A Packing‐Density Metric for Exploring the Interior of Folded RNA Molecules

Schwans, Jason P.; Li, Nan‐Sheng; Piccirilli, Joseph A.

doi:10.1002/anie.200353575

Cited by 11 publications

(10 citation statements)

References 32 publications

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“…To demonstrate the utility of the new deoxyribozymes for synthesis of biologically derived RNAs, we used 7DE5 to prepare the Tetrahymena group I intron P4−P6 domain, a representative and often-studied RNA. ,, As shown in Figure A, synthesis of P4−P6 by 7DE5 was readily achieved in good yield, even though P4−P6 is completely unrelated to the short RNA substrates that were used during the selection procedure that led to the identification of 7DE5. The P4−P6 synthesized by 7DE5 was shown conclusively to have a native 3‘−5‘ linkage at the ligation junction created by the deoxyribozyme (Figures S6 and S7), and the synthetic P4−P6 folds like wild-type P4−P6 as assayed by nondenaturing PAGE (Figure B). , To further demonstrate ligation generality, we used both 9DB1 and 7DE5 to prepare the 72-nucleotide core of the xpt G-riboswitch (Figure C).…”

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General Deoxyribozyme-Catalyzed Synthesis of Native 3‘−5‘ RNA Linkages

et al. 2005

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Deoxyribozymes (DNA enzymes) are DNA catalysts for a variety of chemical reactions that typically involve nucleic acid substrates. 1 Our laboratory has focused on the in vitro selection of DNA enzymes for RNA ligation. 2 A highly challenging goal has been deoxyribozymes that synthesize native 3′-5′ RNA linkages rapidly and in high yield for a wide variety of RNA sequences, rather than for only a limited set of substrates. 2f We recently described a selection strategy that favors native RNA ligation by incorporating a stringently 3′-5′-selective step into the selection rounds. 2h We have now applied this strategy to identify two RNA ligase deoxyribozymes that rapidly form high yields of 3′-5′ linkages with modest sequence requirements for the two RNA substrates, thereby fulfilling all requirements for useful RNA ligase reagents. Because RNA ligation by protein enzymes 3 does not always provide acceptable yields, 4,5 the identification of general DNA enzymes for 3′-5′ RNA ligation enables alternative synthetic routes that will be useful for practical biochemistry.Our recently described selection methodology 2a was used to identify deoxyribozymes that join a 2′,3′-diol to a 5′-triphosphate (Figure 1). Previously, such ligations led to 2′,5′-branched RNA by reaction of an internal 2′-hydroxyl group, 2b,e or they led to linear 3′-5′ RNA but with restrictive and impractical sequence requirements. 2f Here, 3′-5′ selectivity during ligation was enforced by incorporating the RNA-cleaving 8-17 deoxyribozyme 6 into the selection procedure, 2h starting at either round 2 (for selections using 40 mM Mg 2+ ) or round 5 (for selections using 1 mM Zn 2+ ). In both cases, >95% of the ligation products from each uncloned selection pool had 3′-5′ linkages ( Figure S1). When the ligation activities had stopped increasing, individual deoxyribozymes were cloned. On the basis of a preliminary survey of activities, two clones, named 9DB1 (from round 9 of the Mg 2+ selection) and 7DE5 (from round 7 of the Zn 2+ selection) were examined further. By cleaving the ligation products from each of the two new deoxyribozymes with 8-17, which is highly selective for 3′-5′ RNA linkages, 2a both 9DB1 and 7DE5 were verified to create 3′-5′ linkages ( Figure S1).The 9DB1 and 7DE5 deoxyribozymes ligate RNA under practical in vitro incubation conditions. As shown in Figure 2, 9DB1 provides 60-70% yield of ligated RNA with k obs ∼ 0.04 min -1 (t 1/2 ∼ 15 min) at 40 mM Mg 2+ , pH 9.0, and 37°C. Similarly, 7DE5 has 40-50% yield of ligated RNA with k obs ∼ 0.02 min -1 (t 1/2 ∼ 30 min) at 1 mM Zn 2+ , pH 7.5, and 23°C. The 9DB1 deoxyribozyme is also effective at pH 7.5, where k obs is ∼0.2 h -1 (t 1/2 ∼ 4 h; data not shown). Incubation at the lower pH value of 7.5 instead of 9.0 should be useful for synthesis of larger RNAs that may experience more nonspecific degradation during an overnight incubation period, particularly at higher pH.In the selection design that led to 9DB1 and 7DE5, only one RNA nucleotide of each substrate was not base-paired with ...

