A high-throughput cell-based screening method for Zika virus protease inhibitor discovery.

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Structural Basis for Parallel G-Quadruplex Recognition by an Ankyrin Protein.

G-Quadruplex (G4) structures formed by guanine-rich DNA and RNA sequences are implicated in various biological processes. Understanding the mechanisms by which proteins recognize G4 structures is crucial for elucidating their functional roles. Here we present the X-ray crystal structure of an ankyrin protein bound to a parallel G4 structure. Our findings reveal a new specific recognition mode in which a bundle of α-helices and loops of the ankyrin form a flat surface to stack on the G-tetrad core. The protein employs a combination of hydrogen bonds and hydrophobic contacts to interact with the G4, and electrostatic interaction is used to enhance the binding affinity. This binding mechanism provides valuable insights into understanding G4 recognition by proteins.

Crystal structures of an HIV-1 integrase aptamer: Formation of a water-mediated A•G•G•G•G pentad in an interlocked G-quadruplex.

93del is a 16-nucleotide G-quadruplex-forming aptamer which can inhibit the activity of the HIV-1 integrase enzyme at nanomolar concentration. Previous structural analyses of 93del using NMR spectroscopy have shown that the aptamer forms an interlocked G-quadruplex structure in K+ solution. Due to its exceptional stability and unique topology, 93del has been used in many different studies involving DNA G-quadruplexes, such as DNA aptamer and multimer design, as well as DNA fluorescence research. To gain further insights on the structure of this unique aptamer, we have determined several high-resolution crystal structures of 93del and its variants. While confirming the overall dimeric interlocked G-quadruplex folding topology previously determined by NMR, our results reveal important detailed structural information, particularly the formation of a water-mediated A•G•G•G•G pentad. These insights allow us to better understand the formation of various structural elements in G-quadruplexes and should be useful for designing and manipulating G-quadruplex scaffolds with desired properties.

Formation of RNA G-wires by G4C2 repeats associated with ALS and FTD.

In the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), expansion of the G4C2 hexanucleotide repeat in the gene C9orf72 is a most common known cause of the disease. Here we use atomic force microscopy (AFM) and gel electrophoresis to visualize the formation of higher-order structures by RNA G4C2 repeats in physiologically relevant conditions. For the RNA sequence r[G4C2G4], we observed G-wires with left-handed undulating features of 4.4-nm periodicity and a uniform height which is consistently higher than that of a duplex B-DNA. These higher-order structures were not degraded fully when treated with a mixture of RNase A and RNase T1. Similarly, higher-order structures were observed for sequences containing three or four G4C2 repeats, pointing towards their potential formation in longer sequence contexts. Our observations suggest that RNA G-quadruplex blocks and G-wires can accumulate in cells containing G4C2 repeat transcripts.

Bulges in left-handed G-quadruplexes.

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures.

Cyclization of a G4-specific peptide enhances its stability and G-quadruplex binding affinity.

G-quadruplexes (G4) are non-canonical nucleic acid structures with important implications in biology. Based on an α-helical fragment of the RHAU helicase that displays high specificity for parallel-stranded G-quadrplexes, herein we demonstrate its head-to-tail cyclization by a high-efficiency ligase. The cyclic peptide exhibits superior stability and binding affinity to a G-quadruplex, and can serve as an excellent investigational tool for chemical biology applications.