Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.

Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.

Assessment and comparison of thermal stability of phosphorothioate-DNA, DNA, RNA, 2'-F RNA, and LNA in the context of Phi29 pRNA 3WJ.

The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, thermodynamically stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2'-F nucleotides (2'-F), and locked nucleic acids (LNA) on the thermal and in vivo stability of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2'-F RNA < 2'-F RNA/2'-F RNA < 2'-F RNA/LNA < LNA/LNA. This proposition is supported by studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures from 21.2°C to over 95°C. The 3WJLNA was resistant to boiling temperature denaturation, urea denaturation, and 50% serum degradation. Intravenous injection of fluorescent LNA/2'-F hybrid 3WJs into mice revealed its exceptional in vivo stability and presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2'-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve their pharmacokinetics profile.

Methods for construction and characterization of simple or special multifunctional RNA nanoparticles based on the 3WJ of phi29 DNA packaging motor.

The field of RNA nanotechnology has developed rapidly over the last decade, as more elaborate RNA nanoarchitectures and therapeutic RNA nanoparticles have been constructed, and their applications have been extensively explored. Now it is time to offer different levels of RNA construction methods for both the beginners and the experienced researchers or enterprisers. The first and second parts of this article will provide instructions on basic and simple methods for the assembly and characterization of RNA nanoparticles, mainly based on the pRNA three-way junction (pRNA-3WJ) of phi29 DNA packaging motor. The third part of this article will focus on specific methods for the construction of more sophisticated multivalent RNA nanoparticles for therapeutic applications. In these parts, some simple protocols are provided to facilitate the initiation of the RNA nanoparticle construction in labs new to the field of RNA nanotechnology. This article is intended to serve as a general reference aimed at both apprentices and senior scientists for their future design, construction and characterization of RNA nanoparticles based on the pRNA-3WJ of phi29 DNA packaging motor.

Excited-State Dynamics of Melamine and Its Lysine Derivative Investigated by Femtosecond Transient Absorption Spectroscopy.

Melamine may have been an important prebiotic information carrier, but its excited-state dynamics, which determine its stability under UV radiation, have never been characterized. The ability of melamine to withstand the strong UV radiation present on the surface of the early Earth is likely to have affected its abundance in the primordial soup. Here, we studied the excited-state dynamics of melamine (a proto-nucleobase) and its lysine derivative (a proto-nucleoside) using the transient absorption technique with a UV pump, and UV and infrared probe pulses. For melamine, the excited-state population decays by internal conversion with a lifetime of 13 ps without coupling significantly to any photochemical channels. The excited-state lifetime of the lysine derivative is slightly longer (18 ps), but the dominant deactivation pathway is otherwise the same as for melamine. In both cases, the vast majority of excited molecules return to the electronic ground state on the aforementioned time scales, but a minor population is trapped in a long-lived triplet state.

Peptide ligation and RNA cleavage via an abiotic template interface.

We report herein DNA- and RNA-templated chemical transformation of bifacial peptide nucleic acid (bPNA) fragments directed by an abiotic triplex hybrid interface. Assembly of one bPNA strand with two unstructured oligo T/U strands enables facile insertion of DNA and RNA template sites within partially folded nucleic acids; this template topology is not easily accessed through native base-pairing. Triplex hybridization of reactive bPNA fragments on DNA and RNA templates is shown to catalyze amide bond ligation and controlled bPNA chain extension. RNA-templated oxidative coupling of bPNA fragments is found to result in the emergence of ribozyme cleavage function, thus establishing a connection between engineered and native reaction sites. These data demonstrate the use of new topologies in nucleic acid-templated chemistry that could serve as chemically sensitive DNA and RNA switches.

Bifacial peptide nucleic acid as an allosteric switch for aptamer and ribozyme function.

