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Rapid advancements of genome sequencing (GS) technologies have enhanced our understanding of the relationship between genes and human disease. To incorporate genomic information into the practice of medicine, new processes for the analysis, reporting, and communication of GS data are needed. Blood samples were collected from adults with a PCR-confirmed SARS-CoV-2 (COVID-19) diagnosis (target N = 1500). GS was performed. Data were filtered and analyzed using custom pipelines and gene panels. We developed unique patient-facing materials, including an online intake survey, group counseling presentation, and consultation letters in addition to a comprehensive GS report. The final report includes results generated from GS data: (1) monogenic disease risks; (2) carrier status; (3) pharmacogenomic variants; (4) polygenic risk scores for common conditions; (5) HLA genotype; (6) genetic ancestry; (7) blood group; and, (8) COVID-19 viral lineage. Participants complete pre-test genetic counseling and confirm preferences for secondary findings before receiving results. Counseling and referrals are initiated for clinically significant findings. We developed a genetic counseling, reporting, and return of results framework that integrates GS information across multiple areas of human health, presenting possibilities for the clinical application of comprehensive GS data in healthy individuals.

IntroductionThere is considerable variability in symptoms and severity of COVID-19 among patients infected by the SARS-CoV-2 virus. Linking host and virus genome sequence information to antibody response and biological information may identify patient or viral characteristics associated with poor and favourable outcomes. This study aims to (1) identify characteristics of the antibody response that result in maintained immune response and better outcomes, (2) determine the impact of genetic differences on infection severity and immune response, (3) determine the impact of viral lineage on antibody response and patient outcomes and (4) evaluate patient-reported outcomes of receiving host genome, antibody and viral lineage results.Methods and analysisA prospective, observational cohort study is being conducted among adult patients with COVID-19 in the Greater Toronto Area. Blood samples are collected at baseline (during infection) and 1, 6 and 12 months after diagnosis. Serial antibody titres, isotype, antigen target and viral neutralisation will be assessed. Clinical data will be collected from chart reviews and patient surveys. Host genomes and T-cell and B-cell receptors will be sequenced. Viral genomes will be sequenced to identify viral lineage. Regression models will be used to test associations between antibody response, physiological response, genetic markers and patient outcomes. Pathogenic genomic variants related to disease severity, or negative outcomes will be identified and genome wide association will be conducted. Immune repertoire diversity during infection will be correlated with severity of COVID-19 symptoms and human leucocyte antigen-type associated with SARS-CoV-2 infection. Participants can learn their genome sequencing, antibody and viral sequencing results; patient-reported outcomes of receiving this information will be assessed through surveys and qualitative interviews.Ethics and disseminationThis study was approved by Clinical Trials Ontario Streamlined Ethics Review System (CTO Project ID: 3302) and the research ethics boards at participating hospitals. Study findings will be disseminated through peer-reviewed publications, conference presentations and end-users.

Despite the passage of about 30 years since the discovery of the translational activities of ribosomes and the outlining of the roles of the large and small subunits, the actual molecular basis for the mRNA decoding activities of the small subunit has remained essentially obscure. In this paper, we describe a new approach using oligonucleotide analogs of 16S ribosomal RNA, in which the small ribosomal subunit is effectively deconstructed into a smaller more experimentally tractable form. Specifically, we review the results of experiments using an oligonucleotide analog of the decoding region of 16S ribosomal RNA, suggesting that the decoding region is the functional core of the small subunit, that it contacts both mRNA codons and tRNA anticodons, and that it mediates and probably enhances codon–anticodon base pairing, that is, decoding.Key words: translation, ribosome, 30S, 16S, RNA, decoding, antibiotic.

Replication of RNA viruses, such as the human immunodeficiency virus (HIV), is dependent upon multiple specific interactions between viral RNAs and viral and cellular proteins. A small molecule that interferes specifically with one or more of these RNA-protein interactions could be an efficacious antiviral agent. Here we show that certain aminoglycoside antibiotics, in particular neomycin B, can block binding of the HIV Rev protein to its viral RNA recognition element. Inhibition appears to be highly selective, resulting from competitive binding of the drug to a small viral RNA region within the Rev-binding site. We further demonstrate that neomycin B can specifically antagonize Rev function in vitro and in vivo and can inhibit production of HIV. Our results establish the feasibility for developing antiviral drugs that act by selectively blocking RNA-protein interactions.

IT is now generally accepted that 16S and 23S ribosomal RNA play important roles in the decoding and peptidyl transferase activities of ribosomes1,2. Despite their complex structures and numerous associated proteins it is possible that small domains of these rRNAs can fold and function autonomously, particularly those that appear devoid of protein interactions3. One candidate for such a domain is the decoding region, located near the 3′ end of 16S rRNA (Fig. la, b). Consistent with this hypothesis, aminoglycoside antibiotics that interact with the decoding region in 30S subunits interact with other RNAs in the absence of proteins4–7. In addition, certain activities of self-splicing introns, at least superficially, resemble translational decoding8,9. We report here that an oligo-ribonucleotide analogue of the decoding region interacts with both antibiotic and RNA ligands of the 30S subunit in a manner that correlates with normal subunit function. The activities of the decoding region analogue suggest that the intimidating structural complexity of the ribosome can be, to some degree, circumvented.

