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Accelerating Turnover Frequency in Nucleic Acid Templated Re-actions Dalu Chang, Ki Tae Kim, Eric Lindberg, and Nicolas Winssinger Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00663 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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Bioconjugate Chemistry

Accelerating Turnover Frequency in Nucleic Acid Templated Reactions Dalu Chang†, Ki Tae Kim†, Eric Lindberg† and Nicolas Winssinger*† †

Department of Organic chemistry, NCCR Chemical Biology, Faculty of Science, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland; [email protected]

ABSTRACT: Nucleic acid templated reactions have attracted attention as an important technology to sense oligonucleotides and to translate nucleic acid-based instructions into diverse outputs. Great progresses have been made in accelerating the reaction in order to improve signal amplification, reaching rates where substrate turn-over rather than chemical reaction is rate limiting. Herein we explore the utility of architectures inspired by three-way junction that yield a cleavage of a strand thus accelerating substrate turn-over. We demonstrate that such design can overcome product inhibition in templated reactions and operate close to the rate of hybridization.

INTRODUCTION Templated chemical reactions are promoted by the high effective concentration of reagents achieved upon a supramolecular association. Nucleic acid has been a fertile ground to study such reactions based on the predictability of hybridization and the adjustability of supramolecular interactions (Ka).1, 2 While this area of investigation was triggered by inquiries into prebiotic amplification of oligonucleotides with templated ligations, the potential of such reactions has now been harnessed towards other applications.3-6 Diverse reaction manifolds have been developed to yield fluorescence for nucleic acid sensing, uncaging or synthesis of a bioactive molecule as smart therapeutics or more broadly to translate the instructions embedded into a DNA template into functional polymers and synthetic molecules. Templated reactions have been extended to other biosupramolecular interactions, with ligands targeting dimeric proteins, receptors and bisubstrate proteins.7-9 An underlying question in these advances is the level of output amplification that can be achieved through template turnover.10-13 In the original format of templated ligation, the ligated products have higher affinity for the template thus greatly inhibiting template turnover (Figure 1). While it was shown that this could be alleviated with destabilizing interactions in the ligated product,14, 15 critical analyses of the product dissociation kinetics have not been reported. Subsequently, a number of bioorthogonal reactions that do not yield a ligation were reported, with

efforts to broaden the bioorthogonality, and ultimately, perform the reaction in response to cellular RNA, as well as accelerate the reaction to maximize the turnover frequency.16-29 The catalytic cycle involves hybridization, templated-reaction and dissociation/reagent exchange. The hybridization of small probes generally proceeds near diffusion controlled and have been measured to be k ≈ 106 M-1s-1 across different nucleic acid platforms (DNA, LNA, PNA).29-31 The templated-reaction rates have typically been derived from the half-life or the reaction with stoichiometric amount of template and probes to achieve pseudo-first order kinetics. We have recently shown that the photoreduction of pyridinium immolative linker by Ru(phen)(bpy)2 proceeds with a rate of k = 0.138 s-1, but that the catalytic version of the reaction was limited by the rate of dissociation.29 Taking cues from enzymatic reactions, nature has generally evolved enzymes to have a faster dissociation kinetics for the product than the substrate in order to alleviate product inhibition. In analogy, reactions with a three-way junction such that a strand cleavage at the junction of the three-way should result in accelerated product dissociation and turnover. Herein, we report the results from three different designs (Figure 2). First, a three-way junction where the template would bring the reagents that would hybridize both to the template and themselves in a three-way junction (Figure 2A); second, a template-catalyst duplex with an overhang that

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hybridize the substrate through both duplex to the template and triplex with the DNA-catalyst duplex (Figure 2B); third and template that would bring both probes which would further interact through a well characterized biosupramolecular interaction: vancomycin binding DAla-D-Ala peptide (Figure 2C).

triplex forming PNA; C: reaction leads to a rupture between the PNA and ligand. Py = pyridinium, Cou = coumarin.

