Diffusion‐Ordered NMR Spectroscopy of Guest Molecules in DNA Hydrogels and Related Matrices

Abstract

Diffusion‐ordered NMR spectroscopy was used to monitor the diffusion of guest molecules in DNA hydrogels and related DNA matrices. As guest molecule the highly symmetric hollow‐spherical flavoprotein dodecin was studied. Thermoresponsive hydrogels were formed by self‐assembly via hybridization of linear double‐stranded DNA building blocks of 30 base pairs equipped with sticky ends, i. e. additional overhangs of 15 bases on both ends, which were complementary to each other. This resulted in hydrogels, in which dodecin was freely diffusing. When in contrast self‐assembly was performed with rather short building blocks (9 base pairs + sticky ends of 6 bases), the diffusion of the guest molecule was hampered, but as hybridization was reversible within the timescale of the experiment, the resulting DNA matrix did not behave as a true gel. Apparently true DNA hydrogels with small mesh size can be obtained only, when self‐assembly of short DNA building blocks via hybridization is combined with enzymatic ligation leading to a covalently linked network. In that case, the minimum achievable mesh size should be limited by the diameter of the ligase. While DNA hydrogels are an ideal matrix to host rather large molecules or even living cells, it is a challenge to design pristine DNA hydrogels with mesh sizes sufficiently small to capture guest molecules such as drugs or enzymes in the size of only a few nm.

The dodecin of Mycobacterium tuberculosis : biological function and biotechnical applications

“…The second theme is: the use of dodecins as scaffold proteins or nanoparticles. Here, based on the example of MtDod as a scaffold protein (Bourdeaux et al 2020 [236] ) and HsDod as a diffusion probe (Nöll et al 2018 [237] ), the potential of bacterial dodecins as highly modifiable nanoparticles will be discussed. By comparing MtDod with other scaffold proteins/peptides the strengths and weaknesses will be highlighted.…”

“…the toxicity and distribution of PEG-coated gold nanoparticles in the body depends on its size. [275,276] The importance of size and shape distribution is even greater, when the particles are used as probes to study other objects, like in the here described case of measuring the ability of DNA hydrogels to capture particles (Nöll et al 2018 [237] ).…”

“…In the first subchapter the suitability of dodecins as nanoparticles will be discussed based on the example of HsDod, which was used by Nöll et al 2018 [237] to measure via diffusion-ordered NMR the ability of DNA hydrogels to trap guest particles/molecules. HsDod's only role in this setup was to function as a diffusion probe, making it an ideal system to discuss the main features of a protein nanoparticle: stability and size conformity.…”

“…While the publication of Nöll et al 2018 [237] mainly focuses on DNA hydrogels and the measurement method, here, the HsDod as a diffusion probe will be the centre of the discussion.…”

“…[277] Since mesh size is not the same as pore size in hydrogels, because large pores (compared to the pores formed by the regular gel matrix) can form through irregularities in the hydrogel, also methods need to be considered to judge the quality of the generated hydrogel, which was done by the diffusion of HsDod entrapped in the hydrogel in this case. [237] In a hydrogel with a mesh size below the size of the entrapped particles and no larger pores, the diffusion of the entrapped particle is greatly reduced, while with large pores or larger mesh sizes the particle can diffuse rather freely. In Nöll et al 2018 [237] , three different types of DNA hydrogels were studied this way, two hydrogels based on approx.…”