for gene silencing.

Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.

End-ligation can dramatically stabilize i-motifs at neutral pH.

Stabilizing i-motif structures at neutral pH and physiological temperature remains a major challenge. Here, we demonstrate the use of chemical end-ligation to stabilize intramolecular i-motifs at both acidic and neutral pH. We also demonstrate that combining 2'-deoxy-2'-fluoroarabinocytidine substitutions and end-ligation results in an i-motif with an unparalleled thermal stability of 54 °C at neutral pH. Overall, the ligated i-motifs presented herein may be used in screens for selective i-motif ligands and proteins and could find important applications in nanotechnology.

Small Molecule-Templated DNA Hydrogel with Record Stiffness Integrates and Releases DNA Nanostructures and Gene Silencing Nucleic Acids.

Deoxyribonucleic acid (DNA) hydrogels are a unique class of programmable, biocompatible materials able to respond to complex stimuli, making them valuable in drug delivery, analyte detection, cell growth, and shape-memory materials. However, unmodified DNA hydrogels in the literature are very soft, rarely reaching a storage modulus of 103  Pa, and they lack functionality, limiting their applications. Here, a DNA/small-molecule motif to create stiff hydrogels from unmodified DNA, reaching 105  Pa in storage modulus is used. The motif consists of an interaction between polyadenine and cyanuric acid-which has 3-thymine like faces-into multimicrometer supramolecular fibers. The mechanical properties of these hydrogels are readily tuned, they are self-healing and thixotropic. They integrate a high density of small, nontoxic molecules, and are functionalized simply by varying the molecule sidechain. They respond to three independent stimuli, including a small molecule stimulus. These stimuli are used to integrate and release DNA wireframe and DNA origami nanostructures within the hydrogel. The hydrogel is applied as an injectable delivery vector, releasing an antisense oligonucleotide in cells, and increasing its gene silencing efficacy. This work provides tunable, stimuli-responsive, exceptionally stiff all-DNA hydrogels from simple sequences, extending these materials' capabilities.

i-Motif folding intermediates with zero-nucleotide loops are trapped by 2'-fluoroarabinocytidine via F···H and O···H hydrogen bonds.

G-quadruplex and i-motif nucleic acid structures are believed to fold through kinetic partitioning mechanisms. Such mechanisms explain the structural heterogeneity of G-quadruplex metastable intermediates which have been extensively reported. On the other hand, i-motif folding is regarded as predictable, and research on alternative i-motif folds is limited. While TC5 normally folds into a stable tetrameric i-motif in solution, we report that 2'-deoxy-2'-fluoroarabinocytidine (araF-C) substitutions can prompt TC5 to form an off-pathway and kinetically-trapped dimeric i-motif, thereby expanding the scope of i-motif folding landscapes. This i-motif is formed by two strands, associated head-to-head, and featuring zero-nucleotide loops which have not been previously observed. Through spectroscopic and computational analyses, we also establish that the dimeric i-motif is stabilized by fluorine and non-fluorine hydrogen bonds, thereby explaining the superlative stability of araF-C modified i-motifs. Comparative experimental findings suggest that the strength of these interactions depends on the flexible sugar pucker adopted by the araF-C residue. Overall, the findings reported here provide a new role for i-motifs in nanotechnology and also pose the question of whether unprecedented i-motif folds may exist in vivo.

Crystal Structure of the RNA Lariat Debranching Enzyme Dbr1 with Hydrolyzed Phosphorothioate RNA Product.

The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2'-5' phosphodiester bond in RNA lariats produced by the spliceosome. Here, we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) that contain a 2'-5'-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to cocrystallize a phosphorothioate-branched RNA (PS-bRNA) with wild-type Entamoeba histolytica Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond. The crystal structure revealed EhDbr1 in a product-bound state, with the hydrolyzed 2'-5' fragment of the PS-bRNA mimicking the binding mode of the native bRNA substrate. These findings suggest that product inhibition may contribute to the kinetic mechanism of Dbr1. We show that Dbr1 enzymes cleave phosphorothioate linkages at rates ∼10,000-fold more slowly than native phosphate linkages. This new product-bound crystal structure offers atomic details, which can aid inhibitor design. Dbr1 inhibitors could be therapeutic or investigative compounds for human diseases such as human immunodeficiency virus (HIV), amyotrophic lateral sclerosis (ALS), cancer, and viral encephalitis.

Metal content and kinetic properties of yeast RNA lariat debranching enzyme Dbr1.

In eukaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from Entamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from Saccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression E. coli We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec-1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec-1 We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.

Nucleoside Analogues with a Seven-Membered Sugar Ring: Synthesis and Structural Compatibility in DNA-RNA Hybrids.

Herein we describe results on the pairing properties of synthetic DNA and RNA oligonucleotides that contain nucleotide analogues with a 7-membered sugar ring (oxepane nucleotides). Specifically, we describe the stereoselective synthesis of a set of three oxepane thymine nucleosides (OxT), their conversion to phosphoramidite derivatives, and their use in solid-phase synthesis to yield chimeric OxT-DNA and OxT-RNA strands. The different regioisomeric OxT phosphoramidites allowed for positional variations of the phosphate bridge and assessment of duplex stability when the oxepane nucleotides were incorporated in dsDNA, dsRNA, and DNA-RNA hybrids. Little to no destabilization was observed when two of the three regioisomeric OxT units were incorporated in the DNA strand of DNA-RNA hybrids, a remarkable result considering the dramatically different structure of oxepanes in comparison to 2'-deoxynucleosides. Extensive molecular modeling and dynamics studies further revealed the various structural features responsible for the tolerance of both OxT modifications in DNA-RNA duplexes, such as base-base stacking and sugar-phosphate H-bond interactions. These studies suggest that oxepane nucleotide analogues may find applications in synthetic biology, where synthetic oligonucleotides can be used to create new tools for biotechnology and medicine.

