NMR as a tool for structure

determination of nucleic acids

Teresa Carlomagno EMBL, Heidelberg

DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are polymers of nucleotides linked in a chain

through phosphodiester bonds.

Among the 24000 structures contained in the PDB in February 2004, only 15% have been solved by NMR (Fig.

1). However, for nucleic acids structures, the contribution of NMR is significantly higher (44%). This is due to

the fact that nucleic acids, and in particular RNA, are highly flexible molecules which are difficult to crystallize

in absence of proteins. NMR is the optimal technique to investigate such molecules in solution and to

characterize their dynamic properties, which might be functionally relevant.

Fig. 1

Nucleic acids are constituted by a sugar unit (ribose), an aromatic unit (base) and a phosphate backbone (Fig.

2). The backbone of a nucleic acid is made of alternating sugar and phosphate molecules bonded together in a

long chain. Each of the sugar groups in the backbone is attached to a third type of molecule called a nucleotide

base. There are only five type of different nucleotide bases that can occur in a nucleic acid (Fig. 3).

In most living organisms(except for viruses), genetic information is stored in the molecule deoxyribonucleic

acid, or DNA. DNA gets its name from the sugar molecule contained in its backbone(deoxyribose). There are

four different nucleotide bases that occur in DNA: adenine (A), cytosine (C), guanine (G) and thymine (T).

Ribonucleic acid, or RNA, gets its name from the sugar group in the molecule's backbone - ribose. The primary

structure of RNA and DNA are equal; however instead of the thymine base, the uracil base is found in RNA.

There are four main kinds of ribonucleic acid, each of which has a specific function. 1. Ribosomal RNAs-exist

outside the nucleus in the cytoplasm of a cell in structures called ribosomes. Ribosomes are small, granular

structures where protein synthesis takes place. Each ribosome is a complex consisting of about 60% ribosomal

RNA (rRNA) and 40% protein. 2. Messenger RNAs-are the nucleic acids that "record" information from DNA

in the cell nucleus and carry it to the ribosomes (mRNA). 3. Transfer RNAs-the function of transfer RNAs

(tRNA) is to deliver amino acids one by one to protein chains growing at ribosomes. 4. Ribozymes, namely

RNA enzymes: most of them catalyse an autocatalytic reaction which involves breakage or formation of

phosphodiester bonds.

Fig. 2

Fig. 3

Most DNA exists in the famous form of a double helix, in which two linear strands of DNA are wound around

one another (Fig. 4).

Fig. 4

The major force promoting formation of this helix is complementary base pairing: A's form hydrogen bonds

with T's (or U's in RNA), and G's form hydrogen bonds with C's (Fig. 5).

Major groove

Minor groove

Hbond acceptor

Hbond donor

A-T G-C

Fig. 5

G-C base pairs have 3 hydrogen bonds, whereas A-T base pairs have 2 hydrogen bonds: one consequence of

this disparity is that it takes more energy (e.g. a higher temperature) to disrupt GC-rich DNA than AT-rich

DNA.

The two strands of DNA are arranged antiparallel to one another: viewed from left to right the "top" strand is

aligned 5' to 3', while the "bottom" strand is aligned 3' to 5'. This is always the case for duplex nucleic acids.

RNAs are usually single stranded, but many RNA molecules have secondary structure in which intramolecular

loops allow to invert the backbone direction and a double helix is formed by complementary base pairing. Base

pairing in RNA follows exactly the same principles as with DNA: the two regions involved in duplex formation

are antiparallel to one another, and the base pairs that form are A-U and G-C.

The double helix of DNA has been shown to exist in several different forms, depending upon sequence content

and ionic conditions of crystal preparation (Fig. 6). A- form DNA occurs when the amount of water in the

surrounding medium is about 75%. The B- form occurs at much higher moisture content, 92%. Z-DNA, or left-

handed DNA occurs in regions of high GC content. Notice that an RNA/DNA hybrid has the same structural

parameters as A form DNA.

