ACS Bio & Med Chem Au

Non-enzymatic, chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields range from 30% to 95% for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation using a well-defined bimolecular test system and water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results reveal interplay between template-substrate stability and the rates of the chemical steps of ligation, which can cause yields to increase or decrease with increasing temperature. Phosphate location at the nick site also exhibits a strong influence on ligation rates and yields, with a 3 phosphate providing yields near 100% after 24 hours for particularly favourable reaction conditions, while comparable reactions with the phosphate on the 5 position of the nick site only reach 40% ligation even after 48 hours. Ligation rates are also shown to be sensitive to the identity of base pairs flanking a nick site, with some varying by more than three-fold. Finally, DNA substrate modification by EDC can, in some cases, make long reaction times and repeated addition of EDC an ineffective strategy for increasing ligation yields.

Supercoiled (Sc) circular DNA, such as plasmids, has shown therapeutic potential since the 1990s, but is limited by bacterial modifications, unnecessary DNA sequences, and contaminations that may trigger harmful responses. To overcome these challenges, we have developed two novel scalable biochemical methods to synthesize unmodified Sc circular DNA. Linear DNA with two loxP sites in the same orientation is generated via PCR or rolling circle amplification. Cre recombinase then converts this linear DNA into relaxed circular DNA. After T5 exonuclease removes unwanted linear DNA, topoisomerases are employed to generate Sc circular DNA. We have synthesized EGFP-FL, a 2,002 bp mini-circular DNA carrying essential EGFP expression elements. EGFP-FL transfected human HeLa and mouse C2C12 cells with much higher efficiency than E. coli-derived plasmids. These new biochemical methods can produce unmodified Sc circular DNA, in length from 196 base pairs to several kilobases and in quantities from micrograms to milligrams, providing a promising platform for diverse applications.

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extension on a subset of kinetically trapped partially base-paired configurations, followed by shuffling of these configurations to enable continued oligonucleotide elongation. Here we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of length 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides, and investigated factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system, since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is pre-activated. We discuss the necessity of ongoing in-situ activation chemistry for continuous and accurate VCG replication.

Cytotoxic T lymphocytes recognize infected or transformed cells through peptide antigens presented by major histocompatibility complex class I (MHC-I) molecules. Although antigen recognition is typically defined by peptide sequence, chemical modifications to amino acid sidechains can generate structurally distinct epitopes that alter immune recognition. Here, we investigated how endogenous and exogenous electrophiles can install non-enzymatic post-translational modifications (PTMs), thereby influencing antigen presentation and T cell activation. Notably, these structural modifications are typically irreversible and can persist through protein processing and subsequent peptide presentation. We comprehensively mapped out these potential modifications found that peptide variants bearing non-enzymatic PTMs altered MHC-I stability and disrupted T cell recognition, particularly when modifications occurred at TCR-contact residues. To identify such species on MHC-I of cells, we developed a chemical enrichment strategy using an alkyne-tagged probe to capture non-enzymatically acylated peptides associated with MHC-I. Finally, we show that electrophilic environmental chemicals and dietary isothiocyanates (ITCs) can covalently modify antigenic peptides and abolish T cell activation despite preserved MHC-I binding. Together, these findings demonstrate that endogenous and exogenous chemical modifications can reshape the immunopeptidome and generate chemically distinct peptide antigens that alter adaptive immune recognition.

Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m7GpppX cap present at the 5' terminus of coding mRNAs, with an m7G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technologys potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

Phosphorothioated (PST) oligonucleotides are increasingly being used in RNA silencing, antisense, and biosensing applications. However, the possibilities and consequences of their desultory interactions with other possible nucleic acids and DNA polymerases inside the cell remain inadequately characterized. In this study, we report the discovery of an unusual terminal mismatch bypass activity involving 3'-PST containing DNA primers and certain strand displacement DNA polymerases. Using rolling circle DNA amplification, we have identified that strand displacement DNA polymerases such as phi29 and BST large fragment (LF) can bypass 3'-terminal PST mismatches upto 1 - 20 nt length. Next, we explore the length and sequence dependence of this unusual attribute, incubation in near-ambient and 60 - 65{degrees}C temperatures, and measures to blockade or modulate this mismatch bypass activity to create a binary fully nucleic acid-based and non-photocontrolled molecular switch (the first of its kind). After proposing possible underlying mechanisms for this activity, we discuss its potential consequences and applications.

