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Automated and assisted RNA resonance assignment using NMR chemical shift statistics.

Aeschbacher T, Schmidt E, Blatter M, Maris C, Duss O, Allain FH, Güntert P, Schubert M - Nucleic Acids Res. (2013)

Bottom Line: Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts.The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt.The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, and Frankfurt Institute of Advanced Studies, 60438 Frankfurt am Main, Germany and Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.

ABSTRACT
The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and (1)H and (13)C chemical shifts. Statistics of resonances from regularly Watson-Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding (13)C resonances, which provides an important step toward automated structure determination of RNAs.

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1H and 13C chemical shift statistics for central nucleotides of Watson–Crick base-paired triplets in dependence of the RNA sequence displayed in form of box plots. Chemical shifts of residue i are given for a trinucleotide sequences consisting of residues i – 1, i and i + 1. Residue i is underlined. In addition, categories for nucleotides at the 5′ and 3′ terminus are displayed. The number of data points is given next to each box plot. (a) 1H chemical shifts of H6 and H8. (b) 1H chemical shifts of H5 and H1′. (c) 13C chemical shifts of base carbons C6 and C8. (d) 13C chemical shifts of C5 of uracils. (e) 13C chemical shifts of C5 of cytosines. (f) 13C chemical shifts of C1′. (g) Definition of the box plots.
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gkt665-F1: 1H and 13C chemical shift statistics for central nucleotides of Watson–Crick base-paired triplets in dependence of the RNA sequence displayed in form of box plots. Chemical shifts of residue i are given for a trinucleotide sequences consisting of residues i – 1, i and i + 1. Residue i is underlined. In addition, categories for nucleotides at the 5′ and 3′ terminus are displayed. The number of data points is given next to each box plot. (a) 1H chemical shifts of H6 and H8. (b) 1H chemical shifts of H5 and H1′. (c) 13C chemical shifts of base carbons C6 and C8. (d) 13C chemical shifts of C5 of uracils. (e) 13C chemical shifts of C5 of cytosines. (f) 13C chemical shifts of C1′. (g) Definition of the box plots.

Mentions: Chemical shift statistics of H2 and C2 were generated for each individual triplet category, for example the triplet GAU means an adenine succeeding a guanidine and preceding an uracil. Chemical shift statistics of H5, H6, H8, H1′, C5, C6, C8 and C1′ were generated for combined triplet categories in which the base type of nucleotide i + 1 is ignored, e.g. GAX, in which X can be any nucleotide. The same chemical shifts were also analysed for terminal nucleotides, e.g. for the categories 5′GX, CC3′ and 5′GGX. 1D statistical parameters for each chemical shift category were calculated using MicroCal OriginPro 8.5G (Microcal Software Inc.). The mean value, standard deviation, cluster size, skewness, minimum value, 25th percentile (1st quartile), median, 75th percentile (3rd quartile) and maximum value for each category are given in Supplementary Table S2. 1D statistical values are represented by box plots displaying the minimum, 5th percentile, 25th percentile, median, mean, 75th percentile, 95th percentile and maximum value (Figure 1).Figure 1.


Automated and assisted RNA resonance assignment using NMR chemical shift statistics.

Aeschbacher T, Schmidt E, Blatter M, Maris C, Duss O, Allain FH, Güntert P, Schubert M - Nucleic Acids Res. (2013)

1H and 13C chemical shift statistics for central nucleotides of Watson–Crick base-paired triplets in dependence of the RNA sequence displayed in form of box plots. Chemical shifts of residue i are given for a trinucleotide sequences consisting of residues i – 1, i and i + 1. Residue i is underlined. In addition, categories for nucleotides at the 5′ and 3′ terminus are displayed. The number of data points is given next to each box plot. (a) 1H chemical shifts of H6 and H8. (b) 1H chemical shifts of H5 and H1′. (c) 13C chemical shifts of base carbons C6 and C8. (d) 13C chemical shifts of C5 of uracils. (e) 13C chemical shifts of C5 of cytosines. (f) 13C chemical shifts of C1′. (g) Definition of the box plots.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794610&req=5

gkt665-F1: 1H and 13C chemical shift statistics for central nucleotides of Watson–Crick base-paired triplets in dependence of the RNA sequence displayed in form of box plots. Chemical shifts of residue i are given for a trinucleotide sequences consisting of residues i – 1, i and i + 1. Residue i is underlined. In addition, categories for nucleotides at the 5′ and 3′ terminus are displayed. The number of data points is given next to each box plot. (a) 1H chemical shifts of H6 and H8. (b) 1H chemical shifts of H5 and H1′. (c) 13C chemical shifts of base carbons C6 and C8. (d) 13C chemical shifts of C5 of uracils. (e) 13C chemical shifts of C5 of cytosines. (f) 13C chemical shifts of C1′. (g) Definition of the box plots.
Mentions: Chemical shift statistics of H2 and C2 were generated for each individual triplet category, for example the triplet GAU means an adenine succeeding a guanidine and preceding an uracil. Chemical shift statistics of H5, H6, H8, H1′, C5, C6, C8 and C1′ were generated for combined triplet categories in which the base type of nucleotide i + 1 is ignored, e.g. GAX, in which X can be any nucleotide. The same chemical shifts were also analysed for terminal nucleotides, e.g. for the categories 5′GX, CC3′ and 5′GGX. 1D statistical parameters for each chemical shift category were calculated using MicroCal OriginPro 8.5G (Microcal Software Inc.). The mean value, standard deviation, cluster size, skewness, minimum value, 25th percentile (1st quartile), median, 75th percentile (3rd quartile) and maximum value for each category are given in Supplementary Table S2. 1D statistical values are represented by box plots displaying the minimum, 5th percentile, 25th percentile, median, mean, 75th percentile, 95th percentile and maximum value (Figure 1).Figure 1.

Bottom Line: Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts.The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt.The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, and Frankfurt Institute of Advanced Studies, 60438 Frankfurt am Main, Germany and Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.

ABSTRACT
The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and (1)H and (13)C chemical shifts. Statistics of resonances from regularly Watson-Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding (13)C resonances, which provides an important step toward automated structure determination of RNAs.

Show MeSH
Related in: MedlinePlus