RSC Chemical Biology

The potential of therapeutically targeting RNA with small molecules continues to grow yet progress is hindered by difficulties in determining specific mechanisms of action, including impacts on RNA-protein binding. RNA G-quadruplexes (rGQs) are a particularly promising target due to their range of biological functions, structural stability, and hydrophobic surfaces, which promote small molecule and protein interactions alike. Challenges arise due to 1) the low structural diversity among rGQs, thereby limiting binding selectivity, and 2) a lack of knowledge regarding how small molecules can manipulate rGQ-protein binding on a global scale. We first leveraged a small molecule library privileged for RNA tertiary structures that displayed differential binding to rGQs based on loop length, consistent with computational predictions for DNA GQs. We next utilized an RT-qPCR-based assay to measure stability against enzymatic readthrough, expected to be a common mechanism in rGQ function. We discovered small molecules with significant, bidirectional impacts on rGQ stability, even within the same scaffold. Using Stability of Proteins from Rates of Oxidation (SPROX), a stability-based proteomics method, we then elucidated proteome level impacts of both stabilizing and destabilizing rGQ-targeting molecules on rGQ-protein interactions. This technique revealed small molecule-induced impacts on a unique subset of rGQ-binding proteins, along with proteins that exhibited differential changes based on the identity of the small molecule. The domain and peptide-level insights resulting from SPROX allow for the generation of specific hypotheses for both rGQ function and small molecule modulation thereof. Taken altogether, this methodology helps bridge the gap between small molecule-RNA targeting and RNA-protein interactions, providing insight into how small molecules can influence protein binding partners through modulation of target RNA structures.

Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.

Coronaviruses initiate translation through recognition of the viral RNA 5 m7GpppAm cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E-cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5 UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E-RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E-cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F-cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.

mRNA technology has emerged as a powerful new class of medicines. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, including treatments for cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. The 3'-polyadenylated (poly(A)) tail of mRNA is required for ribosome initiation, translation, and mRNA stability and is considered a critical quality attribute. In this study, novel direct mass spectrometry approaches were used for the analysis of both the DNA template and corresponding mRNA generated via in vitro transcription. Nucleotide resolution of the poly(A/T) sequence of the DNA template and mRNA poly(A) tail was achieved. The results show that the mRNA poly(A) tail length and heterogeneity is impacted by the heterogeneity of the DNA template, the DNA template design and RNA manufacturing conditions, including relative NTP concentrations. These results provide further important mechanistic insight into the poly(A) tail length and heterogeneity of mRNAs synthesised in vitro, including the identification of 3'-end additions of cytidine to mRNA poly(A) tails. The ability to rapidly assess DNA template quality, combined with monitoring mRNA poly(A) tail length and heterogeneity, is important as part of the characterisation of mRNA precision medicines and ensuring consistent quality of mRNA from manufacturing processes.

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.

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.

Replication-initiating HUH-endonucleases (Reps) are enzymes that form covalent bonds with single-stranded DNA (ssDNA) in a sequence specific manner to initiate rolling circle replication. These nucleases have been co-opted for use in biotechnology as sequence specific protein-ssDNA bioconjugation fusion partners dubbed HUH-tags. Here, we describe the engineering and in vitro characterization of a series of laboratory evolved HUH-tags capable of forming robust sequence-directed covalent bonds with unmodified RNA substrates. We show that promiscuous Rep-RNA interaction can be enhanced through directed evolution from nearly undetectable levels in wildtype enzymes to robust reactivity in final engineered iterations. Taken together, these engineered HUH-tags represent a promising platform for enabling site-specific protein-RNA covalent bioconjugation in vitro, potentially mediating a host of new applications and offering a valuable addition to the HUH-tag repertoire.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

Virally-derived ribosomal skipping 2A peptides are a popular tool for protein co-expression. Despite their use in over 9,000 publications, the biochemical and biophysical properties underlying the skipping mechanism remain largely unexplored. We identified 4,218 2A-like peptides originating from non-viral organisms. We developed and utilized the Trifluorescent Reporter fluorescent tool for high-throughput multiplexable analysis of ribosomal skipping, and tested 3,271 2A-like peptide sequences. We identified peptides that skipped, failed to skip, and skipped but failed to restart translation, in addition to peptides that induced a reduction in protein abundance. Peptides that skipped and induced reductions in protein abundance largely originated from eukaryotes. A poly-leucine stretch in an alpha-helix N-terminal to the conserved GDxExNPGP motif drove both skipping and the reduction in protein abundance. Analysis of the native eukaryotic protein contexts revealed that reduction may be harnessed as an expression regulator. The high-throughput approach used in this work greatly expands the functional knowledge of what biophysical and biochemical characteristics lead to ribosomal skipping, including an apparent latent eukaryotic 'leucine stall-helix' motif.

