Optimization and optimality of a short ribozyme ligase that joins non-Watson–Crick base pairings

Robertson, Michael P.; Hesselberth, Jay R.; Ellington, Andrew D.

doi:10.1017/s1355838201002199

Cited by 37 publications

(44 citation statements)

References 29 publications

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“…As expected, most of the individual sequences were not Rev ARM peptide dependent. Instead, there were multiple, apparently inactive variants, and two copies of a highly active variant (8-1) that was nearly identical to a previously isolated ribozyme, JH-1 (Robertson et al 2001) except for a single point mutation (G40A) that converted a U:G wobble to a U:A base pair. Only one Rev ARM peptide-dependent aptazyme (8-4) was identified.…”

Section: Sequence and Activity Of A Peptide-dependent Aptazymementioning

confidence: 96%

“…We have previously selected protein-dependent aptazyme ligases from a random sequence pool based on the L1 ligase (Robertson and Ellington 2001). To generate peptide-dependent ligases, a similar protocol was developed (Fig.…”

Section: In Vitro Selection Of Peptide-dependent Aptazymesmentioning

confidence: 99%

“…Methods have now been developed for the selection of effector-dependent ribozymes, or aptazymes, from random sequence pools (Koizumi et al 1999;Wang et al 2002), and the resultant effector activations have been extraordinary. A selected cGMP-dependent hammerhead variant was activated by 5000-fold (Koizumi et al 1999), and a tRNA synthetase-dependent L1 ligase variant was activated 94,000-fold (Robertson and Ellington 2001). Moreover, these ribozymes have proven to be quite specific for their effectors, and can discriminate between small molecules based on single hydroxyl moieties or between related protein effectors.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, peptide-dependent aptazymes could potentially be used as diagnostic or laboratory reagents (Frauendorf and Jaschke 2001). An advantage of aptazymes over conventional diagnostic reagents, such as antibodies, are that they can signal in homogenous solution without the need for multiple processing steps (Tang and Breaker 1997;Robertson and Ellington 2001). In addition, to the extent that peptide-dependent aptazymes could also recognize protein targets, in vitro selections that targeted peptide epitopes might prove to be a ready means for generating aptazymes against multiple protein targets.…”

Section: Introductionmentioning

confidence: 99%

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In vitro selection of ribozymes dependent on peptides for activity

Robertson¹,

Knudsen²,

Ellington

2003

RNA

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A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide-and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

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Section: Sequence and Activity Of A Peptide-dependent Aptazymementioning

confidence: 96%

Section: In Vitro Selection Of Peptide-dependent Aptazymesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In vitro selection of ribozymes dependent on peptides for activity

Robertson¹,

Knudsen²,

Ellington

2003

RNA

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“…7, which is published as supporting information on the PNAS web site)]. In contrast, the previously reported ligases with the exception of class I can use only limited combinations of the two substrate nucleotides at the reaction site (14,(24)(25)(26).…”

