Comparison of capillary electrophoresis-based methods for the analytical characterization of purity and stability of in vitro transcribed mRNA.

Messenger RNA (mRNA) is rapidly growing as a therapeutic modality for vaccination and the treatment of a wide range of diseases. As a result, there is an increased demand for mRNA-based analytical methods capable of assessing purity and stability, which are considered critical quality attributes (CQAs). In recent decades capillary electrophoresis (CE) has emerged alongside liquid chromatography (LC) as an important tool for the assessment of purity and stability of mRNA therapeutics. CE offers a variety of advantages over conventional LC or gel-based analytical methods, including reduced injection volume, increased resolution, and increased separation efficiency. In this study we compared CE-based analytical methods: the Agilent RNA 6000 Nano Kit, the Revvity RNA Reagent Kit, the Sciex RNA 9000 Purity and Integrity Kit, and the Agilent HS RNA Kit. These methods were evaluated on their vendor-recommended instruments: the Bioanalyzer, LabChip GXII, PA800 Plus, and Fragment Analyzer, respectively. We assessed the ability of these methods to measure mRNA integrity, purity, and stability. Furthermore, several parameters for each method were also assessed: selectivity, precision, resolution, analysis time, and ease of use. Based on our results, all four methods are suitable for use in the characterization of in vitro transcribed (IVT) mRNA, depending on the intended application. The Sciex RNA 9000 Purity and Integrity kit method achieved the highest selectivity and resolving power compared with the other methods, making it the most suitable for high-resolution, in-depth sample characterization. In comparison, the Agilent RNA 6000 Nano Kit, Revvity RNA Reagent Kit, and Agilent HS RNA Kit achieved lower selectivity and resolution, but their faster analysis times make them more suitable for high-throughput and screening applications.

Comprehensive chromatographic assessment of forced degraded in vitro transcribed mRNA.

Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities.

A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines.

The 3' poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.

Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters.

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9 Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.

Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA.

The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.

Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage.

Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ∼23-nucleotide (nt)-long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNA-dependent RNA cleavage. A 2.1 Å-resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5-14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2-23. These findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins.

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.

The X-family DNA polymerases λ (Polλ) and ß (Polß) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polß, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polß preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.

Single cell genomics of uncultured, health-associated Tannerella BU063 (Oral Taxon 286) and comparison to the closely related pathogen Tannerella forsythia.

The uncultivated bacterium Tannerella BU063 (oral taxon 286) is the closest relative to the periodontal pathogen Tannerella forsythia, but is not disease-associated itself. Using a single cell genomics approach, we isolated 12 individual BU063 cells by flow cytometry, and we amplified and sequenced their genomes. Comparative analyses of the assembled genomic scaffolds and their gene contents allowed us to study the diversity of this taxon within the oral community of a single human donor that provided the sample. Eight different BU063 genotypes were represented, all about 5% divergent at the nucleotide level. There were 2 pairs of cells and one group of three that were more highly identical, and may represent clonal populations. We did pooled assemblies on the nearly identical genomes to increase the assembled genomic coverage. The presence of a set of 66 "core" housekeeping genes showed that two of the single cell assemblies and the assembly derived from the three putatively identical cells were essentially complete. As expected, the genome of BU063 is more similar to Tannerella forsythia than any other known genome, although there are significant differences, including a 44% difference in gene content, changes in metabolic pathways, loss of synteny, and an 8-9% difference in GC content. Several identified virulence genes of T. forsythia are not found in BU063 including karilysin, prtH, and bspA. The absence of these genes may explain the lack of periodontal pathogenesis by this species and provides a new foundation to further understand the genome evolution and mechanisms of bacterial-host interaction in closely related oral microbes with different pathogenicity potential.