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G L E N R E S E A R C H A Rapid Method for Atomic Mutagenesis of Nucleic Acids I N T E R F E R E N C E M A P P I N G N ucleotide Analog Interference Mapping is a powerful new approach to perform atomic mutagenesis simultaneously, yet individually, at every position in an RNA using materials easily prepared by in vitro transcription.1 Through the use of a collection of nucleotide analogs now available from Glen Research, it is possible to assess the importance of every 2'-OH, exocyclic amine, or imino group throughout the length of an RNA. By comparing the results of multiple nucleotide analogs, it is possible to determine which ribose rings adopt unusual sugar puckers, which 2'-OH groups serve as hydrogen bond donors and/or acceptors, and which nucleotides have perturbed pKas for potential use in folding or catalysis. Furthermore, this approach can reveal which nucleotides make tertiary contacts, on which nucleotide face the contacts are made, and where allows the magnitude of an metal ions are coordinated within an interference effect at each RNA fold. position in the molecule to be quantified by simply viewing a sequencing gel of This extraordinary level of chemical detail is available the reaction products (Fig. 1A). rapidly using a collection of nucleotide analogs that modify individual functional groups on the RNA base or ribose A complete description of this approach was the subject sugar and are marked with an α-phosphorothioate linkage.2 of a previous Glen Report and can be accessed online at: This simple chemical tag makes it possible to identify the http://www.glenres.com/GlenReports/GR11.24.html sites of analog incorporation within the transcript by Since the original Report, ten additional nucleotide iodine cleavage of the phosphorothioate linkage, which analogs have been added to the nine adenosine analogue triphosphates available in the original collection. successfully studied by NAIM range from less than 50 to Additions have generated the complete set of four regular almost 1000 nucleotides in size, yet in all cases the data nucleotides and four 2’-deoxynucleotides, as well as 5- defines the contribution of a specific functional group at methyl-Uridine, 2’-deoxyUridine and Inosine. the individual nucleotide level. This rapid and inexpensive alternative to single site atomic mutagenesis has been implemented for the study of several I N T E R F E R E N C E S U P P R E S S I O N systems, including RNA folding,3 ribozyme catalysis,4-6 RNA-protein interactions,7,8 and even in vivo RNA Although NAIM provides a great deal of chemical information modification. The method can be applied to the study of about the functional importance of each nucleotide in an any nucleic acid that can be functionally selected from RNA sequence, it can be difficult to reach specific conclusions among a larger pool of less active variants. Selection about the tertiary structure from primary interference data schemes have included native gel mobility shifts, alone. While a single NAIM experiment can identify all of nitrocellulose filter binding, selective radiolabeling by RNA the important 2'-OH groups and exocyclic amines within an ligation, and size selection in a denaturing polyacrylamide RNA, it does not define how these groups interact within gel.3-8 It has been applied towards the study of catalytic the overall RNA fold. However, such information can be RNA reactivity, tRNA-synthetase interactions, mRNA obtained using a variation of NAIM, termed Interference processing, rRNA modification and snoRNA assembly. RNAs Suppression.9 This method, which combines site-specific analog substitution and interference modification suppression of 2'-deoxy and 3-deaza interference at A114 approaches, makes it possible to identify specific tertiary (red atoms in Fig. 2), while similar suppressions were hydrogen-bonding partners within an RNA structure.10 The observed exclusively at A207 if the exocyclic amine of principle behind the approach is that if an interaction is G22 was deleted (yellow atoms in Fig. 2).9 Suppression disrupted by deletion or alteration of one functional group experiments do not necessarily require single atom in an interacting pair, then no additional energetic penalty substitution, as mutation of a specific amino acid residue will result from deletion or alteration of the second in a bound protein or mutation of a nucleotide base functional group in the pair. Interference suppression is involved in a tertiary contact can also accomplish a scored by the reappearance of a specific band on the similar effect. sequencing gel which showed interference in the context of the initial NAIM experiment (Fig. 1B). Nucleic acid biochemistry has entered a new era. The high resolution crystal structure of the ribosome has revealed For example, if every RNA in the population lacks a 2'-OH the vast majority of the structural motifs available to RNA, group that is known to be important for activity, interference as well as many of the rules by which proteins interact with will be suppressed at the specific site with the specific RNA molecules11,12. The next challenge will be to assign functional group that makes a hydrogen bonding interaction biological function to the amazingly complex structures with the deleted group. This type of experiment has that nucleic acids are capable of adopting. NAIM provides provided a series of hydrogen bonding structural restraints a particularly valuable tool in this effort and Glen Research that have made it possible to construct a detailed model of is pleased to make this collection of reagents available to the group I intron active site (Fig. 2).10 Within this the scientific community for the multitude of applications catalytic RNA, deletion of the 2'-OH at G22, resulted in that can be envisioned for using them. FIGURE 2: Tertiary interactions in the group I ribozyme active site. The sets of interacting functional groups identified by interference suppression are shown in yellow and red spheres of larger radius.The sheared A·A pairs in the J4/5 segment of the active site are shown in gray and the G·U wobble pair in the P1 helix is shown in cyan.The nucleotide numbers within the Tetrahymena group I intron are also indicated. See text for discussion of specific interactions. R E F E R E N C E S : 1. Strobel, S.A. A chemogenetic approach to RNA function/ structure analysis. Curr Opin Struct Biol 9, 346-352 (1999). 2. Gish, G. & Eckstein, F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520-1522 (1988). 3. 4. 7. Batey, R.T., Rambo, R.P., Lucast, L., Rha, B. & Doudna, J.A. Crystal Structure of the Ribonucleoprotein Core of the Signal Recognition Particle. Science 287, 1232-1239 (2000). 8. Basu, S. et al. A specific monovalent metal ion integral to the A-A platform of the RNA tetraloop receptor. Nat. Struct. Biol. 5, 986-992 (1998). Vortler, C.S., Fedorova, O., Persson, T., Kutzke, U. & Eckstein, F. Determination of 2'-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study. RNA 4, 1444-1454 (1998). 9. Strobel, S.A. & Shetty, K. Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. Proc. Natl. Acad. Sci. U.S.A. 94, 2903-2908 (1997). Strobel, S.A., Ortoleva-Donnelly, L., Ryder, S.P., Cate, J.H. & Moncoeur, E. Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site. Nat. Struct. Biol. 5, 60-66 (1998). 10. Strobel, S.A. Ribozyme chemogenetics. Biopolymers 48, 65-81 (1998). 5. Boudvillain, M. & Pyle, A.M. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis. EMBO J. 17, 7091-7104 (1998). 6. Kazanstev, A.V. & Pace, N.R. Identification by modificationinterference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P. RNA 4, 937-947 (1998). GLEN RESEARCH 22825 DAVIS DRIVE STERLING, VIRGINIA 20164 1 • 703 • 437 • 6191 Fax: 1 • 703 • 435 • 9774 Internet: www.glenres.com PRINTED ON RECYCLED PAPER 11. Ban, N., Nissen, P., Hansen, J., Moore, P. & Steitz, T. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905-920 (2000). 12. Wimberly, B.T. et al. Structure of the 30S ribosomal subunit. Nature 407, 327-339 (2000).