Development of a high-throughput G4-FID assay for screening and evaluation of small molecules binding quadruplex nucleic acid structures

ABSTRACTWe describe a high-throughput fluorescence indicator displacement assay (HT-FID) to evaluate the affinity and the selectivity of compounds binding to different DNA structures. We validated the assay using a library of 30 well-known nucleic acid binders containing a variety chemical scaffolds. We used a combination of principal component analysis and hierarchical clustering analysis to interpret the results obtained. This analysis classified compounds based on selectivity for AT-rich, GC-rich and G4 structures. We used the FID assay as a secondary screen to test the binding selectivity of an additional 20 compounds selected from the NCI diversity set III library that were identified as G4 binders using a thermal shift assay. The results showed G4 binding selectivity for only a few of the 20 compounds. Overall, we show that this HT-FID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.

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High Throughput Quantification of Short Nucleic Acid Samples by Capillary Electrophoresis with Automated Data Processing

Analytical Biochemistry ◽

10.1016/j.ab.2021.114239 ◽

2021 ◽

pp. 114239

Author(s):

Tyler L. Dangerfield ◽

Nathan Z. Huang ◽

Kenneth A. Johnson

Keyword(s):

Capillary Electrophoresis ◽

Nucleic Acid ◽

Data Processing ◽

High Throughput ◽

Automated Data Processing

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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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Novel nucleic acid origami structures and conventional molecular beacon–based platforms: a comparison in biosensing applications

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03309-4 ◽

2021 ◽

Author(s):

Noemi Bellassai ◽

Roberta D’Agata ◽

Giuseppe Spoto

Keyword(s):

Nucleic Acid ◽

Structural Properties ◽

Molecular Beacon ◽

Dna Origami ◽

Molecular Beacons ◽

Conformational Polymorphism ◽

Functional Systems ◽

Similarities And Differences ◽

Nucleic Acid Structures ◽

Nucleic Acid Sequences

AbstractNucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Graphical abstract

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Overlapping but distinct: a new model for G-quadruplex biochemical specificity

Nucleic Acids Research ◽

10.1093/nar/gkab037 ◽

2021 ◽

Author(s):

Martin Volek ◽

Sofia Kolesnikova ◽

Katerina Svehlova ◽

Pavel Srb ◽

Ráchel Sgallová ◽

...

Keyword(s):

Nucleic Acid ◽

Drug Design ◽

Solution Structure ◽

Biochemical Data ◽

Biological Regulation ◽

New Model ◽

G Quadruplex ◽

Range Of Functions ◽

Nucleic Acid Structures ◽

Sequence Requirements

Abstract G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanine tetrads. They are capable of a range of functions and thought to play widespread biological roles. This diversity raises an important question: what determines the biochemical specificity of G-quadruplex structures? The answer is particularly important from the perspective of biological regulation because genomes can contain hundreds of thousands of G-quadruplexes with a range of functions. Here we analyze the specificity of each sequence in a 496-member library of variants of a reference G-quadruplex with respect to five functions. Our analysis shows that the sequence requirements of G-quadruplexes with these functions are different from one another, with some mutations altering biochemical specificity by orders of magnitude. Mutations in tetrads have larger effects than mutations in loops, and changes in specificity are correlated with changes in multimeric state. To complement our biochemical data we determined the solution structure of a monomeric G-quadruplex from the library. The stacked and accessible tetrads rationalize why monomers tend to promote a model peroxidase reaction and generate fluorescence. Our experiments support a model in which the sequence requirements of G-quadruplexes with different functions are overlapping but distinct. This has implications for biological regulation, bioinformatics, and drug design.

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Vasopressin-vermittelte Wasserrückresorption im Sammelrohr der Niere

BIOspektrum ◽

10.1007/s12268-021-1544-1 ◽

2021 ◽

Vol 27 (2) ◽

pp. 165-167

Author(s):

Sandrine Baltzer ◽

Enno Klussmann

Keyword(s):

Water Balance ◽

Small Molecules ◽

High Throughput ◽

Molecular Mechanisms ◽

Collecting Duct ◽

Water Channel ◽

Water Reabsorption ◽

Balance Disorders ◽

Water Homeostasis ◽

Balance Disorder

AbstractVasopressin-mediated water reabsorption from primary urine in the renal collecting duct is essential for regulating body water homeostasis and depends on the water channel aquaporin-2 (AQP2).Dysregulation of the process can cause water balance disorders. Here, we present cell-based high-throughput screenings to identify proteins and small molecules as tools to elucidate molecular mechanisms underlying the AQP2 control and as potential starting points for the development of water balance disorder drugs.

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