Combinatorial Mutation Interference Analysis Reveals Functional Nucleotides Required for DNA Catalysis

Wachowius, Falk; Javadi‐Zarnaghi, Fatemeh; Höbartner, Claudia

doi:10.1002/anie.201003940

Cited by 32 publications

(50 citation statements)

References 29 publications

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“…In af irst approachw ec apitalized on combinatorial mutation interference analysis( CoMA), am ethod developedi n our laboratory that reveals the essential nucleotides of any functional DNA of interest. [14] We found that only as ubset of the 39 nucleotides in the catalytic region of 6BX22 is sufficient for catalysis. The results of the CoMAa lso revealed ah ighg uanosine content in the actives equence.…”

Section: Introductionmentioning

confidence: 68%

“…CoMA is an efficient and reliable methodf or simultaneous analysiso fa ll possible point mutants of af unctionalD NA and has been used to identify the nucleotide requirementso fs everal deoxyribozymes. [9,14] The experimental strategy of CoMA consists of four steps. In the first step, four libraries of DNA mutantsa re synthesized by solid-phase synthesis.…”

Section: Combinatorial Mutation Interference Analysis (Coma)mentioning

confidence: 99%

“…Interferencev alues below 2s how that the mutation haso nly as mall influence and is accepted.I nterference valuesb etween 2a nd 5i ndicatet hat the mutation reduced the catalytic rate of the enzyme, but is not detrimental. [14] For the statistical distribution of 2'-OH-tagged mutations (i.e.,r ibonucleotides,r N) within aD NA library,m ixtures of mutantr Na nd parent deoxyribonucleotides (dN) phosphoramiditesa re used in solid-phase synthesis. For each coupling step, either the mutant rN or the parentd Ni si ncorporated.…”

Section: Combinatorial Mutation Interference Analysis (Coma)mentioning

confidence: 99%

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Functional Hallmarks of a Catalytic DNA that Makes Lariat RNA

Javadi‐Zarnaghi

Höbartner

2015

Chemistry A European J

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Catalytic DNAs, also known as deoxyribozymes, are of practical value for the synthesis of structurally or topologically complex RNAs, but little is known about the molecular details of DNA catalysis. We have investigated a deoxyribozyme that catalyzes the formation of a specific intramolecular 2',5'-phosphodiester bond to produce lariat RNA, which is an important biological intermediate in eukaryotic mRNA splicing. The results of combinatorial mutation interference analysis (CoMA) allowed us to shrink the catalytic core to 70 % of its original length and revealed that the essential part of the deoxyribozyme sequence contained more than 50 % guanosines. Nucleotide analogue interference mapping (dNAIM) and dimethyl sulfate interference (DMSi) experiments provided atomic details of individual guanosine functional groups. Additional spectroscopic experiments and structural probing data identified conformational changes upon metal-ion binding and catalysis. Overall, this comprehensive analysis of the DNA-catalyzed reaction has provided specific insights into the synthesis of 2',5'-branched RNA, and suggested the general features of deoxyribozymes that catalyze nucleic acid ligation reactions.

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Section: Introductionmentioning

confidence: 68%

Section: Combinatorial Mutation Interference Analysis (Coma)mentioning

confidence: 99%

Section: Combinatorial Mutation Interference Analysis (Coma)mentioning

confidence: 99%

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Functional Hallmarks of a Catalytic DNA that Makes Lariat RNA

Javadi‐Zarnaghi

Höbartner

2015

Chemistry A European J

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“…Now that the first crystal structure has been reported [114], surely others will follow. Höbartner’s elegant combinatorial minimization methods, which were applied to 9DB1 before its crystallization [121], will likely be invaluable for enabling crystallization of unrelated deoxyribozymes. In parallel, NMR studies of deoxyribozymes are surely possible, although none have yet been reported.…”