Rat Sarcoma (RAS) genes are the most frequently mutated genes in cancer, with KRAS being the most predominant oncogene, yet they have proved extremely difficult to drug because they operate primarily through protein-protein interactions (PPIs) which lack an obvious pocket for small molecules. Sequence-defined synthetic oligomers could combine the precision and customisability of synthetic molecules with the size requirements to address entire protein-protein interaction surfaces. We have adapted the phosphoramidite chemistry of oligonucleotide synthesis to produce a library of nearly one million non-nucleosidic oligophosphoester sequences - phosphoestamers - and used a fluorescent-activated bead sorting (FABS) process to select oligomers that inhibit the interaction between KRASG12D (the most prevalent, and undrugged, mutant) and RAF, a downstream effector of RAS whose activation results in cell proliferation. Hits were identified using tandem mass spectrometry, and validation showed effective inhibition with IC50 values as low as 25 nM, and excellent selectivity for the mutant over the wild type form. These findings could lead to new drugs against cancers driven by mutant RAS, and provided proof-of-principle for the phosphoestamer platform against PPIs in general.

Benzoxaboroles offer unusual reactivity and protein recognition for the development of small molecule drugs. Despite this potential, they are uncommon in drug discovery or in large fragment screening libraries. We synthesized a small series of structurally related benzoxaboroles containing a diazirine/alkyne tag to enable in-cell photoaffinity labeling (PAL) experiments. A subset of this library was found to have high selectivity for eukaryotic translation initiation factor 4E (eIF4E). The benzoxaborole-eIF4E interaction was found to be stereoselective in nature and competitive with the 7-methylguanosine cap of mRNA. Site of labeling experiments revealed that the benzoxaborole fragment interacts with the cap binding pocket of eIF4E. In silico modeling of the modified protein suggests that H-bonding interactions between the main chain of Trp102 and the side chain of Asn155 to the amide carbonyl and anionic boronate of the benzoxaborole, respectively, drive affinity for this challenging to drug pocket.

Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum, and several fungal species of the genus Aspergillus, including Aspergillus terreus and Aspergillus fumigatus.. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus (AfKdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed AfKdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.

Chemical proteomics is a powerful method to track proteins labelled by reactive small molecules in living cells on proteome-wide scale. The strategy relies on reactivity and specificity of bioorthogonal click reactions. Although a variety of bioorthogonal reactions have been developed to facilitate chemical proteomics, their reactivity and specificity might not be comparable with enzymatic reactions. Here we describe an iodoacetamide chloroalkane cysteine reactive probe that is, upon the reaction with nucleophilic cysteine of thioredoxin (TrxA), efficiently and specifically conjugated with HaloTag protein. The TrxA-HaloTag conjugate is utilized for downstream chemoproteomics analysis including in-gel shift assay and mass spectrometry-based proteomics. The TrxA-HaloTag conjugation in whole cell lysate allows fast and efficient pull-down of labelled protein on anti-HaloTag nanobeads resulting in low background after mass spectrometric analysis. The main advantage of the system is its high efficiency and complete biorthogonality due to enzymatic reactivity that is characteristic for HaloTag ligation. The study demonstrates the utility of chloroalkane small compound probes for chemoproteomics applications.

Nuclear transport receptors (NTRs) are the only proteins able to transport large molecular weight payloads into the nucleus. A dominant area of molecular therapeutic research is the extension of the use of NTRs to target the nucleus for the development of pharmaceuticals or as tools for investigating fundamental biological questions. Although several examples of synthesized peptides harbouring nuclear localization signal (NLS) sequences conjugated to various payloads exist in the literature, the assumption has been that transport occurs by classical nuclear localization via the NTRs importin- and importin-{beta}. This assumption is relevant to nuclear-targeted therapeutics that aim for full potential clinical impact. In addition, fundamental research can benefit from unbiased approaches to investigate the role of NTRs. Herein, we report the construction of a novel NLS-modified agent composed of trastuzumab-emtansine (T-DM1) coupled to cell accumulator (Accum), a technology that enables monoclonal antibodies to escape endosome entrapment and accumulate conjugated payloads in the nucleus without abrogating affinity or specificity to target antigens. Accum harbours a classical NLS sequence from SV-40 large T-antigen. We demonstrate that routing T-DM1 to the nucleus successfully increased cytotoxic potency in the HER2-positive cell line SKBR3. More importantly, through the development of a novel bait-prey proteomic approach, we show that the non-classical NTR importin 7 and not importin-/importin-{beta} was required for the cytotoxicity effect. This result was validated by siRNA knock down. Our findings also indicate that by discovering an unanticipated NTR regulator of an NLS-modified agent, this study demonstrates the utility of combining an unbiased proteomic approach to probe NTR function in mammalian cell system and, is a foresight for future NLS-based development initiatives.

