Single-molecule fluorescence resonance energy transfer shows uniformity in TATA binding protein-induced DNA bending and heterogeneity in bending kinetics.

TATA binding protein (TBP) is a key component of the eukaryotic RNA polymerase II transcription machinery that binds to TATA boxes located in the core promoter regions of many genes. Structural and biochemical studies have shown that when TBP binds DNA, it sharply bends the DNA. We used single-molecule fluorescence resonance energy transfer (smFRET) to study DNA bending by human TBP on consensus and mutant TATA boxes in the absence and presence of TFIIA. We found that the state of the bent DNA within populations of TBP-DNA complexes is homogeneous; partially bent intermediates were not observed. In contrast to the results of previous ensemble studies, TBP was found to bend a mutant TATA box to the same extent as the consensus TATA box. Moreover, in the presence of TFIIA, the extent of DNA bending was not significantly changed, although TFIIA did increase the fraction of DNA molecules bound by TBP. Analysis of the kinetics of DNA bending and unbending revealed that on the consensus TATA box two kinetically distinct populations of TBP-DNA complexes exist; however, the bent state of the DNA is the same in the two populations. Our smFRET studies reveal that human TBP bends DNA in a largely uniform manner under a variety of different conditions, which was unexpected given previous ensemble biochemical studies. Our new observations led to us to revise the model for the mechanism of DNA binding by TBP and for how DNA bending is affected by TATA sequence and TFIIA.

Rapid Identity and Quantity CQA Test for Multivalent mRNA Drug Product Formulations.

The COVID-19 pandemic highlighted mRNA as a promising platform for vaccines and therapeutics. Many of the analytical tools used to characterize the critical quality attributes of mRNA are inherently singleplex and are not necessarily optimal from a labor and cost perspective. Here, we demonstrate the feasibility of a multiplexed platform (VaxArray) for efficient identity verification and concentration determination for both monovalent and multivalent mRNA formulations. A model system comprising mRNA constructs for influenza hemagglutinin and neuraminidase was used to characterize the analytical performance metrics for a VaxArray mRNA assay. The assay presented herein had a time to result of less than 2 h, required no PCR-based amplification nor extraction of mRNA from lipid nanoparticles, and exhibited high construct specificity that enabled application to the bivalent mixture. The sensitivity for influenza hemagglutinin and neuraminidase mRNA was sub-µg/mL, which is vaccine-relevant, and the average accuracy (%recovery of a check standard) and precision were 104 ± 2% and 9 ± 2%, respectively.

Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses.

Rapid, sensitive, and precise multiplexed assays for serological analysis during candidate COVID-19 vaccine development would streamline clinical trials. The VaxArray Coronavirus (CoV) SeroAssay quantifies IgG antibody binding to 9 pandemic, potentially pandemic, and endemic human CoV spike antigens in 2 h with automated results analysis. IgG antibodies in serum bind to the CoV spike protein capture antigens printed in a microarray format and are labeled with a fluorescent anti-species IgG secondary label. The assay demonstrated excellent lower limits of quantification ranging from 0.3 to 2.0 ng/mL and linear dynamic ranges of 76 to 911-fold. Average precision of 11 % CV and accuracy (% recovery) of 92.5 % over all capture antigens were achieved over 216 replicates representing 3 days and 3 microarray lots. Clinical performance on 263 human serum samples (132 SARS-CoV-2 negatives and 131 positives based on donor-matched RT-PCR and/or date of collection) produced 98.5 % PPA and 100 % NPA.

FluChip-8G Insight: HA and NA subtyping of potentially pandemic influenza A viruses in a single assay.

BACKGROUND: Global influenza surveillance in humans and animals is a critical component of pandemic preparedness. The FluChip-8G Insight assay was developed to subtype both seasonal and potentially pandemic influenza viruses in a single assay with a same day result. FluChip-8G Insight uses whole gene segment RT-PCR-based amplification to provide robustness against genetic drift and subsequent microarray detection with artificial neural network-based data interpretation. OBJECTIVES: The objective of this study was to verify and validate the performance of the FluChip-8G Insight assay for the detection and positive identification of human and animal origin non-seasonal influenza A specimens. METHODS: We evaluated the ability of the FluChip-8G Insight technology to type and HA and NA subtype a sample set consisting of 297 results from 180 unique non-seasonal influenza A strains (49 unique subtypes). RESULTS: FluChip-8G Insight demonstrated a positive percent agreement ≥93% for 5 targeted HA and 5 targeted NA subtypes except for H9 (88%), and negative percent agreement exceeding 95% for all targeted subtypes. CONCLUSIONS: The FluChip-8G Insight neural network-based algorithm used for virus identification performed well over a data set of 297 naïve sample results, and can be easily updated to improve performance on emerging strains without changing the underlying assay chemistry.