Section: Structural and Mechanistic Studies Of Deoxyribozymesmentioning

confidence: 99%

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

Silverman

2016

Trends in Biochemical Sciences

295

215

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The discovery of natural RNA enzymes (ribozymes) prompted the pursuit of artificial DNA enzymes (deoxyribozymes) by in vitro selection methods. A key motivation is the conceptual and practical advantages of DNA relative to proteins and RNA. Early studies focused on RNA-cleaving deoxyribozymes, and more recent experiments have expanded the breadth of catalytic DNA to many other reactions. Including modified nucleotides has the potential to widen the scope of DNA enzymes even further. Practical applications of deoxyribozymes include their use as sensors for metal ions and small molecules. Structural studies of deoxyribozymes are just beginning; mechanistic experiments will surely follow. From the first report 21 years ago, the field of deoxyribozymes has promise for both fundamental and applied advances in chemistry, biology, and other disciplines.

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“…For example, in a 40 nt long region of interest, the desired one substitution per molecule can be achieved by approximately 5% spacer incorporation. 5 To achieve this desired level of D substitution, we first examined the coupling efficiency of 1 in competition with RNA phosphoramidites (rN). We synthesized a series of pentamer oligonucleotides with single C3-spacer replacement using phosphoramidite mixtures containing 5-25% of 1.…”

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confidence: 99%

Enzymatic combinatorial nucleoside deletion scanning mutagenesis of functional RNA

Wawrzyniak-Turek

Höbartner

2014

Chem. Commun.

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We describe a general and simple method to identify catalytically and structurally important nucleotides in functional RNAs. Our approach is based on statistical replacement of each nucleoside with a non-nucleosidic spacer (C3 linker, D), followed by separation of active library variants and readout of interference effects by analysis of enzymatic primer extension reactions.Many examples of natural and artificial functional RNA motifs have been identified, including ribozymes, aptamers, riboswitches, small interfering RNAs, or protein-binding RNAs, that demonstrate the diversity of RNA functions beyond the transmission of genetic information. 1 Elucidating the structure and sequence requirements of non-coding nucleic acids is crucial for understanding their mechanisms of action. Furthermore, such knowledge facilitates design and engineering of RNA for practical applications in biomolecular chemistry and synthetic biology.Numerous biochemical and biophysical methods are used to analyse nucleic acid architectures at various levels of resolution. 2 Recent additions to the traditional repertoire are geared towards high-throughput analyses on massively parallel arrays, or combine mutations and footprinting studies with computational methods. 3,4 Our laboratory has recently demonstrated combinatorial analysis methods (CoMA, 5 dNAIM, 6 NDS 7 ) that allowed the simultaneous assessment of all possible single point mutants of deoxyribozymes, as well as the identification of catalytically important nucleotides and their functional groups. However, due to the method design, the reported approaches are only applicable for studying functional DNAs. They are based on the combinatorial solid-phase synthesis of DNA oligonucleotide libraries, in which the mutations are marked with a chemical tag: the hydroxyl group resembling the 2 0 -OH group of ribonucleotides. Mutations tagged in this way can be easily decoded by alkaline hydrolysis. This is an asset for the analysis of functional DNAs, but, for obvious reasons, the same strategy cannot be applied to RNA.In the present work we established an analogously versatile combinatorial approach for RNA that reveals functionally important nucleotides in a simple set of experiments that do not require sophisticated instrumentation. We used the threecarbon linker D as non-nucleosidic spacer unit to statistically replace (''delete'') standard ribonucleotides within functional nucleic acids (Fig. 1). Following separation of the functionally active and inactive library variants, the spacer substitutions were decoded by primer extension reactions. Analysis of the primer extension pattern allowed identification of the essential and non-essential nucleotides. We demonstrated our new approach

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Combinatorial Mutation Interference Analysis Reveals Functional Nucleotides Required for DNA Catalysis

Cited by 32 publications

References 29 publications

Functional Hallmarks of a Catalytic DNA that Makes Lariat RNA

Functional Hallmarks of a Catalytic DNA that Makes Lariat RNA

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

Enzymatic combinatorial nucleoside deletion scanning mutagenesis of functional RNA

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