A: Templated ligation

We began our investigation with the three-way junction as shown in Figure 2A, using a DNA template and PNA32, 33 for the substrate and catalyst strands. The probe containing the ruthenium photocatalyst was designed to hybridize to the template with a strong affinity (10-mer PNA), contain the ruthenium photocatalyst at the junction and a 2mer or 4-mer PNA stretch to form the three-way junction. It is important to note that the ruthenium acts photocatalytically and does not need to exchange on the template for the reaction to turnover. The second probe, the substrate was designed with a shorter PNA to hybridize to the template (6-mer) with a coumarin quenched by the pyridinium immolative linker that bridges the second PNA stretch involved in the three-way junction. Previous studies had shown that a 4-mer PNA-PNA duplex was not sufficient to yield fast templated reaction.29 Hence, the interaction arising from the catalyst-substrate alone should not be sufficient to trigger the reaction. We first compared the pseudo-first order kinetics of a three-way junction with the same system lacking the PNA that form the third branch of the three-way junction (Figure 3). This comparison revealed that the reaction was slower in the three-way junction than the simpler format aligning the reagents on the template without a junction (30 fold slower). Mindful of the fact that the three-way junction is a more rigid system than the simpler hybridization along a template, this was interpreted as evidence that the threeway junction is indeed formed but with a reagent preorganization that does not favor the reaction. We next attempted to vary the flexibility of the linker at the junction site in the hope to discover a favorable geometry conductive to catalysis. We investigated the use of a PEG spacer prior or after the immolative linker, the use of different stereochemistry of the lysine connecting the ruthenium catalyst, as well as PEG spacer on either side of the junction to the ruthenium. However, none of the permutations tested provided a reaction rate that was as fast as the parent control reaction (see Table SI-1 for explicit structures and Figure SII for different tested combinations).

B: Templated reaction without ligation

C: Templated reaction with product strand-cleavage

Figure 1. General strategies for nucleic acid templated reactions.

RESULTS AND DISCUSSION

Figure 2. Architectures of nucleic acid templated reactions designed to maximize turnover by facilitating product dissociation following the cleavage of a pyridinium linker by a ruthenium-based photocatalyst. The reactions yield a fluorescence signal upon linker cleavage (coumarin uncaging). A: reaction leads to a rupture of strand involve in a three-way junction; B: reaction leads to a rupture in the strand linking a duplex and


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Figure 3. Templated reaction using three-way junction architecture. Top: general scheme of the reaction; bottom: plot of the fluorescence output (expressed as a yield of reaction) as a function of time. Reactions were performed with stoichiometric quantities of the three components at a final concentration of 100 nM; DNA : 6-mer PNA-Cou-Py : Ru-10-mer-PNA (black curve); DNA : 6-mer PNA-Cou-Py-AT : AT-Ru-10-mer-PNA (red curve); DNA : 6-mer PNA-Cou-Py-ATCT: AGAT-Ru-10mer-PNA (blue curve). Reaction were carried out with 5 mM sodium ascorbate irradiated with a LED at 455 nm (1W), Buffer: PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). Py = pyridinium, Cou = coumarin. See Figure S-1 for detailed chemical structures.

probability that a hybridization event leads to a reaction. We next extended the pyridinium linker with another PNA segment able to hybridize through Watson-Crick interactions to the template and compared the rate of reaction of the duplex-triplex forming chimera relative to the two control reactions involving strictly the triplex and duplex part of the reagent (I, II, III, Figure 4 B). Using stoichiometric amounts of the templates and both PNA probes, the reaction proved to be fastest for the duplex forming architecture III. As for the three-way architecture discussed above and compared in Figure 3, the chimera architecture I (Figure 4B) will be more rigid than either controls (II and III) and could results in a unfavorable preorganization of the ruthenium catalyst relatively to the pyridinium linker. This unfavorable preorganization would be detrimental to the reaction rate. However, given that the reaction of the chimera (I) had comparable pseudo-first order rate (kapp) to the simple triplex reaction (II), we next investigated the reaction using catalytic template wherein the template turnover can be rate limiting (the rate of dissociation / reagent exchange). As shown in 4B, the chimera I indeed performed better under catalytic conditions (kcat) than the triplex II but still underperformed the simple duplex architecture III. Again, small changes in the linker connecting the two duplex and triplex forming PNA within the chimera did not improve the reaction.

