Pieken W A, Olsen D B, Benseler F, Aurup H, Eckstein F
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Science. 1991 Jul 19;253(5017):314-7. doi: 10.1126/science.1857967.
The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.
通过自动化学合成将2'-氟核苷酸和2'-氨基核苷酸掺入锤头状核酶中。2'-氟尿苷、2'-氟胞苷或2'-氨基尿苷的存在并未显著降低催化效率。掺入2'-氨基胞苷使核酶活性降低约20倍。在检测限内，用2'-氟腺苷取代所有腺苷在MgCl₂存在下会消除催化作用，但在MnCl₂存在下仍保留一些活性。这种对催化的影响局限于核酶保守单链区域内特定的腺嘌呤基团。催化效率的降低是由速率常数的降低引起的；米氏常数未改变。2'-氟和2'-氨基修饰赋予了对核糖核酸酶降解的抗性。与未修饰的核酶相比，在所有尿苷和胞苷位置含有2'-氟核苷酸或2'-氨基核苷酸的核酶在兔血清中抗降解能力提高了至少10³倍。