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.

In the evolving landscape of RNA targeting, there is an indisputable need for new screening methodologies to find small molecules targeting relevant tertiary RNA structures, like viral pseudoknots or bacterial riboswitches. Here, we developed a competitive binding high-throughput screening assay to identify ligands for the bacterial PreQ1-I riboswitch. In this assay, ligands compete with a rationally designed quencherlabeled antisense for binding to the riboswitch. The method is validated for the Fusobacterium nucleatum (Fnu), Thermoanaerobacter tengcongensis (Tte), Bacillus subtilis (Bsu) and Enterococcus faecalis (Efa) PreQ1 riboswitches, using the natural riboswitch ligand PreQ1 and various analogues. A commercial RNA-focused library consisting of [~]15,000 compounds was then screened against the Fnu riboswitch, leading to the identification of 4 hits exhibiting competitive binding activity. These hits were evaluated in in vitro translation assays against several PreQ1 riboswitches. The most promising hit 4494 showed competitive binding activity to the Fnu, Tte, Bsu and Efa riboswitches, and was able to inhibit translation of a Tte riboswitch-regulated reporter gene, making it an interesting starting point for the development of new antibiotics. In essence, this HTS assay has the potential to discover highly sought-after RNA targeting small molecules for complex and clinically relevant RNA structures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/595132v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@d7af01org.highwire.dtl.DTLVardef@227e93org.highwire.dtl.DTLVardef@a3f24corg.highwire.dtl.DTLVardef@17883e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ribosome is responsible for assembling proteins using 20 naturally occurring L-handed amino acids. However, incorporating non-natural amino acids into a protein is a challenging process needs improvement. In this study, we report a new possible approach to creating nonnatural peptides using ribozymes inspired by the peptidyl transfer center. These RNA scaffolds, which are approximately 100 nucleotides in length, bind to RNase T1 truncated tRNA-like chimeras and bring them into close proximity to facilitate peptide ligation. We used single-molecule fluorescence resonance energy transfer (smFRET) to show close distances between RNA-RNA, tRNALys-tRNALys, and RNA-tRNALys pairs, which strongly suggests that the mechanism of peptide ligation is due to the proximity of the substrate through dimerization of the enzymes. Mass spectrometry analysis confirmed the detection of oligopeptides from four amino acids, including L-Lysine, D-Lysine, L-Phenylalanine, and D-Phenylalanine. These results indicate that ribozymes have greater flexibility in accommodating nonnatural amino acids. Our findings pave the way for potentially new avenues in the synthesis of nonnatural peptides, beyond the limitations of ribosomal peptide synthesis and other existing methods. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/538729v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@121044corg.highwire.dtl.DTLVardef@a11b2eorg.highwire.dtl.DTLVardef@ffa0b9org.highwire.dtl.DTLVardef@90f512_HPS_FORMAT_FIGEXP M_FIG C_FIG

Linkers in chemical biology provide more than just connectivity between molecules; their intrinsic properties can be harnessed to enhance the stability and functionality of chemical probes. In this study, we explored the incorporation of a peptide nucleic acid (PNA)-based linker into RNA-targeting probes to improve their affinity and specificity. By integrating a PNA linker into a small molecule probe of Riboglow platform, we enabled dual binding events: cobalamin (Cbl)-RNA structure-based recognition and sequence-specific PNA-RNA interaction. We show that incorporating a six-nucleotide PNA sequence complementary to the region of wild type RNA aptamer (env8) results in a 30-fold improvement in binding affinity compared to the probe with nonfunctional PEG linker. Even greater improvements are observed when the PNA probe was tested against truncated versions of the RNA aptamer, with affinity increasing by up to 280-fold. Additionally, the PNA linker is able to rescue Cbl-RNA interaction even when the cobalamin binding pocket is compromised. We demonstrated that PNA probes effectively bind RNA both in vitro and in live cells, enhancing visualization of RNA in stress granules and U-bodies at low concentrations. The modular nature of the Riboglow platform allows for flexible modifications of the PNA linker, fluorophore and RNA tag, while maintaining high specificity and affinity. This work establishes a new approach for enhancing RNA imaging platforms through the use of PNA linkers, highlighting the potential of combining short oligonucleotides with small molecules to improve the affinity and specificity of RNA-targeting probes. Furthermore, this dual-binding approach presents a promising strategy for driving advancements in RNA-targeted drug development. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616516v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@da5652org.highwire.dtl.DTLVardef@5923fborg.highwire.dtl.DTLVardef@494140org.highwire.dtl.DTLVardef@15afaf4_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.