Clinical validation of the FluChip-8G Influenza A+B Assay for influenza type and subtype identification.

BACKGROUND: The FluChip-8G Influenza A+B Assay is a multiplexed influenza RT-PCR and microarray-based assay with same day turnaround time, developed to subtype seasonal A viruses (H1N1pdm2009 and H3N2), distinguish B viruses as Yamagata or Victoria lineage, and is the only FDA cleared assay capable of positive identification of a wide variety of A subtypes as "non-seasonal" A viruses from human nasal specimens. OBJECTIVE: To evaluate clinical performance of the FluChip-8G Influenza A+B Assay for detection of seasonal influenza viruses in nasal and nasopharyngeal swab specimens, and to evaluate performance for detection of non-seasonal influenza viruses using contrived samples. STUDY DESIGN: For seasonal viruses, a multisite study of the FluChip-8G Influenza A+B Assay using prospectively and retrospectively collected nasal and nasopharyngeal swabs was performed using the FDA-cleared CDC Human Flu Dx Panel as the comparator assay. For non-seasonal viruses, testing was performed at a single site using contrived samples from 100 unique non-seasonal strains representing 41 subtypes. RESULTS: Sensitivity (95% CI) and specificity (95% CI) for each target group, respectively, from results of 1689 clinical specimens were: seasonal H1N1pdm2009: 96.4% (87.9-99.0), 99.3% (98.8-99.6), seasonal H3N2: 91.8% (87.7-94.7), 99.7% (99.2-99.9), Influenza B Victoria: 100% (94.0-100.0), 99.9% (99.6-100.0), and Influenza B Yamagata: 95.6% (89.2-98.3), 99.9% (99.6-100.0). The sensitivity and specificity from contrived influenza A non-seasonal viruses was determined to be 99.0% (94.6-99.8) and 100% (96.7-100.0). CONCLUSION: The FluChip-8G Influenza A+B Assay has robust sensitivity and specificity for detecting and identifying all target virus groups, including non-seasonal influenza A, with same day results.

Analytical evaluation of the microarray-based FluChip-8G Influenza A+B Assay.

BACKGROUND: Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8G Influenza A+B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time. OBJECTIVE: To evaluate key analytical performance characteristics of the FluChip-8G Influenza A+B Assay. STUDY DESIGN: Analytical sensitivity, cross-reactivity, and multi-site reproducibility were evaluated. RESULTS: The limit of detection (LOD) for the FluChip-8G influenza A+B Assay ranged from 5.8 × 102-1.5 × 105 genome copies/mL, with most samples ∼2 × 103 genome copies/mL (∼160 genome copies/reaction). Fifty two (52) additional strains were correctly identified near the LOD, demonstrating robust reactivity. Two variant viruses (H1N1v and H3N2v) resulted in dual identification as both "non-seasonal influenza A" and A/H1N1pdm09. No reproducible cross-reactivity was observed for the 34 organisms tested, however, challenges with internal control inhibition due to crude growth matrix were observed. Lastly, samples tested near the LOD showed high reproducibility (97.0% (95% CI 94.7-98.7)) regardless of operator, site, reagent lot, or testing day. CONCLUSION: The FluChip-8G Influenza A+B Assay is an effective new method for detecting and identifying both seasonal and non-seasonal influenza viruses, as revealed by good sensitivity and robust reactivity to 52 unique strains of influenza virus. In addition, the lack of cross-reactivity to non-influenza pathogens and high lab-to-lab reproducibility highlight the analytical performance of the assay as an alternative to real-time RT-PCR and sequencing-based assays. Clinical validation of the technology in a multi-site clinical study is the subject of a separate investigation.

The HMGB1 C-Terminal Tail Regulates DNA Bending.

High mobility group box protein 1 (HMGB1) is an architectural protein that facilitates the formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single-molecule fluorescence resonance energy transfer (FRET), which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full-length (FL) HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the FL protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the FL protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the FL protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similar to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.

Using FRET to monitor protein-induced DNA bending: the TBP-TATA complex as a model system.

Proteins that bind to DNA can elicit changes in DNA conformation, such as bending and looping, which are important signals for later events such as transcription. TATA-binding protein (TBP) is one example of a protein that elicits a conformational change in DNA; TBP binds and sharply bends its recognition sequence, which is thought to facilitate the recruitment of other protein factors. Here we describe the use of fluorescence resonance energy transfer (FRET) to evaluate DNA bending using TBP as a model system. FRET is a useful technique to measure changes in DNA conformation due to protein binding because small changes in the distance between two fluorophores (2-10 nm) translate into large changes in energy transfer.

Real-time quantification of RNA polymerase activity using a "broken beacon".

A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.