Binder-Responsive DNA Strand Displacement Enables High-Throughput and High-Fidelity Discovering of Small Molecular DNA Binders.

High-throughput screening (HTS) is pivotal in the discovery of small molecules that bind to DNA, yet there are limited sensing mechanisms available for designing HTS assays for DNA binders. Herein, we introduce a binder-responsive toehold-mediated DNA strand displacement (BR-TMSD) technique featuring programmable reaction kinetics in response to DNA-binder interactions. When two DNA binders are used, BR-TMSD is initiated through a rapid binder displacement, followed by the DNA strand displacement. The orthogonal displacement reactions of BR-TMSD enables a high-fidelity, dual-channel HTS assay, returning 19 new DNA binders from a library of 1,170 compounds.

Native Characterization of Noncanonical Nucleic Acid Thermodynamics via Programmable Dynamic DNA Chemistry.

Folding thermodynamics, quantitatively described using parameters such as ΔGfold°, ΔHfold°, and ΔSfold°, is essential for characterizing the stability and functionality of noncanonical nucleic acid structures but remains difficult to measure at the molecular level. Leveraging the programmability of dynamic deoxyribonucleic acid (DNA) chemistry, we introduce a DNA-based molecular tool capable of performing a free energy shift assay (FESA) that directly characterizes the thermodynamics of noncanonical DNA structures in their native environments. FESA operates by the rational design of a reference DNA probe that is energetically equivalent to a target noncanonical nucleic acid structure in a series of toehold-exchange reactions, yet is structurally incapable of folding. As a result, a free energy shift (ΔΔGrxn°) is observed when plotting the reaction yield against the free energy of each toehold-exchange. We mathematically demonstrated that ΔGfold°, ΔHfold°, and ΔSfold° of the analyte can be calculated based on ΔΔGrxn°. After validating FESA using six DNA hairpins by comparing the measured ΔGfold°, ΔHfold°, and ΔSfold° values against predictions made by NUPACK software, we adapted FESA to characterize noncanonical nucleic acid structures, encompassing DNA triplexes, G-quadruplexes, and aptamers. This adaptation enabled the successful characterization of the folding thermodynamics for these complex structures under various experimental conditions. The successful development of FESA marks a paradigm shift and a technical advancement in characterizing the thermodynamics of noncanonical DNA structures through molecular tools. It also opens new avenues for probing fundamental chemical and biophysical questions through the lens of molecular engineering and dynamic DNA chemistry.

Coding Intrinsic Disorder into DNA Hybridization Probes Enables Discrimination of Single Nucleotide Variants over Wide and Tunable Temperature Ranges.

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy-compensate probe (Sprobe) design by coding intrinsic disorder into a stem-loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine-tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Enabling programmable dynamic DNA chemistry using small-molecule DNA binders.

The binding of small molecules to the double helical structure of DNA, through either intercalation or minor groove binding, may significantly alter the stability and functionality of DNA, which is a fundamental basis for many therapeutic and sensing applications. Here, we report that small-molecule DNA binders can also be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder. The binder-induced nucleic acid strand displacement (BIND) thus enables innovative technologies to accelerate the discovery and characterization of bioactive small molecules. Specifically, we demonstrate the comprehensive characterization of existing and newly discovered DNA binders, where critical parameters for binding affinity and sequence selectivity can be obtained in a single, unbiased molecular platform without the need for any specialized equipment. We also engineer a tandem BIND system as a high-throughput screening assay for discovering DNA binders, through which 8 DNA binders were successfully discovered from a library of 700 compounds.

Toehold-Exchange-Based Activation of Aptamer Switches Enables High Thermal Robustness and Programmability.

Aptamer switches are attractive nature-inspired tools for developing smart materials and nanodevices. However, the thermal robustness and programmability of current aptamer switches are often limited by their activation processes that are coupled with high reaction enthalpy. Here, we present an enthalpy-independent activation approach that harnesses toehold-exchange as a general framework to design aptamer switches. We demonstrate mathematically and experimentally that this approach is highly effective in improving thermal robustness and thus leads to better analytical performances of aptamer switches. Enhanced programmability is also demonstrated through fine-grained and dynamic tuning of effective affinities and dynamic ranges, as well as the construction of a synthetic DNA network that resembled biological signaling cascades. Our study not only enriches the current toolbox for engineering and controlling synthetic molecular switches but also offers new insights into their thermodynamic basis, which is critical for diverse synthetic biological designs and applications.

DNA Balance for Native Characterization of Chemically Modified DNA.

Chemical modification is a powerful approach to expand the chemical diversity and functionality of natural DNA. However, when chemically modified oligonucleotides are employed in DNA-based reactions or structures, it becomes quite difficult to predict, understand, and control their kinetics and thermodynamics. To address this challenge, we introduce a rationally designed DNA balance capable of measuring critical thermodynamic and kinetic properties of chemically modified DNA in their native environment. Our DNA balance is operated using the principle of toehold-exchange, where a panel of weight probes were designed by tuning the lengths of forward and reverse toeholds. Once placed on the DNA balance, the chemical modification will be interrogated using the weight probes to determine changes in both Gibbs free energy and hybridization rate constant. Using cyclic-azobenzene (cAB)-modified DNA as a model system, we demonstrated that our DNA balance could not only measure stable chemical modifications, but also solve more challenging issues where unstable chemical modifications and transient isomerization reactions were involved. We anticipate that our DNA balance will find wide uses for measuring important thermodynamic and kinetic parameters for DNA carrying various chemical modifications, as well as for probing transient chemical changes in DNA.