Bridging the Gap between Proteins and Nucleic Acids:  A Metal-Independent RNAseA Mimic with Two Protein-Like Functionalities

Perrin, David M.; Garestier, Thérèse; Hélène, Claude

doi:10.1021/ja003290s

Cited by 149 publications

(122 citation statements)

References 51 publications

(75 reference statements)

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“…This has demonstrably translated into considerable improvements in target binding affinity as well as expansion of protein targets accessible to SELEX (9). The incorporation of various functional groups into nucleic acids has also been used to enhance nucleic acid-mediated catalysis (41,42). The cocrystal structures described in this report provide a glimpse into the astonishing array of unique structural themes that can be achieved with modifications to standard nucleotide chemistry.…”

Section: Discussionmentioning

confidence: 96%

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

Davies¹,

Gelinas

Zhang

et al. 2012

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

205

232

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Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional proteinbinding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.ince the advent of SELEX (Systematic Evolution of Ligands by EXponential enrichment) 22 years ago (1, 2), aptamers have been described that bind specifically and with high affinity to many different types of targets, including proteins, peptides, and small molecules (3). Binding interactions between aptamers and their targets are characterized by shape complementarity, polar contacts, hydrogen bonding interactions, and chargecharge interactions (3-5). Other than base stacking interactions, hydrophobic contacts, which are known to make key contributions to protein-protein interactions (6-8), have been notably limited, reflecting the lack of such moieties in nucleic acid libraries typically used in SELEX.We have recently shown that augmenting the diversity of randomized libraries with functional groups absent in natural nucleic acids can dramatically improve the success rate of SELEX, especially against difficult protein targets (9, 10). We have named this unique class of binding reagents SOMAmers (Slow Off-rate Modified Aptamers), to account for their distinct composition and binding properties. Among the different types of modifications we have tested, functional groups with hydrophobic character have typically yielded SOMAmers with the highest binding affinity. Although the contribution of such functional groups to the outcome of SELEX experiments has been quite apparent (9), the structural basis for the effect of these "side chains" on folding and binding has been unclear. Here, we report two cocrystal structures of related SOMAmers bound to a protein target, platelet-derived growth factor B (PDGF-BB), solved at a resolution of 2.2 Å and 2.3 Å. The structures elucidate the striking impact of the hydrophobic aromatic functional groups in creating novel intramolecular motifs and their extensive participation in shaping the contact surface with the native protein. By combining nucleic acid secondary st...

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Section: Discussionmentioning

confidence: 96%

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

Davies¹,

Gelinas

Zhang

et al. 2012

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

205

232

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“…Therefore, the modified DNA, in which many kinds of functionalities are incorporated, can be synthesized more efficiently compared with the previously described method of direct PCR amplification of modified DNA. Indeed, this has enabled SELEX to be used with libraries of doubly or triply modified DNA prepared using PEX [Perrin et al, 2001;Sidorov et al, 2004;Hollenstein et al, 2009].…”

Section: Polymerase Reaction Involving Modified Nucleotidesmentioning

confidence: 99%

Nucleic Acid Aptamers as Molecular Tags for Omics Analyses Involving Sequencing

Kuwahara¹,

Sugimoto²

2012

DNA Sequencing - Methods and Applications

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“…5-(3-Aminoallyl) Deoxyuridine * Sequenase 2.0 DNA Polymerase * RNase DNAzyme, sequence directed (Perrin et al, 2001) This RNase DNAzyme contains both the 8-[2-(4-Imidazolyl)ethylamino] deoxyadenosine and the 5-(3-aminoallyl) deoxyuridine modified nucleotides and selfcleaved the internal rC. This is the first example of a metalindependent DNAzyme (Perrin et al, 2001).…”

Section: Literature Citedmentioning

confidence: 99%

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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Bridging the Gap between Proteins and Nucleic Acids: A Metal-Independent RNAseA Mimic with Two Protein-Like Functionalities

Cited by 149 publications

References 51 publications

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets

Nucleic Acid Aptamers as Molecular Tags for Omics Analyses Involving Sequencing

In Vitro Selection Using Modified or Unnatural Nucleotides

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