We describe a programmable microfluidic system with onboard pumps and valves that has the ability to process reaction volumes in the sub-microlitre to hundred microlitre range. The flexibility of the architecture is demonstrated with a commercial molecular biology protocol for mRNA amplification, implemented without significant modification. The performance of the microchip system is compared to conventional bench processing at each stage of the multistep protocol, and DNA microarrays are used to assess the quality and performance of bench- and microchip-amplified RNA. The results show that the microchip system reactions are similar to bench control reactions at each step, and that the microchip- and bench-derived amplified RNAs are virtually indistinguishable in differential microarray analyses.

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690–720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5′-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.

Chemical probing methods have been used to "footprint" 16S ribosomal RNA (rRNA) at each step during the in vitro assembly of twenty 30S subunit ribosomal proteins. These experiments yield information about the location of each protein relative to the structure of 16S rRNA and provide the basis for derivation of a detailed model for the three-dimensional folding of 16S rRNA. Several lines of evidence suggest that protein-dependent conformational changes in 16S rRNA play an important part in the cooperativity of ribosome assembly and in fine-tuning of the conformation and dynamics of 16S rRNA in the 30S subunit.

We have investigated in detail the higher-order structure of 16 S ribosomal RNA, both in its naked form and in 30 S ribosomal subunits. Each base in the 16 S rRNA chain has been probed using kethoxal (which reacts with guanine at N1 and N2), dimethylsulfate (which reacts with adenine at N1 and cytosine at N3) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (which reacts with uracil at N3 and guanine at N1). The sites of reaction were identified by primer extension with reverse transcriptase using synthetic oligodeoxynucleotide primers. These results provide a detailed and rigorous experimental test of a model for 16 S rRNA secondary structure, which was derived mainly from comparative sequence analysis. Our data also provide information relevant to tertiary and quaternary structure of 16 S rRNA. Data obtained with naked 16 S rRNA show reasonably close agreement with the proposed model, and data obtained with 30 S subunits show nearly complete agreement. Apart from an apparent overall "tightening" of the structure (in which many weakly reactive bases become unreactive), assembly of the proteins with 16 S rRNA to form 30 S subunits brings about numerous local structural rearrangements, resulting in specific enhancements as well as protections. In many instances, the ribosomal proteins appear to "tune" the 16 S rRNA structure to bring it into accordance with the phylogenetically predicted model, even though the RNA on its own often seems to prefer a different structure in certain regions of the molecule. Extensive protection of conserved, unpaired adenines upon formation of 30 S subunits suggests that they play a special role in the assembly process, possibly providing signals for protein recognition.

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.

We have used rapid probing methods to follow the changes in reactivity of residues in 16 S rRNA to chemical and enzymatic probes as ribosomal proteins S2, S3, S10, S13 and S14 are assembled into 30 S subunits. Effects observed are confined to the 3′ major domain of the RNA and comprise three general classes. 1. (1) Monospecific effects, which are attributable to a single protein. Proteins S13 and S14 each affect the reactivities of different residues which are adjacent to regions previously found protected by S19, S10 effects are located in two separate regions of the domain, the 11201150 stem and the 1280 loop; both of these regions are near nucleotides previously found protected by S9. Both S2 and S3 protect different nucleotides between positions 1070 and 1112. In addition, S2 protects residues in the 11601170 stem-loop. 2. (2) Co-operative effects, which include residues dependent on the simultaneous presence of both proteins S2 and S3 for their reactivities to appear similar to those observed in native 30 S subunits. 3. (3) Polyspecific effects, where proteins S3 and S2 independently afford the same protection and enhancement pattern in three distal regions of the domain: the 960 stem-loop, the 10501200 stem and in the upper part of the domain (nueleotides 1070 to 1190). Proteins S14 and S10 also weakly affect the reactivities of several residues in these regions. We believe that several of the protected residues of the first class are likely sites for protein-RNA contact while the third class is indicative of conformational rearrangement in the RNA during assembly. These results, in combination with the results from our previous study of proteins S7, S9 and S19, are discussed in terms of the assembly, topography and involvement in ribosomal function of the 3′ major domain.