The success of SARS-CoV-2 mRNA vaccines demonstrated that rapid, large-scale manufacturing of synthetic mRNA is necessary for an effective and timely response to a pandemic. Innovations in areas such as template design and manufacturing processes are being implemented to facilitate more simple, cost-effective and scalable mRNA synthesis. In this study, for the first time, we demonstrate that the enzymatic steps in mRNA production (including DNA template linearization, RNA synthesis, 5' capping and methylation) can be carried out using enzymes immobilized to a solid support. Specifically, we demonstrate efficient IVT template DNA linearization using immobilized BspQI, where the linearized template DNA can be directly used in IVT without the need of purification. We also showed that immobilized T7 RNA polymerase, Faustovirus RNA capping enzyme (FCE), vaccinia cap 2'-O-methyltransfease (2'OMTase) and a novel FCE::T7RNAP fusion enable efficient enzymatic synthesis of Cap-1 RNA in a one-pot format. This solid-phase enzymatic platform may enable highly efficient, seamless and continuous mRNA synthesis workflows that minimizes sample loss and units of operation in biopharmaceutical manufacturing.

Circularization is at the frontier of therapeutic messenger RNA (mRNA) enhancements. Currently available enzymatic and ribozymatic methods for generating circular RNAs (circRNAs) face several challenges related to sequence limitations, purification, and sub-optimal biological activity. The chemical circularization of synthetic RNA fragments potentially overcomes these limitations but is applicable only to extremely short sequences. Here, we report a novel approach for accessing circular RNAs based on the chemical circularization of in vitro transcribed RNA. We efficiently accessed chemically circularized RNAs (chem-circRNAs) by making in vitro transcribed precursor RNAs modified at the 5' end with an ethylenediamine moiety, which undergoes an intramolecular reaction with the periodate-oxidized RNA 3' end under reductive amination conditions. We demonstrate that this method is modification-compatible and applicable to various sequences. Additionally, we report methods for the effective separation of chem-circRNAs from their linear precursors. Using this approach, we prepared multiple chemically-obtained circular RNAs (chem-circRNAs; 35-1500 nt long) with circularization efficiencies reaching up to 60%. We show that protein-coding chem-circRNAs are translationally active in living cells and exhibit increased durability, similar to enzymatically circularized mRNAs. We also demonstrate that this approach enables unprecedented access to chemically modified circRNAs, such as circ-mRNAs incorporating a functional endocyclic N7-methylguanosine cap or modified with N1-methylpseudouridine within the RNA body. Notably, circRNAs containing an endocyclic cap structure engage in the most efficient, cap-dependent mechanism of translation. Our approach makes chemically-modified circularized full-length protein-coding RNAs easily accessible, thereby opening new avenues for the design, modification, and functionalization of circular mRNAs.

Therapeutic gene expression can address many of the challenges associated with the controlled delivery of intracellularly active biologics, such as enzymes that degrade RAS for treatment of RAS-driven cancers. Here, we demonstrate that an optimized synthetic nonviral gene delivery platform composed of poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-PPS) can block copolymers conjugated to a dendritic cationic peptide (PPDP2) for nontoxic delivery and therapeutic expression of mRNA within human pancreatic cancer cells and tumors. The naturally occurring bacterial enzyme RAS/RAP1-specific endopeptidase (RRSP) is a potent RAS degrader that specifically targets all RAS isoforms. Using PPDP2, rrsp-mRNA is delivered to human pancreatic cells resulting in RRSP protein expression, degradation of RAS, and loss of cell proliferation. Further, pancreatic tumors are reduced with residual tumors lacking detectable RAS and phosphorylated ERK. Using structural modeling, we further demonstrate that a noncatalytic RAS-binding domain of RRSP provides high specificity for RAS. These data support that the synthetic nanocarrier PPDP2 can deliver rrsp-mRNA to pancreatic tumor cells to interrupt the RAS signaling system.

Eukaryotic mRNAs undergo co-transcriptional 5-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am - a common epitranscriptomic mark unique to the mRNA 5-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analog of the m6Am-cap - m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as an RP-HPLC purification handle, allowing separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic setting as anti-cancer vaccines. The biochemical characterization suggests several phenomena underlying the biological properties of AvantCap: (i) increased competitiveness of the mRNA 5-end for eIF4E protein by reducing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of the m6Am in mRNA.

