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Structural Aspects of Nucleic Acid Analogs and Antisense Oligonucleotides

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Structural Aspects of Nucleic Acid Analogs and Antisense Oligonucleotides REVIEWS Structural Aspects of Nucleic Acid Analogs and Antisense Oligonucleotides Martin Egli" Hybridization of complementary launch an extensive search for oligonu- tions at selected sites in quantities suit- oligonucleotides is essential to highly cleotide analogs with improved binding able for three-dimensional structure in- valuable research tools in many fields properties for hybridization with RNA vestigations. Such studies should reveal including genetics, molecular biology, and higher resistance toward nuclease the structural origins of the observed and cell biology. For example, an anti- degradation. During the last years this changes in affinity and specificity of sense molecule for a particular segment research has resulted in a flurry of new binding for particular modifications and of sense messenger RNA allows gene ex- chemical analogs of DNA and RNA may guide the development of second- pression to be selectively turned off, and with modifications in the sugar-phos- and third-generation antisense mole- the polymerase chain reaction requires phate backbone as well as in the nucleo- cules. In addition, the availability of a complementary primers in order to pro- base sites. However, to date little effort previously unimaginable variety of ceed. It is hoped that the antisense ap- has been directed toward uncovering the modified building blocks and the inves- proach may lead to therapeutics for exact origins of the gain or loss in stabil- tigation of their structures provides the treatment of various diseases including ity when nucleic acid analogs bind to basis for a deeper understanding of the cancer. Areas of active research in the RNA. Although large amounts of native DNA and RNA structures. This antisense field focus on the mechanisms thermodynamic data have been collect- contribution will summarize the results of cellular uptake of antisense molecules ed, the structural perturbations induced of X-ray crystallographic structure de- and their delivery to specific cell sites, an by the modifications in hybrid duplexes terminations of modified nucleic acid improved understanding of how these are only poorly understood. For many fragments conducted in our laboratory molecules inhibit the production of modified oligonucleotides the compati- during the last three years and the in- proteins. as well as the optimization of bility of protection, coupling, and de- sights gained from them. the chemical stability of antisense mole- protection chemistry with standard cules and the thermodynamic stability DNA and RNA synthesis protocols Keywords: crystal structure analysis of the duplexes they form with the makes it now possible to generate modi- DNA - hydrogen bonding - RNA mRNA targets. The last two issues in fied nucleic acid fragments or mixed particular have prompted chemists to oligonucleotides containing modifica- 1. Introduction and viruses hybridization between sense RNA and antisense RNA is a common mechanism for regulation as well as inhibi- The precisely ordered strings formed by the four nucleotide tion of gene activity. Both replication and transcription were bases adenine (A). thymine (T), guanine (G), and cytosine (C), shown to be controlled by such mechanisms in prokaryotes." -41 and the formation of complementary, hydrogen-bonded A-T In order to control replication of the E. coli plasmid ColEl , a and G-C base pairs provide the foundation for gene structure sense -antisense RNA complex is formed between two comple- and expression. In chromosomes the DNA usually exists in the mentary loop structure^!^' These initial observations of a nat- form of an antiparallel double helix composed of the sense ural mechanism to suppress genetic information via nucleic strand and the antisense strand. Pairing specificity is the under- acids were followed by a decade of intense exploration of the lying principle of all biological information transfer-replica- possibilities and limits of the antisense approach. tion, recombination, transcription, and translation. In bacteria Antisense RNA need not necessarily be administered through expression vectors, but can be injected into cells in the form of [*] Prof M Eglt short synthetic oligonucleotides. In addition, it was first shown Department of Molecular Pharmacology and Biological Chemistry Northwestern Uniwrsiry Medical School by Zamecnik and coworkers that DNA can also be utilized to 303 &st Chicago Avenue effectively inhibit protein expre~sion.~'. Messenger RNA in Chicago. 11. 6061 1-3008 (USA) Fax: Int. code +(312)503-0796 the unprocessed and processed state is not the only possible e-mil I 1 . in-egl I (n 11 w u .ed u target; interference with the protein expression pathway can in A?f::w ('hcwi. 1171. C/.En~ql. 1996. 