We next turned our attention to the second design involving a substrate binding to the template and ruthenium probe through a duplex and triplex respectively (Figure 2B). For the triplex formation, we made use of the recently developed nucleobases that extend the Hoogsteen triplet to any permutation of nucleobase (M•G-C and P•CG).34, 35 We first validated that the triplex formation was functional in templated reactions (Figure 4A). To this end, we synthesized a PNA able to form a triplex with the template-PNA duplex containing the ruthenium catalyst. We observed a distinct rate acceleration that was proportional to the length of the triplex and dependent on the presence of the three oligonucleotide strands. A control experiment lacking DNA had negligible rate attesting to the fact that the two PNA probes cannot interact in other ways than through the Hoogsteen-based triplex. The fact that the pseudo-first order reaction rate increases as a function of the length of the triplex simply reflect the fact that the templated reaction is slower than the reagent dissociation and that reactions are performed at concentrations below the KD of hybridization; the longer the probe, the longer the half-life of a transient hybridization and the higher the



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the reactions, catalytic versions were performed with 10 nM concentration of DNA. Reaction were carried out with 5 mM sodium ascorbate irradiated with a LED at 455 nm (1W), Buffer: PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). Py = pyridinium, Cou = coumarin. See Figure S-1 for detailed chemical structures.

The third strategy investigated is similar to the aforementioned three-way junction but involves a different biosupramolecular interaction for the arms of the three-way junction: the molecular recognition of L-Lys-DAla-D-Ala (KAA) by vancomycin.36 The affinity of the tripeptide for vancomycin is ca. 1 µM.37 While this is akin to the three-way junction shown in Figure 2A, we reasoned that it would provide a different reagent alignment than the purely PNA-based three-way junction and, as such, may not suffer from the same shortcomings. The first system tested made use of a longer PNA coupled to the ruthenium catalyst and vancomycin, providing a stable duplex with the template, and a short PNA substrate (4mer) with the pyridinium linker and profluorescent coumarin connecting to the KAA peptide (Figure 5). The 4mer PNA segment of the substrate is too short to give fast templated reaction by itself. Likewise, the vancomycin:KAA complex was anticipated to be too weak in affinity to yield templated reaction by itself. Comparing the reaction kinetics for a templated-reaction of PNA-Py-CouKAA + Van-Ru-PNA to PNA-Cou-KAA + Ru-PNA under stoichiometric conditions showed that the former was significantly faster (Figure 5).

Figure 4. Templated reaction for PNA:PNA:DNA triplex. A. Top: general scheme of the reaction; bottom: plot of the fluorescence output (expressed as a yield of reaction) as a function of time. Reactions were performed with stoichiometric quantities of the three components at a final concentration of 100 nM using different length of PNA for the Py-CouPNA substrate (9-mer: red curve; 7-mer: blue curve; 5-mer: green curve; 9-mer without DNA template: black curve). B. Top: general scheme of reaction I-III; bottom kinetic analysis of

Figure 5. Templated reaction for PNA-KAA:van-PNA:DNA architecture. Top: general scheme of the reaction; Bottom: plot of the fluorescence output (expressed as a yield of reaction) as a function of time. Reactions were performed with stoichiometric quantities of the three components at a final concentration of 100 nM using different length of PNA for the PNA-Py-Cou-KAA substrate with Van-Ru-PNA (red curve) and compared to the PNA-Py-Cou substrate with RuPNA (blue curve). Reactions were carried out with 5 mM sodium ascorbate irradiated with a LED at 455 nm (1W), Buffer: PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). Py = pyridinium, Cou = coumarin. See Figure S-1 for detailed chemical structures.