We have examined the effect of binding ribosomal protein S4 to 16 S rRNA on the susceptibility of the RNA to a variety of chemical and enzymatic probes. We have used dimethyl sulfate to probe unpaired adenines (at N-1) and cytosines (at N-3), kethoxal to probe unpaired guanines (at N-1 and N-2) and cobra venom (V1) ribonuclease as a probe of base-paired regions of 16 S rRNA. Sites of attack by the probes were identified by primer extension using synthetic oligodeoxynucleotides. Comparison of probing results for naked and S4-bound rRNA shows: 1. (1) Protein S4 protects a relatively compact region of the 5′ domain of 16 S rRNA from chemical and enzymatic attack. This region is bounded by nucleotides 27 to 47 and 394 to 556, and has a secondary structure characterized by the junction of five helical elements. Phylogenetically conserved irregular features (bulged nucleotides, internal loops and flanking unpaired nucleotides) and helical phosphodiester bonds of four of the helices are specifically protected in the S4-RNA complex. We conclude that this is the major, and possibly sole region of contact between 16 S rRNA and S4. Many of the S4-dependent changes mimic those observed on assembly of 16 S rRNA into 30 S ribosomal subunits. 2. (2) Binding of S4 causes enhanced chemical reactivity coupled with protection from V1 nuclease outside the S4 junction region in the 530, 720 and 1140 loops. We interpret these results as indicative of loss of structure, and suggest that S4 binding causes disruption of adventitious pairing in these regions, possibly by stabilizing the geometry of the RNA such that these interactions are prevented from forming.

The herpes simplex virus transactivator VP16 participates in the formation of a multiprotein-DNA complex with the ubiquitous octamer-motif-binding factor Oct-1. Complex formation is dependent on specific amino acids in the Oct-1 homoeodomain which are in positions analogous to positive control mutations in helix 2 of the lambda phage repressor helix-turn-helix motif, indicating that this structure is an ancient target for protein-protein interactions mediating transcriptional control.

Publisher Summary This chapter focuses on the structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Chemical and enzymatic probing, monitored by primer extension, has become a powerful tool for the analysis of RNA structure. The reactivities of individual nucleotides composing large RNA molecules may be determined rapidly by utilizing a series of primers spaced at approximately 200 nucleotide intervals. In addition, numerous chemical reagents and nucleases may be employed as probes, since the only requirement is that they modify the template so as to produce pauses or stops in the progress of reverse transcriptase. The RNA, either alone or complexed with proteins and/or ligands, is incubated under suitable conditions with chemical or enzymatic probes. Dimethyl sulfate (DMS), kethoxal (KE) and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluene sulfonate (CMCT) are employed for chemical probing, while ribonucleases A and T 1 , and V l nuclease are employed as enzymatic probes. The extent of the reactions is limited so that no more than a few stops are present within 300 nucleotide stretches in a given RNA molecule.

Our understanding of the structure and function of ribosomal RNA has evolved rapidly during the past few years. Complete primary structures for 16S, 23S, and 5S rRNA (and their analogs) from a wide range of organisms and organelles are now available, as are accurate secondary structure models that are helping us to understand the higher order folding of these molecules (reviewed in Woese et al., 1983; Brimacombe et al., 1983; Ebel et al., 1983; Noller, 1984). Current efforts in this area are focused on understanding tertiary and quaternary structure of rRNA and the nature of its involvement in protein synthesis.

We have derived a model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA, using interactive computer graphic methods. It is based on (1) the secondary structure derived from comparative sequence analysis, (2) the three-dimensional co-ordinates for the centers of mass of the 30 S subunit proteins, and (3) the locations of sites in 16 S rRNA that interact with specific ribosomal proteins, from footprinting and crosslinking studies. We present a detailed description of the derivation of the model. About 75% of the RNA chain is sufficiently constrained to provide a useful model. This contains most of the universally conserved core of the molecule. In all but a few instances, protected and crosslinked sites can be placed within or very close to their cognate proteins, while obeying stereochemical rules. The overall shape of the model and locations of specific regions of the RNA correspond well to data derived from electron micrographs of 30 S subunits, although such data were not used to construct the model. Phylogenetic variations in the structure are readily accommodated; as an example, we have modeled the 950-nucleotide mammalian mitochondrial 12 S rRNA by superimposing it on the E. coli structure. The three major RNA domains, as defined by secondary structure, appear to exist as autonomous structural units in three dimensions, for the most part. There is an extensive interface between the 5' and central domains, whereas the 3' major domain has relatively little apparent contact with the rest of the structure. The 5', central and 3' major domains form structures that resemble the body, platform and head, respectively, seen in electron micrographs of 30 S subunits. We discuss possible roles for the ribosomal proteins in stabilizing specific structural features of the RNA during ribosome assembly. The decoding site, as deduced from footprinting and crosslinking studies involving the tRNA anticodon stem-loop, is well-localized. Bases protected from chemical probing by the anticodon stem-loop line the cleft of the subunit. The conserved loop at position 530, which contains some of the bases protected by A site-bound tRNA, is remote (approx. 80 A) from the decoding site. Protection of these bases by the anticodon stem-loop is thus unlikely to be due to direct contact.