Abstract
Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.
Highlights
The ability to detect and modify the genome of living organisms is important for the diagnosis, prevention, and treatment of many diseases [1]
Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1aminium iodide (N+ azide) or p-toluenesulfonyl azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for tosyl sulfonyl phosphoramidate (Ts) modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs)
The incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes
Summary
The ability to detect and modify the genome of living organisms is important for the diagnosis, prevention, and treatment of many diseases [1]. The site-specific targeting and manipulation of genomic DNA or RNA using chemically modified short oligodeoxynucleotides (ONs) is considered to be a viable therapeutic strategy [2,3,4,5]. Apart from strategies that use modular enzymes such as zinc-finger nucleases [6] or transcription activator-like effector nucleases (TALENs) [7] to recognise and cut DNA sequences, or CRISPR-CAS9 [8,9,10] and CAS9-constructs [11,12,13,14] which rely on large proteins to open the target duplex, triplex-forming oligonucleotides (TFOs) [15] can be designed to bind in a sequence-specific manner to double-stranded DNA (dsDNA) [16]. In forming the parallel triple-helix structure, a polypyrimidine TFO binds to dsDNA through Hoogsteen base-pairing [17], in which the cytosine bases in the TFO are protonated at the N3 atom (Figure 1B)
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