Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA nanostructures in real time

Abstract

We describe a method for the real-time and high-throughput monitoring of the self-assembly and disassembly of complex DNA superstructures, using temperature-dependent Förster resonance energy transfer (FRET) spectroscopy. Compared with other spectroscopic approaches, such as UV-visible and circular dichroism, the method described has advantages in terms of sensitivity, feasibility for high-throughput analysis and applicability to virtually any kind of supramolecular structure. To this end, two oligonucleotides out of the entire set building up the superstructure are labeled with a fluorescein and a tetramethylrhodamine, as FRET donor and acceptor, respectively. Correct assembly of the superstructure induces maximum FRET efficiency, whereas complete dissociation leads to minimal FRET. Monitoring of temperature-dependent FRET efficiency yields a thermal profile that is used for thermodynamic analysis. In the case of reversible and cooperative assembly/disassembly of the DNA superstructure, application of the van't Hoff law allows for the determination of the thermodynamic parameters of the process. Owing to slow temperature ramping, the entire assay requires about 17 h. The protocol allows to simultaneously analyze up to 384 samples with only 30 microl sample volume each.

Trinucleotide duplex formation inside a confined nanospace under supercooled conditions

“…In a bulk water system, d-T 10 /a-A 10 pair exhibited clear FRET response (Fig. 4a), and analysis of the FRET response on the basis of a conventional donor-quenching approach 23 (Supplementary Note 2) confirmed that y of d-T 10 /a-A 10 pair was almost 1 below 278 K (Fig. 4b).…”

“…F A /F D values were used for an estimation of assembled fraction (y) of the FAM-and TAMRAmodified DNA fragments on the basis of a simple two-state model 23 , which is constructed assuming that only the DNA duplex exhibits a FRET response. In the two-state model, the relationship between F A /F D and y is described by equation (1) (1) are described in the Supplementary Note 1.…”

“…In the simple DNA-DNA hybridization model, the enthalpy (DH) and entropy (DS) changes for the duplex formation can be estimated from y values 23 :…”

DNA duplex stabilization in crowded polyanion solutions

“…DNA melting was thus monitored by fluorescence enhancement. [15][16][17][18][19] The melting curves in the presence of increasing concentrations of NaPAA1200 are shown in Figure 2A, where typical DNA melting transitions are observed. The temperature corresponding to the maximal of the first 65 derivative of a melting curve is Tm.…”

Analysis of the Self‐Assembly of 4×4 DNA Tiles by Temperature‐Dependent FRET Spectroscopy

“…[6][7][8] These techniques are usually destructive and allow for visualization of the structures only after their formation, thereby precluding any kind of on-line detection and/or manipulation of the assembly process. We recently described a new method, [9,10] based on fluorescence resonance energy transfer (FRET) spectroscopy, [11][12][13][14][15][16][17][18] to overcome these limitations. This microplate-based method allows one to monitor in real time and with high throughput the formation of DNA tiles and nanoarrays.…”