Czech National Centre of the European Research Infrastructure Consortium INSTRUCT ERIC

(D–F) Ribbon diagrams for the complete cryo-EM structures. Shown are (D) the A/H7N9 TRX structure stalled by CMPcPP, (E) T1106-stalled and backtracked A/H7N9 structure, and (F) T1106-stalled and backtracked FluB structure. The polymerase color code is as follows: PA endonuclease (forest green), PA-C-terminal domain (green), PB1 (cyan), PB2-N (red), PB2-midlink domain (magenta), PB2-cap-binding domain (orange), PB2-627 domain (deep pink), and PB2-NLS domain (firebrick). The RNA is colored as in (A)–(C), with (D) the CMPcPP shown in red and (E and F) the observed backtracked nucleotides shown in purple-blue.

(G–I) Determined structures of the RNA moieties. Shown are (G) the A/H7N9 TRX structure stalled by CMPcPP (red) with template (orange), 18-mer capped product (slate blue), and 5′ end (violet); (H) T1106-stalled and backtracked A/H7N9 structure with singly incorporated T1106 (magenta), template (pale orange), 21-mer capped product (pale blue), and 5′ end (violet); and (I) the T1106-stalled and backtracked FluB structure with doubly incorporated T1106 (magenta), template (yellow), 21-mer capped product (blue), and 5′ end (violet). In each case, the conformation of the priming loop residues 632–660 in A/H7N9 (631–659 in FluB) are shown in cyan, fully extruded in (G) and (I), partially extruded in (H), and with the position of Tyr657 (Tyr656 in FluB) highlighted.

Stephen Cusack Research Group

Significance

The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favourable overall configuration.

Kouba, T., et.al.: Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106 Cell Rep. 2023, 42, 111901, https://doi.org/10.1016/j.celrep.2022.111901

a, Schematic illustration of the in silico design of libraries of de novo and unevolved random-sequence proteins. A de novo library (DN) was built from putative de novo proteins identified in human and fly. Subsequently, a library of unevolved random sequences (R) was designed to mirror the length and amino acid frequencies of library DN. The two libraries were synthesized by oligonucleotide library synthesis ready for experimental study. b, Approaches used to profile solubility and structure content of each library. Following amplification, each library was expressed in a chaperone-assisted cell-free format and structural content was quantified using a proteolytic assay. In parallel, subcloned libraries were expressed in E. coli to screen for soluble and folded variants that did not disrupt periplasmic export.

Klára Hlouchová and Erich Bornberg-Bauer Research Groups

Significance

De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterize a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioinformatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit moderately higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic random sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system than random expectation, given their higher solubility.

Brennen, H. et. al.: Experimental characterization of de novo proteins and their unevolved random-sequence counterparts Nat. Ecol. Evol. (2023), 7, 570-580: https://doi.org/10.1038/s41559-023-02010-2

Detection of SARS-CoV-2 N protein. (A) UCNP label: Alkyne-PEG-neridronate strongly binds via two phosphonate groups to surface lanthanide ions of UCNPs, and a click reaction binds the conjugate to azide-modified streptavidin. (B) Scheme of sandwich ULISA: A microtiter plate is coated with two monoclonal antibodies that capture the N protein. Then, two biotinylated detection antibodies bind to the N protein. The sandwich immune complex is finally detected by using the UCNP label.

Hans H. Gorris Research Group

Significance

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

Brandmeier, J.C., Jurga, N., Grzyb, T., Hlaváček, A., Obořilová, R., Skládal, P., Farka, Z., and Gorris, H.H.: Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay, Anal. Chem. 2023 95, 51, 4753 – 4759, https://doi.org/10.1021/acs.analchem.2c05670