A-form B-form Z DNA

20 Angtroms26 Angtroms 18 Angtroms

A-form B-form Z DNAA-form B-form Z DNAA-form B-form Z DNA

20 Angtroms26 Angtroms 18 Angtroms

Fig. 6

The parameter that characterize each helix form are listed in the following table:

Parameters A Form B Form Z-FormDirection of helical rotation Right Right LeftResidues per turn of helix 11 10 12 base pairs

Rotation of helix per residue(in degrees) 33 36 -30

Base tilt relative to helix axis(in degrees) 20 6 7

Major groove narrow anddeep wide and deep Flat

Minor groove wide andshallow narrow and deep narrow and deep

Orientation of N-glycosidicBond Anti Anti Anti for Py, Syn for Pu

Comments - most prevalent within cells

occurs in stretches of alternating purine-pyrimidine base pairs

Parameters A Form B Form Z-FormDirection of helical rotation Right Right LeftResidues per turn of helix 11 10 12 base pairs

Rotation of helix per residue(in degrees) 33 36 -30

Base tilt relative to helix axis(in degrees) 20 6 7

Major groove narrow anddeep wide and deep Flat

Minor groove wide andshallow narrow and deep narrow and deep

Orientation of N-glycosidicBond Anti Anti Anti for Py, Syn for Pu

Comments - most prevalent within cells

occurs in stretches of alternating purine-pyrimidine base pairs

ParametersParameters A FormA Form B FormB Form Z-FormZ-FormDirection of helical rotationDirection of helical rotation RightRight RightRight LeftLeftResidues per turn of helixResidues per turn of helix 1111 1010 12 base pairs12 base pairs

Rotation of helix per residue(in degrees)

Rotation of helix per residue(in degrees) 3333 3636 -30-30

Base tilt relative to helix axis(in degrees)

Base tilt relative to helix axis(in degrees) 2020 66 77

Major grooveMajor groove narrow anddeep

narrow anddeep wide and deepwide and deep FlatFlat

Minor grooveMinor groove wide andshallow

wide andshallow narrow and deepnarrow and deep narrow and deepnarrow and deep

Orientation of N-glycosidicBond

Orientation of N-glycosidicBond AntiAnti AntiAnti Anti for Py, Syn for PuAnti for Py, Syn for Pu

CommentsComments -- most prevalent within cells

most prevalent within cells

occurs in stretches of alternating purine-pyrimidine base pairs

occurs in stretches of alternating purine-pyrimidine base pairs

A- DNA B- DNA

Major-Groove

Major-Groove

minor-Groove

minor-Groove

Wide &shallow

Narrow &deep

Wide &deep

Narrow &deep

Fig. 7

The A form is preferred by RNA (Fig. 7). From now on we will focus on the determination of the conformation

of RNA. However, many of the concepts and of the methods proposed here can be applied to DNA structure

determination as well.

Six torsion angles define the conformation of the RNA backbone, five torsion angles define the sugar

conformation and one torsion angle defines the orientation of the nucleotide base with respect to the ribose (Fig.

8).

Fig. 8

Sugar conformation.

The sugar furanose rings are twisted out of plane to minimize non-bonded interactions between their

substituents. This is called puckering (Fig. 9) and is a displacement of the C2’ and C3’ from the median plane

of C1’-O4’-C4’. If the out of plane atom is to the same side as the C5’ substituent, the sugar is in the endo

conformation; if on the opposite side the sugar is exo. The C2’-endo pucker is preferred in standard B-DNA, the

C3’-endo pucker occurs in A-RNA.

