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Evaluation of the nuclease resistance of intramolecular DNA four‐way junction composed of phosphorothioate bonds
Author(s) -
Klein Mitchell,
Parker Elliott,
Troisi Maria,
Smith Samara,
Wade Elizabeth,
Moorehead Gaston,
Bell Anthony Jerome
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.471.11
Subject(s) - nuclease , exonuclease , dna , endonuclease , exonuclease iii , chemistry , oligonucleotide , microbiology and biotechnology , biology , biochemistry , biophysics , gene , dna polymerase , escherichia coli
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 target proteins to control gene expression. One strategy uses double stranded DNA as “decoy” sequences that are directed against transcription factors such as NF‐kB and specificity protein 1. More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, cruciform and bent DNA are directed against the damage‐associated molecular pattern molecule, High Mobility Group B1 (HMGB1). We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of immobilized 4WJs for ex vivo studies, the helical termini are modified to generate the intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the 4WJ lattice to enhance nuclease resistance. In this study, the nuclease resistance of five intramolecular 4WJs and the DNA control, J1 are evaluated. J1 is a classic immobilized 4WJ that is composed of four asymmetric strands of DNA. Three nucleases are used: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I is an endonuclease that cleaves dsDNA and ssDNA in a largely non‐specific manner. Exo III has expanded functionally. Here, we focus on the 3′®5′ exonuclease activity of the enzyme. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction. The nuclease protection data shows that each intramolecular 4WJ has significantly higher nuclease resistance values than J1 against DNase I and Exo III. Circular dichroism studies are being developed to investigate the minor groove binding properties of J1 vs . the intramolecular 4WJs. We hypothesize that perturbations within the minor groove of intramolecular 4WJs may enhance the nuclease resistance of these constructs vs . J1. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .