Directing in Vitro Selection towards G‐quadruplex‐forming Aptamers to Inhibit HMGB1 Pathological Activity

Napolitano, Ettore; Criscuolo, Andrea; Riccardi, Claudia; Esposito, Carla L.; Catuogno, Silvia; Coppola, Gabriele; Roviello, Giovanni N.; Montesarchio, Daniela; Musumeci, Domenica

doi:10.1002/anie.202319828

“…The difference in the migrations can be attributed to variations in the loop region. , In addition, 10SS, TAA, and CAA showed minor bands migrating very slowly. These bands indicate that there are high-order structures, such as multimeric G4s and G-wire, , although it is difficult to determine the structure. We further performed CD melting experiments (Figure S2) and SVD analysis of the CD spectra at different temperatures to identify the major structure by the use of KinTek Explorer (see Experimental Details).…”

Section: Resultsmentioning

confidence: 99%

Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes

Nakata,

Kosaka,

Kawauchi

et al. 2024

ACS Omega

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The thermal stability of G-quadruplexes is important for their biological roles. G-quadruplexes are stable in the presence of cations such as K + and Na + because these cations coordinate in the G-quartet of four guanine bases. It is well known that the number of G-quartets and the configuration of the guanine bases affect the binding affinity of the cation. Recently, structures formed in the loop regions connecting the guanine stretches have attracted significant attention, because the loop region affects Gquadruplex properties, such as topology, thermal stability, and interactions with proteins and small molecules. Considering these effects, the loop region can also affect the binding affinity of the cations. Here, we designed a series of G-quadruplex-forming DNA sequences that contain a hairpin in a loop region and investigated the effects of the sequence and structure of the loop region on the cation binding affinity as well as the thermal stability of the Gquadruplex as a whole. First, structural analysis of the DNA sequences showed that the hairpin at the loop plays a key role in determining G4 topology (strand orientation). Second, in the case of the G-quadruplexes with the hairpin-forming loop region, it was found that a longer loop length led to a higher thermodynamic stability of the G-quadruplex as well as higher cation binding affinity. In contrast, an unstructured loop region did not lead to such effects. Interestingly, the cation binding affinity was correlated to the thermodynamic stability of the hairpin structure at the loop region. It was quantitatively demonstrated that the stable loop region stabilized the whole G-quadruplex structure, which induced higher cation binding affinity. These systematic and quantitative results showed that the loop region is one of the determinants of cation binding and expanded the possibilities of drug development targeting G4s by stabilizing the loop region.

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Fighting Mutations with Mutations: Evolutionarily Adapting a DNA Aptamer for High‐Affinity Recognition of Mutated Spike Proteins of SARS‐CoV‐2

Wang,

Li,

Zhang

et al. 2024

Angewandte Chemie

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An on‐going challenge with COVID‐19, which has huge implications for future pandemics, is the rapid emergence of viral variants that makes diagnostic tools less accurate, calling for rapid identification of recognition elements for detecting new variants caused by mutations. We hypothesize that we can fight mutations of the viruses with mutations of existing recognition elements. We demonstrate this concept via rapidly evolving an existing DNA aptamer originally selected for the spike protein (S‐protein) of wildtype SARS‐CoV‐2 to enhance the interaction with the same protein of the Omicron variants. The new aptamer, MBA5SA1, has acquired 22 mutations within its 40‐nucleotide core sequence and improved its binding affinity for the S‐proteins of diverse Omicron subvariants by >100‐fold compared to its parental aptamer (improved from nanomolar to picomolar affinity). Deep sequencing analysis reveals dynamic competitions among several MBA5SA1 variants in response to increasing selection pressure imposed during in vitro selection, with MBA5SA1 being the final winner of the competition. Additionally, MBA5SA1 was implemented into an enzyme‐linked aptamer binding assay (ELABA), which was applied for detecting Omicron variants in the saliva of infected patients. The assay produced a sensitivity of 86.5 % and a specificity of 100 %, which were established with 83 clinical samples.

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Fighting Mutations with Mutations: Evolutionarily Adapting a DNA Aptamer for High‐Affinity Recognition of Mutated Spike Proteins of SARS‐CoV‐2

Wang,

Li,

Zhang

et al. 2024

Angew Chem Int Ed

0

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An on‐going challenge with COVID‐19, which has huge implications for future pandemics, is the rapid emergence of viral variants that makes diagnostic tools less accurate, calling for rapid identification of recognition elements for detecting new variants caused by mutations. We hypothesize that we can fight mutations of the viruses with mutations of existing recognition elements. We demonstrate this concept via rapidly evolving an existing DNA aptamer originally selected for the spike protein (S‐protein) of wildtype SARS‐CoV‐2 to enhance the interaction with the same protein of the Omicron variants. The new aptamer, MBA5SA1, has acquired 22 mutations within its 40‐nucleotide core sequence and improved its binding affinity for the S‐proteins of diverse Omicron subvariants by >100‐fold compared to its parental aptamer (improved from nanomolar to picomolar affinity). Deep sequencing analysis reveals dynamic competitions among several MBA5SA1 variants in response to increasing selection pressure imposed during in vitro selection, with MBA5SA1 being the final winner of the competition. Additionally, MBA5SA1 was implemented into an enzyme‐linked aptamer binding assay (ELABA), which was applied for detecting Omicron variants in the saliva of infected patients. The assay produced a sensitivity of 86.5 % and a specificity of 100 %, which were established with 83 clinical samples.

show abstract

Directing in Vitro Selection towards G‐quadruplex‐forming Aptamers to Inhibit HMGB1 Pathological Activity

Cited by 5 publications

References 64 publications

Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes

Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes

Fighting Mutations with Mutations: Evolutionarily Adapting a DNA Aptamer for High‐Affinity Recognition of Mutated Spike Proteins of SARS‐CoV‐2

Fighting Mutations with Mutations: Evolutionarily Adapting a DNA Aptamer for High‐Affinity Recognition of Mutated Spike Proteins of SARS‐CoV‐2

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