Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding

Dieckmann, Thorsten; Butcher, Samuel E.; Sassanfar, Mandana; Szostak, Jack W.; Feigon, Juli

doi:10.1006/jmbi.1997.1329

Cited by 46 publications

(53 citation statements)

References 27 publications

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“…The binding affinity of aptRNA for ADP and AMP was very similar. The data partially agree with previous publications showing that the ATP-binding RNA aptamer binds ATP, ADP, AMP, and adenosine equally (26,35,40,41). Nevertheless, the binding affinity of aptRNA to ADP and AMP was lower than that for ATP (Fig.…”

Section: Resultssupporting

confidence: 92%

A Viral RNA That Binds ATP and Contains a Motif Similar to an ATP-binding Aptamer from SELEX

Shu

Guo

2003

Journal of Biological Chemistry

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The intriguing process of free energy conversion, ubiquitous in all living organisms, is manifested in ATP binding and hydrolysis. ATPase activity has long been recognized to be a capability limited to proteins. However, the presence of an astonishing variety of unknown RNA species in cells and the finding that RNA has catalytic activity have bred the notion that RNA might not be excluded from the group of ATPases. All DNA-packaging motors of double-stranded DNA phages involve two nonstructural components with certain characteristics typical of ATPases. In bacterial virus phi29, one of these two components is an RNA (pRNA). Here we report that this pRNA is able to bind ATP. A comparison between the chemically selected ATP-binding RNA aptamer and the central region of pRNA reveals similarity in sequence and structure. The replacement of the central region of pRNA with the sequence from ATPbinding RNA aptamer produced chimeric aptRNA that is able to both bind ATP and assemble infectious viruses in the presence of ATP. RNA mutation studies revealed that changing only one base essential for ATP binding caused both ATP binding and viral assembly to cease, suggesting that the ATP binding motif is the vital part of the pRNA that forms a hexamer to drive the phi29 DNApackaging motor. This is the first demonstration of a natural RNA molecule that binds ATP and the first case to report the presence of a SELEX-derived RNA aptamer in living organisms.One common feature in the assembly of all linear doublestranded (ds) 1 -DNA viruses including herpes virus, pox virus, adenovirus, and all of the linear dsDNA phages is that the lengthy viral genome is translocated with remarkable velocity into a limited space within a preformed protein shell and packaged into crystalline density (1-3). This energetically unfavorable DNA motion process is accomplished by an ATP-driven motor (4 -7). Careful scrutiny of the well studied dsDNA viruses reveals a striking commonality: all DNA-packaging motors involve two nonstructural components with certain characteristics typical of ATPases (8). For example, gpA and gpNuI of the DNA-packaging motors of -phage (9, 10) contain consensus ATP-binding domains and are involved in ATP hydrolysis. Both gp16 and gp17, which constitute the terminase of bacteriophage T4, are involved in ATP hydrolysis (11). In bacterial virus phi29, one of the nonstructural components for DNA packaging is an RNA molecule called pRNA (Fig. 1) (12). One ATP is used to package two base pairs of phage DNA of the phi29 (8) and the T 3 system (13).The phi29-encoded 120-base pRNA binds the connector and leaves the DNA-filled capsid after completing the DNA-packaging task (14). Six pRNAs form a hexagonal complex to gear the DNA-translocating machinery (14 -18). This DNA-packaging motor has been reported recently (19,20) to be the strongest nanometer motor with a stalling force of Ͼ50 picoNewtons. It stuffs the viral procapsid with DNA at an initial speed of 100 base pairs/second under the extra load. The crystal structure of one of ...

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Section: Resultssupporting

confidence: 92%

A Viral RNA That Binds ATP and Contains a Motif Similar to an ATP-binding Aptamer from SELEX

Shu

Guo

2003

Journal of Biological Chemistry

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“…Thus, multiple alterations to the sugar-phosphate backbone may be required to disrupt both opportunities for binding. In contrast, our conditions did not retrieve any sequences similar to a previously isolated RNA aptamer that binds ATP (33), as expected because this aptamer requires at least two specific 2′-hydroxyls at defined locations for detectable activity (35)(36)(37). Similarly, our selection for GTP aptamers from the MNA pool did not yield any sequences related to those identified in previous selections from an RNA pool (25,26) suggesting that 2′-hydroxyls are required for activity in these aptamers.…”

Section: Discussionsupporting

confidence: 64%

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity

Trevino

Zhang

Elenko

et al. 2011

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

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Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 1∶1 mixture of deoxy-and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy-and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.abiogenesis | genetic takeover | RNA progenitor | origin of life G iven that RNA is likely to have played a key role early in the evolution of life (1), understanding the origins of the RNAbased biosphere is a critical aspect of understanding the origin of life. The difficulties associated with the prebiotic synthesis of a macromolecule as complicated and fragile as RNA have stimulated interest in the hypothesis that RNA was preceded by simpler and/or more stable progenitor nucleic acids (2-4). The systematic exploration of nucleic acids that are structurally related to RNA has revealed that the formation of stable WatsonCrick base-paired, antiparallel duplex structures is compatible with a surprising degree of variation in the sugar-phosphate backbone (5). These studies imply that many distinct nucleic acids are in principle capable of mediating the inheritance of genetic information. On the other hand, recent advances in the prebiotic chemistry leading to the pyrimidine ribonucleotides (6) have revived the prospects of the RNA-first model, with the attendant advantage of avoiding a difficult genetic takeover. Both RNAfirst and RNA-late models assume that life began with a single, well-defined genetic polymer. Perhaps the greatest problem implicit in this assumption is the challenge of explaining the origin of pure nucleotide monomers on the early Earth. Indeed, the supp...

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“…The more informationally complex Class I aptamer has two platforms of stacked base-triples and several other base-base contacts that stabilize the core of the RNA. In contrast, the recognition bulge of the Sassanfar ATP aptamer has no base-triples and only two stabilizing base-base interactions (Dieckmann et al 1996(Dieckmann et al , 1997Jiang et al 1996).…”

Section: Discussionmentioning

confidence: 99%

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Carothers¹,

Davis²,

Chou³

et al. 2006

RNA

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Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K d = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K d of 6 mM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex ($45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K d . These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.

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Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding

Cited by 46 publications

References 27 publications

A Viral RNA That Binds ATP and Contains a Motif Similar to an ATP-binding Aptamer from SELEX

A Viral RNA That Binds ATP and Contains a Motif Similar to an ATP-binding Aptamer from SELEX

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

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