Nature-Inspired Nanoprobes for the Point-Of-Care Detection of Clinically Relevant Biomarkers for Early Tumor Diagnosis

Project location: ITALY, Rome
Project start date: January 2013 - Project end date: January 2015
Project number: 2012-079
Beneficiary: Università di Roma Tor Vergata, Dipartimento di Scienze e Tecnologie Chimiche

TIMELINE OF THE PRESENT ACTIVITY REPORT: From January 2013 to June 2013


The major goal of this project is to mimic nature to exploit the “designability” of nucleic acids to design and develop molecular nanodevices that undergo binding-induced conformational changes (switches) upon target binding and, in doing so, can signal the presence of a cancer marker.

These “nature-inspired” nucleic acid based nanoswitches that signal the presence of specific tumor markers by undergoing a binding induced conformational change will be based on different nucleic acid secondary structures (pseudoknots, triplex, G-quadruplex, etc.) and different switching mechanisms (“sliding switch”, stem-loop, etc).
 
During the first reporting period the thermodynamic optimization of a specific clamp-like switch was performed using optical reporters (fluorophore/quencher pairs). This switching mechanism is also used by Nature to improve the affinity and specificity of biomolecular recognition elements. This strategy can be thus of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines. This clamp-like DNA-based switch is designed so that it recognizes a target oligonucleotide via both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. This clamp-switch is composed of two recognition elements separated by an unstructured, 10-base loop. The first recognition element, a 15-base polypyrimidine sequence binds the target, a polypurine sequence, via Watson-Crick base pairing. The second recognition element, a polypyrimidine sequence, then binds the so-formed duplex via sequence-specific Hoogsteen base pairing. The formation of this triplex structure occurs through a conformational switch mechanism that leads to the switch’s closure.

Clamp-switch probes bind to their targets with greater affinity than do the equivalent linear probes. Unfortunately, however, the resultant difference in binding free energy between the clamp-switch and the reference linear probe is great enough that we cannot measure it directly. That is, there is no single target for which both probes produce a measurable dissociation constant. For example, while the clamp-switch exhibits micromolar affinity with a target as short as 8 bases (KD_clamp (8-base) = 1.4 mM; Figure 3, right), the linear non-switching probe does not exhibit any detectable binding with this same target at even the highest concentrations we have tested (100 mM). Using extrapolations from data it was possible to estimate the difference in the free energy with which each probe would bind a specific, 10-base target. For example, by observing that the affinity of the clamp-switch for a 10-base target matches the affinity with which the linear non-switching probe binds a longer, 12-base target (KD = 20 nM; Figure 3) it is possible to predict the energetic improvement due to the clamp mechanism. A nearest-neighbor model predicts that these two extra G-C Watson-Crick base pairs should provide an additional 3.3 kcal/mol in binding energy, suggesting that this represents the extra stabilization provided by the Hoogsteen base pairing between the clamp-switch and the 10-base target.

Clamp-switches also provide a significant affinity advantages over other mechanisms of coupling binding to a large-scale conformational change. Specifically, the affinity of clamp switches is greater than that of the equivalent (i.e., same recognition site) switch using an engineered distorted state. To show this the clamp-switch was compared with the equivalent molecular beacon, a commonly employed optical or electrochemical approach for the detection of specific DNA sequences. A molecular beacon is a fluorophore-and-quencher-modified DNA strand that forms a low-emissive stem-loop conformation due to hybridization of its complementary ends. This structure opens - thus producing enhanced fluorescence - when a target hybridizes to the loop, breaking the stem and segregating the fluorophore/quencher pair. In contrast to the nanomolar affinity that the clamp-switch shows for targets as short as 10 bases, the molecular beacon does not reach this affinity until targets of at least 15 bases are used. This occurs because molecular beacons employ an engineered distorted state, (here a stem-loop structure) the stabilization energy of which competes with target binding. This effect appears obvious if we compare the affinity of the linear probe and the equivalent molecular beacon (experimental limitations once again do not allow us to compare directly the affinities of the switch-clamp and molecular beacon probes for a single target of constant length). While the linear non-switching probe shows nanomolar affinity for a 13-base target, the same target gives 1000-fold poorer affinity with the molecular beacon.

In addition to improving binding affinity, the clamp-switch mechanism also enhances specificity. To explore this, the affinities of the model systems were compared against a perfectly matched and a single-base mismatched target (see Figure 2 for mismatch location). In order to describe specificity quantitatively the discrimination factor, Q, which is the ratio of the output signal produced by the perfectly matched target (cPM) to that of the mismatched target (cMM) was used (Figure 4) and the specificity window, defined here as the range of target concentration at which a value of Q equal or above 5 is observed (thus representing a 20% interfering signal). The specificity window of the simple linear non-switching probe spans about an order of magnitude in target concentration (Figure 4, bottom). The specificity window of the clamp-switch, in contrast, is 10 times wider (Figure 4, top). Due to the experimental limitations described above, however, the specificity of the clamp-switch probe was determined using a shorter target (10-base) than that employed to test the specificity of the linear non-switching probe (13-base) which could, in theory, also lead to higher specificity. To rule this out we performed simulations using the nearest–neighbor model, which confirms that the small difference in target length is not responsible for the observed large specificity variation.

Comparison of the specificity windows of the two probes provides insights into how the clamp-switch mechanism leads to enhanced specificity. Specifically, the difference in affinity between the perfectly matched and a single-base mismatched target for the clamp-switch (138-fold) suggests that the mismatch is 3.10 kcal/mol less stable than the perfect-match target. For the linear-probe/target complex the destabilization provided by the mismatch is only 1.66 kcal/mol. The extra Hoogsteen interactions in the clamp mechanism thus improve the specificity of the clamp-switch by ca. 1.44 kcal/mol.

The triplex-forming DNA clamp-switch probe, characterized during this first reporting period, recognizes its specific target through two sequential binding events which sum up to provide a higher binding free energy and greater discrimination efficiency than those observed for either the equivalent, non-switching linear probe or a switch based on the engineering of a distorted state. These advantages most likely explain why evolution so often employs the clamp-like mechanism. The clamp-switch strategy thus provides a route towards engineering switches with improved affinity and specificity.

The enhanced performances of the DNA clamp-switch enables the detection of very short targets with high affinity and specificity. This characteristic obviously appears particularly important for applications in which both affinity and specificity play a crucial role, such as sensing and therapeutic purposes (PCR, in vivo imaging of small repeats, interfering DNAs), and in the building of DNA nanostructures, such as DNA nanomachines and DNA origami, where the ability to specifically and tightly bind short DNA sequences could lead to improved structural control. For these reasons these routes will be tested in the next reporting period together with the characterization of other DNA-based switching mechanisms.
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