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Toehold Probes for Nucleic Acid Detection

Highly accurate and sensitive DNA and RNA-detecting probes for clinical diagnostics and basic research

The accurate detection of specific DNA or RNA sequences is important for many research and diagnostic applications, and unspecific detection of similar sequences that can differ by only a single nucleotide can give false positive results. In addition, researchers and clinicians would like to accurately test for presence or absence of multiple single base changes in a patient sample in order to, for example, help them guide therapy. Conventional probes, however, are often unable to provide the necessary selectivity for their target sequences; and they are optimized to work only under specific temperature and salt conditions preventing them from being easily combined in multiplexed analysis.

As a solution, Wyss Institute researchers have developed “Toehold Probes,” a DNA and RNA detection technology that is based on specifically engineered nucleic acid sequences. By programming DNA and RNA probes with highly predictable thermodynamic hybridization behaviors, the approach enables the detection of sequences of interest with single-nucleotide precision. Toehold Probes also exhibit high temperature robustness, which allows them to be multiplexed in a single reaction.

This animation explains how toehold probes consisting of a “probe strand” and a “protector strand” are assembled and how they leverage thermodynamic principles to allow the specific detection of a correct target sequence, or to prevent them from detecting a spurious target sequence that can differ from the correct target sequence by only a single non-matching nucleotide. Credit: Wyss Institute at Harvard University

Their unique advantages are based on the Toehold principle: Toehold Probes contain two strands of DNA that are hybridized to each other due to complementary of their nucleotide sequences. One, the “probe strand” is also complementary to a target sequence, for example, in the human genome, while the second “protector strand” copies part of the target DNA. Toeholds—short sequences at the ends of the probe strand that are either complementary to the target sequence or the protector strand—initiate two exchange reactions. These either result in the probe strand being specifically bound to its target DNA/RNA (to allow its detection) and the protector strand being released; or, in reverse, in the probe strand re-engaging with the protector strand and leaving the target DNA/RNA behind. The two competing exchange reactions lead to an equilibrium that is highly predictable and highly sensitive to perturbations such that the presence of a single non-matching nucleotide (a variant) in the target sequence prevents its detection. In addition, Toehold Probes can robustly operate in much broader temperature windows than conventional probes, which allows them to be multiplexed.

Toehold probes with their robust single-nucleotide specificities can be leveraged in various diagnostic and research applications, including polymerase chain reaction (PCR), DNA/RNA sequencing-based methods and single cell fluorescence in situ hybridization (FISH) techniques for the detection of rare pathological and other sequence variants. The Wyss Institute-launched start-up companies NuProbe and Torus Biosystems are developing toehold probe technology as new precision diagnostics for, respectively, cancer and infectious disease medicine.

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