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A control experiment using a PNA-KAA with a shorter PNA (2-mer) revealed no measurable reactions under the same conditions indicating that the KAA:vancomycin interaction is indeed not sufficient to yield a templated reaction at this concentration (100 nM). Taken together, this suggest that both interactions (PNA:template and KAA:vancomycin) contribute to the stability of the ternary complex to yield a templated reaction. As expected, extending the length of the PNA accelerates the pseudofirst order rate of reaction (Figure 5), in agreement with the fact that slower template dissociation kinetics maximize the probability of a reaction upon hybridization up to the point where the dissociation rate is slower than the reaction rate. We next compared the pseudo-first order rate (kapp) and the rate using catalytic template (kcat) across the different length of PNA length in the PNA-KAA substrate or PNA alone substrate (Figure 6). It is interesting to note that the increase of pseudo-first order rate saturates at a shorter PNA length for the PNA-KAA substrate (8mer, Figure 6a) relative to the PNA alone substrate (9mer) but both reach the same value (k ≈ 0.025 s-1). Taken together, these experiments clearly demonstrate that KAA:vancomycin interaction participate in a ‘three-way’ architecture that contributes to a slower dissociation kinetics but contrarily to the prior architectures (Figure 2A and B) does not result in an unfavorable conformation of the reagents. We had previously shown that there was a tradeoff between the pseudo-first order rate (kapp) and the rate of catalytic templated reaction (kcat) since longer probes accelerate the reaction but slow down turnover.29 Based on the pseudo first order rate measured, the inflection point for the PNA alone substrate would be at 9-mer. Indeed, comparing the reaction kcat, it is clear that the catalytic reactions with PNA alone, have a maximum efficiency at the 9-mer. The 10-mer probe is 7 times slower, consistent with probe dissociation being the rate limiting step. However, in the case of the PNA-KAA, this dramatic reduction of rate as probe length increase is not observed. Indeed, while the kcat of the 9-mer PNA is comparable to the one of 9-mer PNA-KAA, the kcat of the 10mer PNA is 6.4 times lower than that of 10-mer PNA-KAA. Furthermore, the kcat of 10-mer PNA-KAA (0.016 s-1) is close to the pseudo-first order rate (kapp) calculated using stoichiometric quantities (0.023 s-1). These observations are consistent with a scenario where substrate dissociation is slower than the reaction rate yet turn-over is not inhibited by the product. Given the fact that the 10-mer PNA itself has dissociation rate that are slower than the reaction rate, this suggest that following the reaction, there is a fast dissociation of KAA which is no longer linked to the PNA; vancomycin binding to another PNA-KAA substrate creates a high effective concentration of PNA that

Figure 6. Comparison of reaction rate under stoichiometric and catalytic conditions across different PNA length and template architecture. A: kapp of template reaction using 100 nM : 100 nM : 100 nM DNA : PNA-Py-Cou-KAA : PNA-Ru-VAN ( or PNA-Ru) with different lengths of PNA-Py-Cou-KAA. 5 mM sodium ascorbate, LED 455 nm 1W, Buff: PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). See Figure S-1 for detailed chemical structures; B: kcat of template reaction using 10 nM : 10 nM DNA: PNA-Ru-VAN or PNA-Ru at varying concentrations of PNA-Py-Cou-KAA with different lengths. 5 mM sodium ascorbate, LED at 455 nm (1W), Buffer: PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). Py = pyridinium, Cou = coumarin. See Figure S-1 for detailed chemical structures.

