Nucleic Acids Research, 2006, Vol. 34, No. 8 2445–2454 doi:10.1093/nar/gkl276 Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection Kenny Schlosser, Jeffrey C.F. Lam and Yingfu Li* Department of Biochemistry and Biomedical Sciences and Department of Chemistry, McMaster University, Hamilton, Canada L8N 3Z5 Received March 6, 2006; Revised March 27, 2006; Accepted April 4, 2006 ABSTRACT We previously conducted an in vitro selection experi- ment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nuc- leotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short 14–15 nt catalytic DNA motif commonly known as 8–17. Herein, we report extensive truncation experi- ments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8–17 and to determine the effect of excess sequence ele- ments on the activity of this motif and the outcome of selection. Although we observed beneficial, detri- mental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2-fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activ- ity, called G15–30, was 2.5-fold larger and experi- enced a 4-fold greater inhibitory effect from exc- ess sequence elements than the average 8–17 motif. Our results suggest that 8–17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a pre- viously unappreciated selective advantage that may contribute to its widespread recurrence. INTRODUCTION In vitro selection or SELEX (1–3), is based on the fundamental assumption that functional DNA or RNA molecules can be found in a pool of random nucleic acid sequences. This assumption has been experimentally validated on numerous occasions since 1990, with the isolation of hundreds of deoxyribozyme, ribozyme and aptamer sequences. However, there are still unanswered questions regarding the distribution of functional nucleic acids in sequence space, and more importantly, how best to access them. Every new in vitro selection experiment begins with a com- binatorial library of single-stranded DNA molecules. The pre- vailing view supported by several theoretical studies is that longer libraries offer greater access to larger modular motifs with greater structural complexity (4–6), which in turn may be correlated with greater functional activity (7). However, the tangible beneﬁts of longer random libraries are somewhat ambiguous given the fact that both long and short libraries (from >200 nt to <30 nt; nt: nucleotide) have yielded func- tional molecules. Assessing the true merits of longer random libraries is made even more difﬁcult by the presence of excess sequence elements that often mask a shorter functional motif, often with equal or greater activity than the full-length pre- cursor. Examples of this scenario are abundant in the literature, suggesting that this is both a signiﬁcant and general phe- nomenon that applies to different types of reactions for both functional DNA (8–13) and RNA (14–26). The presence of excess sequence elements has been espe- cially relevant to the selection of one very well known RNA- cleaving deoxyribozyme, called 8–17. The 8–17 motif meas- ures just 14–15 nt in length, but has been repeatedly isolated in several independent selection experiments using random libraries greater than or equal to 40 nt (27–32). Previously, we conducted an in vitro selection experiment using a DNA library that featured the longest random-sequence domain (80 nt) ever used for the isolation of RNA-cleaving deoxyri- bozymes (33). Our primary motivation for choosing this length was to increase the probability of isolating a diverse collection of deoxyribozymes. For the same reason, we used several reaction times during the course of selection (from as long *To whom correspondence should be addressed. Tel: 1 905 5259140; Fax: 1 905 522 9033; Email: liying@mcmaster.ca The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org