Phosphorylation is a highly regulated protein post-translational modification catalyzed by kinases. Kinases and phosphorylated proteins are key players in a myriad of cellular events, including cell signaling. When cell signaling networks are improperly regulated by kinases, various pathologies can arise, such as cancers and neurodegenerative disease. With critical roles in normal and disease biology, kinase-substrate interactions must be thoroughly characterized. Previously, the chemoproteomic method, kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP), was developed to identify the kinases of a phosphoprotein substrate of interest. Here, K-CLIP was modified to profile the substrates of a kinase of interest. Specifically, the substrate profile of cAMP-dependent protein kinase (PKA) was studied with K-CLIP using a new ATP analog, ATP-alkyne aryl azide. Kinase-focused K-CLIP discovered SMC3 as a PKA substrate. With versatility for any kinase or phosphoprotein substrate of interest, K-CLIP will expand our understanding of kinase-mediated cell biology in healthy and diseased states.

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with the hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 {+/-} 0.58 %ID g-1 (n = 3; 2 h post-administration) and was reduced by [~]50% in the blocking group (2.47 {+/-} 0.42 %ID g-1; n = 3; P-value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

A modified protein fragment complementation assay has been designed and validated as a gain-of-signal biosensor for nucleic acid:nucleic acid interactions. The assay uses fragments of NanoBiT, the split luciferase reporter enzyme, that are esterified at their C-termini to steramers, sterol-modified oligodeoxynucleotides. The Drosophila hedgehog autoprocessing domain, DHhC, served as a self-cleaving catalyst for these bioconjugations. In the presence of ssDNA or RNA with segments complementary to the steramers and adjacent to one another, the two NanoBiT fragments productively associate, reconstituting NanoBiT enzyme activity. NanoBiT luminescence in samples containing nM ssDNA or RNA template exceeded background by 30-fold and as high as 120-fold depending on assay conditions. A unique feature of this detection system is the absence of a self-labeling domain in the NanoBiT bioconjugates. Eliminating that extraneous bulk broadens the detection range from short oligos to full-length mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/572427v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@f0706dorg.highwire.dtl.DTLVardef@1653d01org.highwire.dtl.DTLVardef@18843bcorg.highwire.dtl.DTLVardef@1170e47_HPS_FORMAT_FIGEXP M_FIG C_FIG

We developed a series of nitro reduction-reversible acylating reagents. Following optimization of the acylation conditions, these reagents were tested for deacylation with sodium dithionite in vitro. We applied this reversible acylation to modulate RNAzyme-mediated pre-tRNA maturation, demonstrating its ability to regulate RNA-RNA interactions. Furthermore, the in vitro reversible acylation of EGFP mRNA indicated effective control of its translational activity. To explore cellular applications, we validated NQO1-mediated deacylation in vitro and then induced hypoxia in HepG2 cells using cobalt chloride, thereby reactivating the function of acylated EGFP mRNA via endogenous NQO1. Overall, this study highlights the potential for developing nitro reduction-reversible acylation as a new strategy for RNA functional control and RNA-based drug modification.

Organelle specific protein identification is essential for understanding how cell functions on a subcellular level. Here, we report a light mediated proximal labeling (LIMPLA) strategy for organelle specific protein profiling in living cells. In this strategy, various commercial mitochondria-localized fluorescent trackers, such as Mitoview 405 and Rhodamine 123, can activate 2-Propynylamine (PA) to label proximal proteins in mitochondria under illumination. PA tagged proteins are subsequently derivatized via click chemistry with azido fluorescent dye for imaging or with azido biotin for further enrichment and mass-spec identification. This strategy can be generalized to other organelles specific protein labeling. For example, proteins in nucleus are labeled by utilizing the commercial nucleus tracker DRAQ5. As compared with other chemical strategies for subcellular protein labeling, there are several advantages for this LIMPLA strategy. First, this approach allows minimal interference to the cells status by avoiding exogenous gene tansduction and some special treatment such as hydrogen peroxide or serum starvation. Second, all reagents used in this strategy are commercially available without additional synthesis work. Further, this strategy holds the potential for analyzing proximal proteins of specific macromolecules that can be tagged with fluorescent dye by metabolic labeling strategy.

The human major histocompatibility complex (MHC) plays a crucial role in the presentation of peptidic fragments from proteins; these peptides can be derived from self-proteins or from non-human antigens, such as those produced by viruses or bacteria. To prevent cytotoxicity against healthy cells, thymocytes expressing T cell receptors (TCRs) that recognize self-peptides are removed from circulation in a process called negative selection. However, post-translational modifications (PTMs) are largely excluded from negative selection; this feature opens the door to the possibility that PTMs directly contribute to the development of autoreactive T cells and subsequent autoimmune diseases. Despite it being well-established that PTMs are prevalent in peptides presented on MHCs, the exact mechanisms by which PTMs influence the antigen presentation machinery remains poorly understood. In our work, we introduce chemical modifications mirroring PTMs onto peptides to systematically investigate their impact on MHC binding and TCR recognition. Our findings reveal the numerous ways PTMs alter antigen presentation, which could have implications for tumor neoantigen presentation.

