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Department of Bioorganic Chemistry

Use of Non-Uniformly Deuterated and/or 13C- or 15N-Labelled Nucleotides to Create the NMR-Window for Structural Studies of Large DNA and RNA by NMR (the "Uppsala NMR-Window" approach)

(1) Introduction

One of the major problems of the structure elucidation of functional oligo-RNA by NMR is the severe overlap of the sugar proton resonances,1 most of which normally appear within a very small stretch of ~1.0 ppm or less centered at ~4.5 ppm. In order to tackle this, Danyluk et al introduced the deuteration approach2a-f (~90 atom % 2H incorporation) as early as 1972 by preparing perdeuterated building blocks2 from RNA digest of blue-green algae grown in D2O for assignment purposes, which turned out to be inadequate for any high resolution NMR studies because of residual proton signals. In 1986, we introduced stereospecific chemical methodologies to introduce deuterium on the sugar carbons (>97 atom % 2H incorporation) to suppress specific NMR resonances in nucleosides.3 Subsequently, we have developed the Uppsala "NMR-window" concept,4 in which partially-deuterated sugar residues are non-uniformly incorporated into either oligo-DNA4a-c,e,i or -RNA4f by the solid-phase synthesis protocol or enzymatic means4g for simplification of the spectral crowding4a-c,e-h,j and coupling patterns,4e-h,j increasing NOE intensities,4c-e,j probing dynamics by selective T1 and T2 measurements,4k-m reducing the spin diffusion4e,j as well as the line-broadening4d associated with 1H dipolar relaxation. Non-uniform 2H labelling (with or without 13C enriched blocks)4l,m at specific sites in an oligonucleotide molecule ("NMR-window") can be easily achieved today in a desired manner using chemospecifically synthesised phosphoramidite or H-phosphonate derivatives of deuterated nucleoside blocks by solid-phase chemistry.

 The importance of specific non-uniformly deuterium labelled oligo-DNA and -RNA at the sugar has been subsequently recognized in other laboratories: Stereoselectively 2'(R)-deuterated 2'-deoxynucleoside blocks5 have been used for extraction of the 3JH1',H2' and 3JH1',H2" coupling constants from COSY-type experiments.6 Incorporation of isotopomeric 5'(R/S) mixture of 2H-labelled nucleosides7 facilitated the 3JHH determination and unambiguous NOE assignment of the diastereotopic H5'/5" methylene resonances in oligo-DNAs,8 whereas 5'- 2H/13C double-labelled nucleoside incorporated oligo-DNA gave information regarding vicinal 1H-31P coupling constants.9 Methods for stereoselective 5'-labelling with deuterium have also been developed for 2'-deoxynucleosides.10 Deuteration of C5/C6 of pyrimidines or 5-methyl of thymine and C8 of purine nucleobases removed unessential crosspeaks in the NOESY spectra of oligo-DNA11a,b,d or -RNA.11c Sequence specific incorporation of C1'-deuterated nucleosides into an RNA duplex12 decreased the spectral overcrowding of the aromatic-H1' region in the NOESY spectra. 3',4',5',5"-2H4-Labelled nucleosides4n,13 were uniformly incorporated into RNA and the effects of this site-specific deuteration on the spectral crowding and relaxation behaviour were studied.13
 

(2) What is Uppsala "NMR-window" approach?


Our exploitation of deuterium labelling is based on the concept [Tetrahedron 48, 9033 (1992)] of the creation of NMR visible short (the "NMR-window") and invisible longer sequences (Fig. 1) within large oligo-DNA and -RNA upon incorporating site specifically deuterated nucleoside building blocks into them in a sequence specific manner. In order to determine the solution structure of these oligomers by multidimensional NMR spectroscopy, the structurally relevant NMR data (J-couplings, nOe volumes or relaxation times) might be extracted from a spectrum emerging from the much shorter sequence of the "NMR-window".
 

(2.1) Deuteration Approach

We have developed synthetic methods for various site-specific deuteration of nucleosides [Tetrahedron 48, 9033 (1992), J. Biochem. Biophys. Methods 26, 1 (1993)]. Upon their sequence specific (invisible part) incorporation into 21mer RNA [Nucleic Acids Res. 24, 1187 (1996) (PDF:256)] via synthetic chemistry and 31mer RNA [Nucleic Acids Res. 24, 2022 (1996) (PDF:257)] using labelled CTP and T7 polymerase [Nucleosides & Nucleotides 16, 517 (1997)], 2',3',4'(50 atom%),5',5"-d5 (type A, R=OH) blocks with additional C5 deuteration at pyrimidines resulted in significant spectral simplification increasing the number of

