Quantitative design and experimental validation for a single-molecule DNA nanodevice transformable among three structural states

Abstract

In this work, we report the development and experimental validation of a coupled statistical thermodynamic model allowing prediction of the structural transitions executed by a novel DNA nanodevice, for quantitative operational design. The efficiency of target structure formation by this nanodevice, implemented with a bistable DNA molecule designed to transform between three distinct structures, is modeled by coupling the isolated equilibrium models for the individual structures. A peculiar behavior is predicted for this nanodevice, which forms the target structure within a limited temperature range by sensing thermal variations. The predicted thermal response is then validated via fluorescence measurements to quantitatively assess whether the nanodevice performs as designed. Agreement between predictions and experiment was substantial, with a 0.95 correlation for overall curve shape over a wide temperature range, from 30 C to 90 C. The obtained accuracy, which is comparable to that of conventional melting behavior prediction for DNA duplexes in isolation, ensures the applicability of the coupled model for illustrating general DNA reaction systems involving competitive duplex formation. Finally, tuning of the nanodevice using the current model towards design of a thermal band pass filter to control chemical circuits, as a novel function of DNA nanodevices is proposed.

Figure 1.

Fluorescence bistable DNA system. A bistable DNA, CH employed for competitive hairpin formation was encoded to have a sequence of the form: - - - - (5′–3′). The subsequence of length 13 nucleotides and its fully complementary subsequence are indicated by shading. and are poly-T subsequences of length 29 and 28 nt, respectively. Vertical lines represent base pairings. The fully melted coil form is depicted simply as a curved line. FAM and TAMRA fluorophores, attached for discrimination of the targeted hairpin structure, are indicated by F and T in gray and black circles, respectively. Upon formation of the targeted hairpin structure, the emission from FAM attached to the 5′ end of CH is preferentially quenched by the proximal TAMRA attached to the 3′ end.

Figure 2.

Tuning by variation of the lengths of elementary structures. The difference in the lengths of stem regions between the targeted suboptimal hairpin and the inhibitory optimal hairpin, was varied for simulation. The total length and length of the stem region of the targeted suboptimal hairpin structure, were fixed to 95 nt and 12 bp, respectively (the same values as CH).

Figure 3.

Efficiency behavior of the targeted hairpin formation. Solid curve depicts the predicted efficiency behavior of CH, in terms of the fractional occupancy of the target structure, which shows a non-symmetric and singly peaked curve. The efficiency, reaches a maximum value of 0.20 at = 62.3C. For comparison, the dash-dotted curve indicates , the predicted occupancy of the target hairpin structure when predictions are made via an isolated equilibrium model, along with the corresponding melting temperature, . The characteristic temperatures of the two models are each indicated, with dashed lines added for clarity. The above prediction for , for a model system restricted to form the single target hairpin only, was experimentally validated in (28).

Figure 4.

Simulated and measured fluorescence footprints. (A) The predicted FP shown in Figure 3 was converted to the simulated fluorescence footprint (thermal profile of ), by taking into account the unintended 14%-FRET quenching upon formation of the inhibitory hairpin. (B) The measured fluorescence footprint exhibited good agreement with the simulated fluorescence footprint, with a correlation of 0.95.

Figure 5.

Simulated FP curves. (A) Simulated FP curves for various values of are shown as semi-log plots. The width (FWHM) and maximum value obtained for FP decreased with increasing . (B) Stringently limited formation of the targeted suboptimal hairpin was found after tuning. The predicted maximum value for FP and peak temperature were 0.0050% and 79.4°C, respectively, for 24 bp.