Evaluation of the nuclease resistance of DNA four‐way junctions.

Abstract

Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also control gene expression by targeting proteins. One popular approach uses double stranded DNA as transcription factor decoy sequences that are directed against transcription factors such as NF‐kB and specificity protein 1 (Sp1). More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, nucleic acids are directed against the damage‐associated molecular pattern (DAMP) molecule, High Mobility Group B1 (HMGB1). We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. To enhance the ex vivo stability of 4WJs, the helical termini of a DNA junction are modified to generate the ODN, i‐J1. The objective of this study is to investigate the nuclease resistance of i‐J1, a control 4WJ (J1) and the intramolecular 4WJ, J4 against three nucleases. The three nucleases are: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I cleaves dsDNA and ssDNA in a largely non‐specific manner to generate mono‐ and oligodeoxyribonucleotides. Exo III has expanded functionally; the enzyme acts as a 3′®5′ exonuclease, 3′‐repair diesterase, 3′‐phosphomoesterase and ribonuclease. Here, we focus on the 3′®5′ exonuclease activity of Exo III. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction to release mono‐, di‐, tri‐nucleotides and oligonucleotides. The nuclease protection data shows that i‐J1 and J4 have significantly higher nuclease than J1 against DNase I and Exo III. We suspect the increase in nuclease resistance in linked to changes in the minor groove dimensions of i‐J1 and J4. A binding model is presented to explain the increase in nuclease resistance of i‐J1 and J4.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.