structural constraints obtained from the visible "1H-NMR window". For studying DNA structure, the use of similar 2',2",3',4'(~50 atom%),5',5"-d6 2'-deoxynucleosides (type A, R=D) decreased the spectral overlap drastically except for the aromatic-H1' and H1'-H4' regions [Nucleic Acids Res. 21, 5005 (1993)] and decreased the relaxation rates of the residual protons within the deuterated nucleosides [Nucleic Acids Res. 22, 1404 (1994)]. We have recently employed 2'(R/S[~15:85]),3',4' (~50 atom%),5',5"-d5-2'-deoxynucleotide residues (type B+C) in a 20-mer DNA duplex in a non-uniform manner as NMR visible part. This new labelling resulted in considerable simplification of the spectral overlap providing both the J-coupling and the nOe information by our new NMR-window concept [Tetrahedron51, 10065 (1995) (PDF:251)]. This modified concept is based on partial deuteration of C2' along with full suppression of H3' resonance by deuteration creating a C2' isotopomeric mixture. The residual proton at C2' shows a ca 2-fold increase of T2 compared to the natural non-deuterated residue. A HAL-NOESY experiment has been used to filter off all proton resonances belonging to the non-deuterated nucleotides in the 20-mer DNA duplex. It has been found that the error in NOE volumes extracted from the HAL-NOESY experiment compared to a standard NOESY experiment is negligible thereby allowing the extraction of quantitative interproton distance information in the NMR-window concept. The use the diastereomeric mixture of 2'(R/S[~15:85]),3', 5'(R/S[~50:50]-d3 (type D+E) blocks [Tetrahedron 54, 14487 (1998) (PDF:287)] in the visible part made the extraction of 3JH1'H2", 3JH4'H5', 3JH4'H5", 3JPH4', 3JPH5' and 3JPH5" values possible [Tetrahedron 54, 14515 (1998) (PDF:288)]. Due to the reduced cross peak overlap, a HAL-NOESY experiment provided H1'-H5'/5", H1'-H4', H1'-H2", H2"-H4', H2"-H5'/H5", H4'-H5'/H5", H1'i-H5'/5"i+1, H1'i-H4'i+1, Ar-H5'/5" nOe volumes. These data are sensitive indeed to changes in the sugar or backbone conformations and the nOe data obtained are much less affected by spin diffusion as a result of the reduced number of relaxation pathways available for the residual protons. The incorporation of (B+C) or (D+E) 2'-deoxynucleosides into 12mer DNAs [J. Chem. Soc., Perkin Trans. 2 2689 (1998) (PDF:289)] allowed the measurement of T1 relaxation time of the C2' carbon at natural abundance, giving information about the dynamics of the 2'-methylene fragment. Further NMR work with a 55mer RNA [J. Biochem. Biophys. Methods 42, 153 (2000) (PDF:308)] modelling RNA three-way junctions frequently formed by natural RNAs led to the conclusions that although the use of blocks A (R=OH) is capable to provide convincing evidences

for the presence of helical regions, the low and varying level of deuterium incorporation at C4' (~85-~50 atom%) results in overcrowding of important regions of NOESY spectra. To remedy this problem, we developed first the diastereospecific chemical synthesis of 3',4',5',5"-d4 ribonucleosides F [Tetrahedron 55, 4747 (1999) (PDF:292)]. Then a new method was found for the efficient deuteration of C2' [Collect. Czech. Chem. Commun. Symp. Ser. 2 47 (1999) (PDF:303), Nucleosides, Nucleotides & Nucleic Acids 20, 1333 (2001) (PDF:325)]. A combination of these two achievements resulted in our subsequent synthesis of 2',3',4',5',5"-d5 ribonucleosides G [J. Org. Chem.66, 6560 (2001) (PDF:326)].
 

(2.2) NMR-window with the 13C labelled nucleoside blocks


We have also developed methodologies for non-uniform 13C labelling of oligo RNA [Tetrahedron 55, 6603 (1999) (PDF:293)] with uniformly sugar 13C-labelled nucleosides H probing the relaxation properties of specific stretches in oligo-RNA, whereas the recently developed non-uniform 13C/2H double-labelling technique using blocks I (main 2'-isotopomer is shown) [Nucleosides & Nucleotides18, 1377 (1999)] allowed us to investigate the relaxation properties of labelled oligo-DNAs [Magn. Reson. Chem. 37, 203 (1999) (PDF:290), Magn. Reson. Chem. 38, 403 (2000) (PDF:309)]. These double labelled nucleosides have also been used to study their complexes with enzymes by high-field NMR spectroscopy [J. Chem. Soc., Perkin Trans. 2 2199 (2000) (PDF:312)]. Work has been initiated for 13C and 15N labelling where the sugar moieties are uniformly labelled with 13C whereas the nucleobases are 15N labelled with additional deuteration in case of pyrimide nucleosides [J. Labelled. Cpd. Radiopharm. 44, 763 (2001) (PDF:328)].

Our present status of NMR-window concept thus demonstrates that a judicious synthetic strategy for non-uniform deuterium labelling opens new possibilities to minimize spectral overlap and reduce line-broadening enabling NMR structure determination of large DNA or RNA molecules using a relatively low-field NMR spectrometer (ie. a 500 or a 600 MHz spectrometer).
 
 

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