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General Deoxyribozyme-Catalyzed Synthesis of Native 3‘−5‘ RNA Linkages

et al. 2005

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“…Additionally, G NHMe and G NMe 2 may serve as useful analogues to define the functional contribution of hydroxyl groups by application of Quantitative Structure Activity Relationship (QSAR) analysis 2g or to evaluate the packing density around individual hydroxyl groups. 4a,22 .…”

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confidence: 99%

Synthesis of 2′-N-Methylamino-2′-deoxyguanosine and 2′-N,N-Dimethylamino-2′-deoxyguanosine and Their Incorporation into RNA by Phosphoramidite Chemistry

et al. 2011

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The 2′-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2′-substituents, including −OCH3, −NH2, −NHMe, and −NMe2, to identify hydroxyl groups within RNA that donate functionally significant hydrogen bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other guanosines whose 2′-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2′-methylamino-2′-deoxyguanosine (GNHMe) and 2′-N,N-dimethylamino-2′-deoxyguanosine (GNMe2) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved SN2 displacement of 2′-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the GNMe2 phosphoramidite and incorporated it into RNA. However, the GNHMe phosphoramidite posed a significantly greater challenge due to lack of a suitable -2′-NHMe protecting group. After testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2′-N-methylamino group during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine’s 2′-hydroxyl group within RNA.

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“…A measure for atomic PD in conjunction with an analysis of hydrogen bonds was used to evaluate flexible sites of the ribosomal 16S RNA identified by ultraviolet-induced photocrosslinking (15). To reveal sites of high PD biochemically, changes to the three-dimensional structure of RNA upon mutation of residues to analogs with higher volumes were investigated (16). A statistical analysis of geometric features was used to analyze the shape and packing of interface contacts in RNA–protein complexes (17,18).…”

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Voronoia4RNA—a database of atomic packing densities of RNA structures and their complexes

Ismer¹,

Rose²,

Tiemann³

et al. 2012

Nucleic Acids Research

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Voronoia4RNA (http://proteinformatics.charite.de/voronoia4rna/) is a structural database storing precalculated atomic volumes, atomic packing densities (PDs) and coordinates of internal cavities for currently 1869 RNAs and RNA–protein complexes. Atomic PDs are a measure for van der Waals interactions. Regions of low PD, containing water-sized internal cavities, refer to local structure flexibility or compressibility. RNA molecules build up the skeleton of large molecular machineries such as ribosomes or form smaller flexible structures such as riboswitches. The wealth of structural data on RNAs and their complexes allows setting up representative data sets and analysis of their structural features. We calculated atomic PDs from atomic volumes determined by the Voronoi cell method and internal cavities analytically by Delaunay triangulation. Reference internal PD values were derived from a non-redundant sub-data set of buried atoms. Comparison of internal PD values shows that RNA is more tightly packed than proteins. Finally, the relation between structure size, resolution and internal PD of the Voronoia4RNA entries is discussed. RNA, protein structures and their complexes can be visualized by the Jmol-based viewer Provi. Variations in PD are depicted by a color code. Internal cavities are represented by their molecular boundaries or schematically as balls.

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A Packing‐Density Metric for Exploring the Interior of Folded RNA Molecules

Cited by 11 publications

References 32 publications

General Deoxyribozyme-Catalyzed Synthesis of Native 3‘−5‘ RNA Linkages

General Deoxyribozyme-Catalyzed Synthesis of Native 3‘−5‘ RNA Linkages

Synthesis of 2′-N-Methylamino-2′-deoxyguanosine and 2′-N,N-Dimethylamino-2′-deoxyguanosine and Their Incorporation into RNA by Phosphoramidite Chemistry

Voronoia4RNA—a database of atomic packing densities of RNA structures and their complexes

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