RNA-RNA and RNA-DNA ligation with the sTobRV(+) hammerhead ribozyme

The sTobRV(+) ribozyme consists of a small catalytic domain and two wing sequences(1). By changing its wing sequences, the ribozyme can cleave many different RNAs in a site-specific manner, functioning as an RNA restriction enzyme(1). Although relatively strong ligase activity is known to be associated with sTobRV(+) RNA(2,3), the sTobRV(+) ribozyme itself has been claimed to have no ligase activity. Here, we show the evidence that the sTobRV(+) ribozyme has the ability to rejoin its digestion products at low temperatures such as 4 degrees C. In contrast, little or no ligation product can be produced at 50 degrees C, the temperature giving the maximum digestion activity. The ligation reaction requires Mg++ ion. The first substrate (P1, see Fig.1), possessing 2',3' cyclic phosphate, must be RNA, but the second substrate (P2), required to have 5'OH, can be replaced by DNA counterparts, equal to or longer than dimer, thus making it possible to generate RNA-DNA chimeric molecules. We also show the resultant RNA-DNA chimera to be digestable by the sTobRV(+) ribozyme. RNase digestion indicates the phosphodiester linkage thus generated to be exclusively 3'-5'.