can strand displace the PNA in a process akin to toe-hold strand displacement. It should be noted that toe-hold strand displacements have been shown to operate with second order rate constant of 105 M-1s-1.36 The dissociation constants (KD) for the 9-mer and 10-mer were measured to 62 and 1.4 nM, respectively. Given a 106 M-1s1 rate constant of association (kon), the rate constant of dissociation (koff) are 0.062 s-1and 0.0014 s-1for the 9-mer and 10-mer respectively. These finding are consistent with the observation that the 10-mer PNA catalytic turnover is limited by product dissociation. We next compared product inhibition for the reaction with PNA-KAA vs PNA alone. As shown in figure 7, performing the reaction in the presence of 10-fold excess of product had virtually no impact of the kinetics of PNA-KAA whereas it dramatically slowed down the reaction of the PNA (45-fold difference). Collectively, this data supports the fact that adding a secondary biosupramolecular interaction (vancomycin:KAA) that is cleaved following the templated reaction achieves the goal of facilitating substrate turnover and dramatically reducing product inhibition.



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PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). Py = pyridinium, Cou = coumarin. See Figure S-1 for detailed chemical structures.

CONCLUSION

Figure 7. Kinetic of template reactions in the presence of competitor. 100 nM : 100 nM : 100 nM DNA :10-mer PNA-PyCou-KAA : 12-mer PNA-Ru-Vancomycin ( or PNA-Ru) and 1 uM 10 mer competitor. 5 mM sodium ascorbate, LED at 455 nm (1W), Buffer: PBS 0.1 M, pH 7.4, 0.05% tween (polyoxyethylene). Py = pyridinium, Cou = coumarin. See Figure S-1 for detailed chemical structures.

We next tested the suitability of this system for the detection of templates at low concentration (100 pM) and compared the reaction of a perfect match template with that of a mismatched template. Gratifyingly, we observed 5% conversion in 30 min reaction time (25 turn over) whereas the mismatched template yielded less than 1% product. This corresponds to a rate constant k = 0.0138 s1 , close to the value measured at 100 fold higher catalyst concentration. It is noteworthy that under these conditions the rate of reaction is close (> 4 fold) to the rate of hybridization (pseudo-first order k = 0.05 s-1) and further significant improvements in the system would be limited by the rate of hybridization.

In summary, we have developed a novel architecture for nucleic acid templated reaction. To the best of our knowledge, this is the first example of a templated reaction designed to yield a product with lower affinity for the catalyst than the substrate. This was achieved using a photocatalytic cleavage of a substrate with a pyridinium linker bridging two different entities, each contributing to the affinity of the substrate to the catalyst. The successful design employed two orthogonal biosupramolecular interactions: PNA hybridizing to a DNA template and vancomycin binding to its tripeptide ligand. We demonstrated that the addition of the second biosupramolecular interaction between the substrate and the catalyst enhanced turnover by a 7-fold. We further showed that this design reduced product inhibition by a 40-fold. The findings are applicable to other types of templated reaction and offer a framework to design catalytic cycles devoid of product inhibition.

ASSOCIATED CONTENT Supporting Information Details on the experimental protocols and characterization data. This information is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *Nicolas Winssinger Phone: +41 22 379 61 05, Fax: +41 22 379 32 15, Email: [email protected] ORCID Nicolas Winssinger : 0000-0003-1636-7766 Dalu Chang : 0000-0002-9245-5576 Ki Tae Kim : 0000-0003-2479-6093 Eric Lindberg : 0000-0002-4258-5583

Figure 8. Comparison of perfect match (PM) vs mismatched (MM) templated reaction using low templated loading (100 pM, 0.002 eq of template). PNA-Py-Cou-KAA : 10-mer PNA-PyCou-KAA : 12-mer PNA-Ru-Vancomycin (50 nM : 0.1 nM : 0.1 nM) 5 mM sodium ascorbate, LED at 455 nm (1W), Buffer:

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT


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This work was supported by the Swiss National Science Foundation and NCCR Chemical Biology.

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9 ACS Paragon Plus Environment

Accelerating Turnover Frequency in Nucleic Acid ... - ACS Publications

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