Challenges and Opportunities for Nucleic Acid Therapeutics.

After decades overcoming difficult problems, antisense oligonucleotide (ASO), duplex RNA (siRNA), and messenger RNA (mRNA) nucleic acid therapeutic strategies are finally demonstrating clinical benefits. This success presents new challenges. What goals remain for basic research? Will there be an explosion of clinical applications that benefit many patients with different diseases, or will success be restricted to diseases that are ideal for the application of current technologies? The aim of this perspective is to describe a selection of the major goals for the next decade.

Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5' handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2'-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2'-hydroxyl sensitivity. Modified 5' pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5' pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5' pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Design and enhanced gene silencing activity of spherical 2'-fluoroarabinose nucleic acids (FANA-SNAs).

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) - nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior - have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.

2'-Fluoro-arabinonucleic Acid (FANA): A Versatile Tool for Probing Biomolecular Interactions.

This Account highlights the structural features that render 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) an ideal tool for mimicking DNA secondary structures and probing biomolecular interactions relevant to chemical biology.The high binding affinity of FANA to DNA and RNA has had implications in therapeutics. FANA can hybridize to complementary RNA, resulting in a predominant A-form helix stabilized by a network of 2'F-H8(purine) pseudohydrogen bonding interactions. We have shown that FANA/RNA hybrids are substrates of RNase H and Ago2, both implicated in the mechanism of action of antisense oligonucleotides (ASOs) and siRNA, respectvely. This knowledge has helped us study the conformational preferences of ASOs and siRNA as well as crRNA in CRISPR-associated Cas9, thereby revealing structural features crucial to biochemical activity.Additionally, FANA is of particular use in stabilizing noncanonical DNA structures. For instance, we have taken advantage of the anti N-glycosidic bond conformation of FANA monomers to induce a parallel topology in telomeric G-quadruplexes. Subsequent single-molecule FRET studies elucidated the mechanism by which these parallel G-quadruplexes are recognized and extended by telomerase. Similarly, we have utilized FANA to stabilize elusive telomeric i-motifs in the presence of concomitant parallel G-quadruplexes and under physiological conditions, thereby reinforcing their potential relevance to telomere biology. In another study, we adapted microarray technology and used FANA substitutions to enhance the binding affinity of the G-quadruplex thrombin-binding aptamer to its thrombin target.Finally, we discovered that DNA polymerases can synthesize FANA strands from DNA templates. On the basis of this property, other groups demonstrated that FANA, like DNA, can store hereditary information. They did so by engineering polymerases to efficiently transfer genetic information from DNA to FANA and retrieve it back into DNA. Subsequent studies showed that FANA could be evolved to acquire ribozyme-like endonuclease or ligase activity and to form high-affinity aptamers.Overall, the implications of these studies are remarkable because they promise a deeper understanding of human biochemistry for innovative therapeutic avenues. This Account summarizes past achievements and provides an outlook for inspiring the increased use of FANA in biological applications and fostering interdisciplinary collaborations.

Recent progress in non-native nucleic acid modifications.

While Nature harnesses RNA and DNA to store, read and write genetic information, the inherent programmability, synthetic accessibility and wide functionality of these nucleic acids make them attractive tools for use in a vast array of applications. In medicine, antisense oligonucleotides (ASOs), siRNAs, and therapeutic aptamers are explored as potent targeted treatment and diagnostic modalities, while in the technological field oligonucleotides have found use in new materials, catalysis, and data storage. The use of natural oligonucleotides limits the possible chemical functionality of resulting technologies while inherent shortcomings, such as susceptibility to nuclease degradation, provide obstacles to their application. Modified oligonucleotides, at the level of the nucleobase, sugar and/or phosphate backbone, are widely used to overcome these limitations. This review provides the reader with an overview of non-native modifications and the challenges faced in the design, synthesis, application and outlook of novel modified oligonucleotides.

Small nucleic acids and the path to the clinic for anti-CRISPR.

CRISPR-based therapeutics have entered clinical trials but no methods to inhibit Cas enzymes have been demonstrated in a clinical setting. The ability to inhibit CRISPR-based gene editing or gene targeting drugs should be considered a critical step in establishing safety standards for many CRISPR-Cas therapeutics. Inhibitors can act as a failsafe or as an adjuvant to reduce off-target effects in patients. In this review we discuss the need for clinical inhibition of CRISPR-Cas systems and three existing inhibitor technologies: anti-CRISPR (Acr) proteins, small molecule Cas inhibitors, and small nucleic acid-based CRISPR inhibitors, CRISPR SNuBs. Due to their unique properties and the recent successes of other nucleic acid-based therapeutics, CRISPR SNuBs appear poised for clinical application in the near-term.