RESUMO
Understanding how oxidatively damaged RNA interacts with ribonucleases is important because of its proposed role in the development and progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers, is fundamental. We explored the reactivity of RNase A, RNase T1, and RNase H toward oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful for understanding (1) how these RNases can be used to characterize the structural impact that this lesion has on RNA and (2) how oxidatively modified RNA may be handled intracellularly. 8-OxoG was incorporated into 10-16-mers of RNA, and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and the use of other C8-purine-modified analogues (8-bromoguanosine, 8-methoxyguanosine, and 8-oxoadenosine). RNase T1 does not recognize sites containing 8-oxoG, while RNase A recognizes and cleaves RNA at positions containing this lesion while differentiating if it is involved in H-bonding. The selectivity of RNase A followed the order C > 8-oxoG ≈ U. In addition, isothermal titration calorimetry showed that an 8-oxoG-C3'-methylphosphate derivative can inhibit RNase A activity. Cleavage patterns obtained from RNase H displayed changes in reactivity in a sequence- and concentration-dependent manner and displayed recognition at sites containing the modification in some cases. These data will aid in understanding how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA.
Assuntos
Oligonucleotídeos/genética , Ribonuclease H/genética , Ribonuclease T1/genética , Ribonuclease Pancreático/genética , Dicroísmo Circular , Guanosina/análogos & derivados , Guanosina/química , Guanosina/genética , Humanos , Oligonucleotídeos/química , Estresse Oxidativo/genética , RNA/química , RNA/genética , Ribonuclease H/química , Ribonuclease T1/química , Ribonuclease Pancreático/química , Ribonucleases/química , Ribonucleases/genética , Especificidade por SubstratoRESUMO
Dodecamers of RNA [CUACGGAAUCAU] were functionalized with C2'-O-2-thiophenylmethyl groups to obtain oligonucleotides 10-14 and 17. The modified nucleotides were incorporated into RNA strands via solid-phase synthesis. The biophysical properties of these ONs were used to quantify the effects of this modification on RNA:RNA and RNA:DNA duplexes. A combination of UV-vis and circular dichroism were used to determine thermal stabilities of all strands, which hybridized into A-form geometries. Destabilization of the double stranded RNA was measured as a function of number of consecutive modifications, reflected in decreased thermal denaturation values (ΔTm, ca. 2.5-11.5 °C). Van't Hoff plots on a duplex containing one modification (10:15) displayed a ca. ΔΔG° of +4 kcal/mol with respect to its canonical analogue. Interestingly, hybridization of two modified strands (13:17, containing a total of eight modifications) resulted in increased stability and a distinct secondary structure, reflected in its CD spectrum. Molecular modeling based on DFT calculations shed light on the nature of this stability, with induced changes in the torsional angle δ (C5'-C4'-C3'-O3) and phosphate-phosphate distances that are in agreement with a compacted structure. The described synthetic methodology and structural information will be useful in the design of thermodynamically stable structures containing chemically reactive modifications.
