We previously used in vitro selection to identify Mg(2+)-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5'-hydroxyl group with a 2',3'-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2'-5' phosphodiester bonds rather than native 3'-5' linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2'-5' linkages. This includes deoxyribozymes with an arrangement that favors 3'-5' linkages for a different chemical reaction, that of a 2',3'-diol plus 5'-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2'-5' RNA linkages upon opening of a 2',3'-cyclic phosphate with a 5'-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2',3'-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.