Section: ϫ3mentioning

confidence: 99%

De novo synthesis and development of an RNA enzyme

Ikawa

Tsuda²,

Matsumura³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

117

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Arbitrary manipulation of molecular recognition at the atomic level has many applications. However, systematic design and de novo synthesis of an artificial enzyme based on such manipulation has been a long-standing challenge in the field of chemistry and biotechnology. In this report, we developed an artificial RNA ligase by implementing a synthetic strategy that fuses a series of 3D molecular modelings based on naturally occurring RNA-RNA recognition motifs with a small-scale combinatorial synthesis of a modular catalytic unit. The resulting ligase produces a 3-5 linkage in a template-directed manner for any combinations of two nucleotides at the reaction site. The reaction rate is 10 6 -fold over that of the uncatalyzed reaction with a yield higher than those of previously reported ligase ribozymes. The strategy may be applicable to the synthesis and development of a variety of nonnatural functional RNAs with defined 3D structures. Many RNA receptors and enzymes have been selected in vitro from combinatorial libraries that consist of a long random sequence (1). Anatomies of the selected RNAs are difficult to predict because their higher-order structures were not specified before the selection. However, if a precisely designed assemblage of well established modular units is used as a scaffold for preparing such functional RNAs, redesign of the resulting selected RNAs will be facilitated. They will thus enable a variety of artificial evolutionary pathways, starting from the selected molecule.In general, molecular design of biopolymers such as RNAs or proteins is difficult at the 3D level because of their highly complicated folding process. In the 1990s, biochemical and structural analyses revealed that many functional noncoding natural RNAs are organized into modules and fold into defined 3D structures (2-5). Moreover, several commonly used RNA-RNA binding motifs in these RNAs were identified by phylogenetic comparison (6) and high-resolution structural analyses (7-10). Consequently, it has become possible to design self-folding RNAs precisely by employing such motifs and mimicking the modular organization of natural RNAs (11-13). As one such example, we have previously reported the design of a self-folding RNA consisting of standard doublestranded helices connected by the two motifs: a tetraloopreceptor interaction and consecutive base-triples ( Fig. 1 A and B) (13). Results indicated that the constructed RNA folds compactly into the designed 3D structure.In this paper, we report the synthesis and development of an artificial RNA ligase as shown in the scheme (Fig. 1 A). First, a reaction site for RNA-RNA ligation was installed into the designed RNA scaffold, and a different region of the RNA was subject in vitro selection from a small combinatorial library to provide a catalytic center. The ligation reaction was chosen as a convenient target because several RNA ligases had already been obtained from large-scale pools by in vitro selection (ref. 14 and references cited therein). Biochemical characterization a...

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In Vitro Selection Using Modified or Unnatural Nucleotides

Knudsen

Robertson

Ellington

2001

CP Nucleic Acid Chemistry

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Incorporation of modified nucleotides into in vitro RNA or DNA selections offer many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions.In vitro selection is the process by which a pool of nucleic acids is enriched via iterative selection and amplification for those species that are capable of performing a particular task. Nucleic acids have been selected that bind to particular targets (aptamers), catalyze reactions (ribozymes or deoxyribozymes), or act as molecular switches (aptazymes). Similarly, nucleic acids have been found in nature that control gene expression upon binding an analyte (riboswitches).Instructions for carrying out in vitro selection experiments have been detailed elsewhere in this chapter (i.e. UNITS 9.3, 9.4, and 9.5). This unit augments these units by describing how modified nucleotides can potentially be incorporated into a selection. It is strongly recommended that the researcher be conversant with a "normal" in vitro selection experiment prior to attempting selections with modified nucleotides. A normal in vitro selection experiment is already fraught with problems and pitfalls, and the addition of modified nucleotides adds an extra level of difficulty. For simplicity, this unit focuses on in vitro selection using RNA pools; however, similar procedures can be used for DNA pools.CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer. NOTE:Experiments involving RNA require careful precautions to prevent contamination and degradation by RNases (see APPENDIX 2A). The water used to make all solutions and NIH Public Access Author ManuscriptCurr Protoc Nucleic Acid Chem. Author manuscript; available in PMC 2015 March 26. Published in final edited form as:Curr Protoc Nucleic Acid Chem. ; 56: 9.6.1-9.6.33. doi:10.1002/0471142700.nc0906s56. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript buffers should be RNase free or treated with diethylpyrocarbonate (DEPC; APPENDIX 2A). Use sterile, disposable plasticware where possible. STRATEGIC PLANNING Advantages of Using Modified Nucleotides in In Vitro S...

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Optimization and optimality of a short ribozyme ligase that joins non-Watson–Crick base pairings

Cited by 37 publications

References 29 publications

In vitro selection of ribozymes dependent on peptides for activity

In vitro selection of ribozymes dependent on peptides for activity

De novo synthesis and development of an RNA enzyme

In Vitro Selection Using Modified or Unnatural Nucleotides

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