DNA-based nanostructures (DNs) are advantageous for design of functional materials for biology and medicine due to the nanoscale control afforded by their predictable self-assembly. However, the use of DNs <i>in vivo</i> has been limited due to structural instability in biofluids. The standard approach to assess stability is through gel electrophoresis to monitor the rate of digestion by specific nucleases. However, this technique is challenged by high background signal in complex biofluids (e.g., serum, saliva, etc.), requiring DNs to be denatured or extracted from the test matrix prior to analysis. The development of efficient methods to characterize DN stability under diverse biological conditions could accelerate the development of new functional DNs. As such, we have developed a size exclusion chromatography (SEC) method to characterize the degradation profiles of DNs. Our technique measures the stability of an intact DN and does not require denaturation or extraction before analysis. Through direct injection of incubated samples and peak integration, our method can reliably assay DN breakdown by nuclease treatment. Using a standard DNA tetrahedron (TDN) as a model system, we show that DNaseI at a concentration of 1.56 U mL<sup>-1</sup> degrades TDNs with a half-life of 11.63 min, which is consistent with reported literature observations. Furthermore, by exploiting the differential optical properties of DNs and proteins, we demonstrate that the degradation analysis of intact DNs in a biofluid can be performed using a ratiometric monitoring approach, without requiring pre-analysis purification. We demonstrate that a ratio chromatogram technique can be applied to deconvolute the DN signal from a human serum protein matrix for efficient degradation analysis. We found that TDNs exhibit half-lives of 23.9 hours and 10.1 hours in 20% and 50% diluted human serum, respectively, which is consistent with reported stability studies in 10% fetal bovine serum. We anticipate that this method can be broadly applicable to characterize a variety of DNs and serve as an efficient technique toward analysis of the stability of new DN designs in complex biological matrixes.

Development of a biostability characterization method for DNA nanostructures using size exclusion chromatography

ACS Spring 2022

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