35, 1894- 1909 C= VCH VerlaRsgeseIlschrrfi mbH, 0-69451 Wernherm. 1996 0570-0~3333:9613517-1K95J I5.OOi .3'0 1895 REVIEWS M. Egli principle also occur at the gene level. However, this approach is amides.['"] and ~ulfones.['~~The analogs with modifications on thus far restricted largely to homopurine target sites that allow the sugar portion comprise carbocyclic compounds,[5h.571 L-2'- triplex formation with the antigene strand.["- "I More effective deoxyriboses.["I and hexoses.["- ''I Oligonucleotides in which control of gene expression may be exhibited by antisense RNAs the pyrimidine rings have propyne substituents at C5 display with functional properties, for example trcins-acting hammer- promising features for antisense applications.'62J and the pep- head ribozymes that can cleave a target message after the se- tide nucleic acids (PNAs) introduced in 1994 are the analogs quence UH (H = A, C. or U) and will thus not only inhibit but with the highest divergence from the native nucleic acid~.'~~J actively degrade the sequence."'. 131 The abundant number of modifications is in stark contrast to The experience gathered in various areas of the antisense field the handful of detailed structural studies of analogs using either during the last years has indicated several critical points that solution NMR or modeling methods. Among those analyzed must be addressed should the antisense technology become ap- are amide-linked DNA.Ih4.('51 formacetal DNA,Ih6.671 thioform- plicable in the treatment of human diseases. These obstacles for acetal DNA.Ih8' urea-linked DNA,[6y1phosphorodithioate antisense drug development will only be briefly addressed here; DNA."'] 2'-O-methyl RNA,L7'I 2'-5'-linked RNA,["] a PNA- most aspects have been exhaustively treated in a series of excel- RNA and a PNA-DNA In addition. lent recent reviews and book^.^'^-^^] Potential pitfalls to suc- structural investigations conducted with PNAs include the crys- cessful application of antisense therapeutics are the often low tal structure of a triple-stranded nucleic acid complex, consist- cellular uptake and the insufficient chemical stability of natural ing of a homopurine DNA nonamer and two homopyrimidine and chemically modified oligonucleotides against degradation PNA nonamers, in which the latter strands are linked through by various nucleases. Once resistant against nucleases and effi- six amino Much more structural work will be neces- ciently delivered, a modified oligonucleotide may not sufficient- sary to interpret the thermodynamic stability data of oligonucleo- ly discriminate between a chosen target and other sites. Further tide modifications, and to begin to understand the complex complications can arise from the fact that most of the modified interplay between enthalpic and entropic effects, including fac- DNAs and RNAs do not induce RNaseH-mediated cleavage of tors such as conformational preorganization, hydration, salt mRNA,[341that folding of cellular RNAs into secondary and concentration. and cooperativity.I7'. 771 tertiary structures may render the selected target sites inacces- Beyond the search for therapeutically feasible antisense com- sible, and that the antisense strand and target may not be found pounds, recent progress in the chemical alteration of DNA and in the same location within the cell. RNA provides numerous opportunities to probe nucleic acid It is anticipated that appropriate chemical modification of structure[78.7y1 as well as interactions between proteins and oligonucleotides may help overcome some of these problems. In nucleic particular, modifications may lead to improved hybridization properties, nuclease resistance, and cell permeation. The struc- tural framework of DNA and RNA allows a multitude ofchem- 2. The Structures of DNA and RNA ical modifications on the phosphate backbone and on the sugar and base moieties. First-generation analogs based on these RNA differs from DNA by the additional hydroxyl group at three constituents include the phosphor~thioates,[~~.~~lthe the 2'-position in the ribose. This seemingly subtle difference has methylphosphonate~,[~~.381 and a range of 02'-modified important consequences for the chemical and structural proper- oligonucIeotide~.[~~-~~~The current variety of analogs is over- ties of RNA and its biological role. DNA is normally a double- whelming, and the interested reader is referred to other sources stranded molecule, while RNA is single-stranded and can adopt such as the previously mentioned ~eviews.I~~-~~] complex folding motifs with a range of structural elements such Among the newer generation of backbone modifications are as double-helical stems. bulges (with unpaired bases), hairpin 3'-allylethers and 3'-alIylsulfide~,[~~~amide~,[~~] amine~.[~'] car- loops, and pseudoknots.'811 DNA has been observed in two
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