We demonstrate herein that bifacial peptide nucleic acid (bPNA) hybrid triplexes functionally substitute for duplex DNA or RNA. Structure-function loss in three non-coding nucleic acids was inflicted by replacement of a duplex stem with unstructured oligo-T/U strands, which are bPNA binding sites. Functional rescue was observed on refolding of the oligo-T/U strands into bPNA triplex hybrid stems. Bifacial PNA binding was thus used to allosterically switch-on protein and small-molecule binding in DNA and RNA aptamers, as well as catalytic bond scission in a ribozyme. Duplex stems that support the catalytic site of a minimal type I hammerhead ribozyme were replaced with oligo-U loops, severely crippling or ablating the native RNA splicing function. Refolding of the U-loops into bPNA triplex stems completely restored splicing function in the hybrid system. These studies indicate that bPNA may have general utility as an allosteric trigger for a wide range of functions in non-coding nucleic acids.

Bifacial PNA complexation inhibits enzymatic access to DNA and RNA.

FULL STOP: Herein we report the effective in vitro inhibition of transcription, reverse-transcription and exonuclease function by formation of synthetic bPNA-nucleic acid triplex structures. Selective bPNA targeting of both DNA and RNA substrates suggests possible application of bPNAs as synthetic regulators of nucleic acid function.

Supercoiled DNA percentage: A key in-process control of linear DNA template for mRNA drug substance manufacturing.

The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality in vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance.

Bifacial peptide nucleic acid directs cooperative folding and assembly of binary, ternary, and quaternary DNA complexes.

We report herein the structuring of single-stranded thymine-rich DNA sequences into peptide-DNA hairpin triplex structures via designed melamine-thymine nucleobase recognition. Melamine-displaying α-peptides were synthesized with the general form (EM*)n, where M* denotes a lysine residue side chain derivatized with melamine, a bifacial hydrogen bond complement for thymine. We have found that (EM*)n peptides, which we term bifacial peptide nucleic acid (bPNA), function as a noncovalent template for thymine-rich DNA tracts. Unstructured DNA of the general form dTnCmTn are bound to (EM*)n peptides and fold into cooperatively melting 1:1 bPNA-DNA hairpin complexes with dissociation constants in the submicromolar to low nanomolar range for n = 4-10. As the length of the interface (n) is decreased, the melting temperature of the bPNA-DNA complex drops significantly, though Kd increases are less substantial, suggestive of strong enthalpy-entropy compensation. This is borne out by differential scanning calorimetry analysis, which indicates enthalpically driven bPNA-DNA base-stacking that becomes markedly less exothermic as the recognition surface n decreases in size. The recognition interface tolerates a high number of "mismatches" and indicates half-site, or monofacial, recognition between melamine and thymine may occur if only 1 complementary nucleobase is available. Association correlates directly with fractional thymine content, with optimal binding when the number of T-T sites match the number of melamine units. Interestingly, when a DNA host has more T-T sites than melamine sites on bPNA, two or three bPNAs can bind to a single DNA, resulting in ternary and quaternary complexes that have higher thermal stability than the binary (1:1) bPNA-DNA complex, suggestive of cooperative multisite binding. In contrast, when two bPNAs of different lengths bind to the same DNA host, a ternary complex is formed with two melting transitions, corresponding to independent melting of each bPNA component from the complex. These data demonstrate that melamine-displaying bPNA recognize thymine-rich DNA in predictable and multifaceted ways that allow binding affinity, structure stability, and stoichiometry to be tuned through simple bPNA length modification and matching with DNA length. Synthetic bPNA structuring elements may be useful tools for biotechnology.

Discrete assembly of synthetic peptide-DNA triplex structures from polyvalent melamine-thymine bifacial recognition.

We have designed a 21-residue α-peptide that simultaneously recognizes two decadeoxyoligothymidine (dT(10)) tracts to form triplexes with a peptide-DNA strand ratio of 1:2. The synthetic peptide side chain displays 10 melamine rings, which provide a bifacial thymine-recognition interface along the length of the 21-residue peptide. Recognition is selective for thymine over other nucleobases and drives the formation of ternary peptide·[dT(10)](2) complexes as well as heterodimeric peptide·[dT(10)C(10)T(10)] hairpin structures with triplex stems.

Double-stranded RNA reduction by chaotropic agents during in vitro transcription of messenger RNA.

