A series of 10–23 deoxyribozymes (D2–D9) containing single amino-acid-bearing nucleosides (thr6dA, hisam6dA, hisam5dU and ncmnm5dU) at positions 4, 5, 8 or 15 of the catalytic core was obtained by chemical synthesis. The deoxyribozymes were screened for their catalytic efficiency, and in the presence of 1 mM Mg2+ two of them, containing at position 8 either hisam5dU (D8) or ncmnm5dU (D9), were found to be RNA nucleases several times more active than their non-modified precursor. Moreover, in the magnesium-free TRIS or PIPES buffers, these enzymes were able to catalyze the cleavage of the phosphodiester bond located between the 5′-GpU-3′ sequence of the complementary RNA substrate. The cleavage reaction proceeded with the highest efficiency at pH > 7.