We have devised a single pot, low-cost method to modify RNA with sulfinate salts that can directly add almost any desired functional group to nucleobases under mild aqueous conditions. This chemistry modifies the Hoogsteen edge of RNA and DNA nucleobases. It can be applied to RNA or DNA of any size, as well as to individual nucleotides. Existing methods of RNA modification have relatively limited applicability due to constraints on the size of the RNA and the lack of diversity of possible modifications. We have been able to add azide groups for click reactions directly onto the nucleobases of RNA utilizing sulfinate salts. C-H bonds on the nucleobase aromatic rings serve as the sites of attachment, with C-H being replaced with C-R, where R is the azide-containing linker. With the addition of azide functional groups, the modified RNA can easily be reacted with any alkyne-labeled compound of interest, including fluorescent dyes as shown in this work. This methodology enables the exploration of diverse chemical groups on RNA that can potentially confer protection from nucleases, allow for efficient delivery of nucleic acids into cells, or act as new tools for the investigation of nucleic acid structure and function.

Cell-free protein synthesis (CFPS) systems are a powerful platform with immense potential in fundamental research, biotechnology, and synthetic biology. Conventional prokaryotic CFPS systems, particularly those derived from Escherichia coli (E. coli), often rely on complex reaction buffers containing up to thirty-five components, limiting their widespread adoption and systematic optimization. Here, we present an optimized E. coli cell-free protein synthesis (eCFPS) system, which is significantly streamlined for high efficiency. Through systematic screening, we successfully reduced the essential core reaction components from 35 to a core set of 7. The thorough optimization of these seven key components ensured that protein expression levels were not only maintained but even substantially improved. Furthermore, we developed a much simpler procedure for preparing the bacterial cytosolic extracts, a "fast lysate" protocol that eliminates the traditional time-consuming runoff and dialysis steps, thereby enhancing the overall accessibility and robustness of eCFPS. This optimized and user-friendly eCFPS efficiently synthesizes challenging proteins, including functional, self-assembling vimentin, and active restriction endonuclease BsaI despite its strong cytotoxicity, and serves as a powerful tool that will facilitate diverse applications in basic life science research and beyond.

G-quadruplexes (G4s) are four-stranded nucleic acid structures that have gathered a significant attention due to their involvement in key biological processes, including gene regulation, genome stability, and telomeres maintenance. Some G4 antibodies have been developed to selectively recognize these structures over duplex DNA; however, most, even the widely studied BG4 and 1H6, bind G4s in a general manner and lack discrimination between distinct topologies, particularly between parallel and antiparallel conformations. In this study, we report on the development and characterization of a novel antibody selected via phage display method using a constrained antiparallel G4 structure mimicking one of the conformation adopted in vitro by the human telomeric sequence. Our findings demonstrate that this new antibody selectively recognizes the antiparallel topology of the telomeric G4 sequence, a property further validated in cellular models.

We describe the synthesis of C-5 indole-tagged pyrimidine and C-8 indole-tagged purine nucleoside phosphoramidites and their incorporation into double-stranded DNA 15 base pairs in length. Of the 23 sequence modifications tested, two induced the DNA duplex to adopt a Z-like left-handed conformation under physiological salt conditions, bypassing the specific sequences typically required for a left-handed Z-DNA structure. The impact of these modifications varied with the linker type: flexible propyl linkers exhibited distinct effects compared to rigid propargyl linkers. Notably, modifications positioned directly on or near a restriction site emphasized the pivotal role of linker rigidity in controlling DNA conformation. Specifically, the conformational change induced by the flexible linker impacted nuclease and restriction endonuclease cleavage, reducing sequence specificity. In contrast, the rigid linker suppressed this effect. Furthermore, our findings indicate that nucleic acid duplexes modified with indole-linked nucleotides using a flexible propyl linker have a pronounced tendency to form BZ or Z-like regions in longer DNA sequences. A higher density of modifications may even induce a full Z-like conformation throughout the duplex. These modified nucleotides hold potential for the development of novel antisense therapeutics and introducing valuable tools for in vitro screening of small molecules targeting distorted B-DNA, BZ-DNA, and Z-DNA structures.