In-vitro-transcribed messenger RNA (mRNA) has recently shown increasing significance in the development of vaccines and therapeutics. Immunogenic double-stranded RNA (dsRNA) is an undesired byproduct formed during in vitro transcription (IVT), and it is challenging to reduce dsRNA byproduct from mRNA due to their similar sizes and intrinsic characteristics. Removal of dsRNA relies heavily on post-IVT chromatography purifications, such as reverse-phase high-pressure liquid chromatography, which increase manufacturing costs, reduce yield, and often decrease integrity, especially for long mRNA. Thus, it would be ideal to reduce and control the level of dsRNA during IVT. We herein present a simple, scalable, and controllable method to reduce the formation of dsRNA byproducts during IVT. Selected chaotropic agents at optimized concentrations are included during IVT to create a mild denaturing environment to prevent the undesired intermolecular or intramolecular base-pairing that is thought to promote RNA-templated dsRNA formation by RNA polymerase. Compared with regular IVT, our improved method produces mRNA with significantly less dsRNA, much lower immuno-stimulation, and more efficient protein expression. Therefore, this method potentially eliminates dsRNA removal purification steps and does not require reduced magnesium concentration, elevated temperature, or custom reagents, enabling a straightforward, high-yield, and cost-effective scale-up approach for mRNA manufacturing.

Multiplexed Photoactivation of mRNA with Single-Cell Resolution.

We demonstrate sequential optical activation of two types of mRNAs in the same mammalian cell through the sequential photocleavage of small molecule caging groups ("photocages") tethered to the 5'-untranslated region (5'-UTR) of mRNAs. Synthetic photocages were conjugated onto target mRNA using RNA-TAG, an enzymatic site-specific RNA modification technique. Translation of mRNA was severely reduced upon conjugation of the photocages onto the 5'-UTR. However, subsequent photorelease of the cages from the mRNA transcript triggered activation of translation with single-cell spatiotemporal resolution. To achieve sequential photoactivation of two mRNAs in the same cell, we synthesized a pair of photocages that can be selectively cleaved from mRNA upon photoirradiation with different wavelengths of light. Sequential photoactivation of two mRNAs enabled precise optical control of translation of two unique transcripts. We believe that this modular approach to precisely and rapidly control gene expression will serve as a powerful tool in future biological studies that require controlling translation of multiple transcripts with high spatiotemporal resolution.

RNA Nanotechnology to Solubilize Hydrophobic Antitumor Drug for Targeted Delivery.

Small-molecule drugs are used extensively in clinics for cancer treatment; however, many antitumor chemical drugs dissolve poorly in aqueous solution. Their poor solubility and nonselective delivery in vivo often cause severe side effects. Here, the application of RNA nanotechnology to enhance the solubility of hydrophobic drugs, using camptothecin (CPT) for proof-of-concept in targeted delivery for cancer treatment is reported. Multiple CPT prodrug molecules are conjugated to RNA oligos via a click reaction, and the resulting CPT-RNA conjugates efficiently self-assemble into thermodynamically stable RNA three-way junction (3WJ) nanoparticles. The RNA 3WJ is covalently linked with seven hydrophobic CPT prodrug molecules through cleavable ester bonds and a folic acid ligand for specific tumor targeting while remaining soluble in aqueous solutions without detectable aggregation at therapeutic dose. This CPT-RNA nanoparticle exhibits efficient and specific cell binding and internalization, leading to cell apoptosis. Tumor growth is effectively inhibited by CPT-RNA nanoparticles; the targeted delivery, strengthened by tumor ligand, further enhances tumor suppression. Compared with the traditional formulation, solubilization of CPT in aqueous buffer using RNA nanoparticles as a carrier is found to be safe and efficacious, demonstrating that RNA nanoparticles are a promising platform for the solubilization and the delivery of hydrophobic antitumor drugs.

Multiresponsive switchable diarylethene and its application in bioimaging.

A multiresponsive fluorescent switch based on diarylethene and terpyridine units was developed. It exhibits effective switchable fluorescence which can be controlled by UV/visible light or metal ion/EDTA in solution. More importantly, having low toxicity, it can enter live cells as a fluorescent probe and can also serve as a detector for the biological process of metal ion transmembrane transport.