Fig. 9

However a more rigorous designation of the sugar conformation uses the E and T notation. If four of its atoms

lie in a plane, this plane is chosen as a reference plane, and the conformation is described as envelope (E); if

they do not lie in a plane, the reference plane is that of the three atoms that are closest to the five-atom, least-

squares plane, and the conformation is described as twist (T) (Fig. 10). Atoms that lie on the side of the

reference plane from which the numbering of the ring appears clockwise are written as superscripts and precede

the letter (E or T); those on the other side are written as subscripts and follow the letter. These definitions mean

that atoms on the same side of the plane as C5 in D-ribofuranose derivatives are written as preceding

superscripts. The E/T notation has superseded the endo/exo description, in which atoms now designated by

superscripts were called endo, and those now designated by subscripts were called exo. C3'-endo/C2'-exo has

become 3T2; C3'-endo has become 3E. Symmetrical twist conformations, in which both atoms exhibit equal

displacements with respect to the five-atom plane' are denoted by placing the superscript and subscript on the

same side of the letter T, e.g. 23T, 43T, etc.

32T

Fig. 10

The sugar ring conformation has also been described by Altona and Sundaralingam using the concept of

pseudorotation, which has been found advantageous in describing the conformational dynamics of the sugar

ring. Each conformation of the furanose ring can be unequivocally described by two pseudorotational

parameters: the phase angle of pseudorotation, P, which describes which atom(s) lies out of the plane of the

others and on which side, and the degree of pucker, m., which describes the extent to which one or two

atom(s) lie out of the plane of the others. The degree of pucker is correlated with the bond lengths and angles

and is usually ignored. This leaves us with the pseudoration phase P that can be calculated according to the

following equations:

tan P = (ν2+ν4-ν1-ν3)/2ν0(sin36 +sin72)

A standard conformation (P=0o) is defined with a maximally positive C1'-C2'-C3'-C4' torsion angle, i.e. the

symmetrical 23T form. Conformations in the upper or northern half of the circle (P = 0 90o) are denoted N and

those in the southern half of the circle (P = 180 90o) are denoted S conformation. It may be seen that the

symmetrical twist (T) conformations arise at even multiples of 18o of P and the symmetrical envelope (E)

conformations arise at odd multiples of 18o of P.

Glycosidic torsion angle

The base is free to rotate around the glycosidic bond (torsion angle χ). Two main orientations are adopted,

called syn and anti.

In the anti conformation the 6-membered ring of the purines and the oxygens of the pyrimidines point away

from the sugar group, instead the smaller H6 (pyrimidines) or H8 (purines) lie above the sugar ring. The syn

conformer has the larger O2 (pyrimidines) or N3 (purines) above the sugar ring. Pyrimidines adopt a narrow

range of anti conformations whereas purines have a wider range of anti conformations.

The anti conformation is always preferred, except guanine prefers the syn conformation in alternating

oligomers e.g. d(CpGpCpG), in Z-DNA and in mononucleotides. The syn conformation can only be built into

left handed helices. When a purine base adopts the syn conformation with respect to the sugar, as in Z-DNA,

the sugar adopts the C3’-endo conformation to avoid steric clashes (Fig.

11).

synsyn < < ---- > C3> C3‘‘endoendo

antianti < < ---- > C2> C2‘‘endoendo--C3C3‘‘endoendo

Preferred values:

Syn (~40º)

Anti (~200º)

High-anti (~320º)

Fig. 11

Other torsion angles.

The γ torsion angle can assume three conformations, which have different probability of existing, depending on

the sugar conformation and on the nature of the sugar (DNA or RNA).

A plot of the distribution of the backbone torsion angles in the RNA portion of the large ribosome subunit is

shown in the figure 12:

Nucleic Acids Research, 2004

Fig. 12

In general the distribution of the backbone torsion angles in helical regions and in all structural regions is quite

similar (Fig. 13).

Fig. 13

The probability that one torsion angle is found in a certain conformation depends on the values assumed by the

other torsion angles. A complete analysis of the energetically allowed conformations and of the

interdependence of the torsion angles can be found in Murthy et al., JMB 1999 291, 313.

Base pairing

The Watson-Creek base pairing is only one of the several base pairing patterns observed in RNA. Non-

canonical base pairs are essential in stabilizing tertiary interactions between different RNA elements and are

often functionally relevant. In the figure 14 the Watson-Creek base pairs are shown together with three non-

canonical base pairs.

A UA UG CG C

G U G A

A

Gwobble

sheared

Fig. 14

A rigorous classification of the base pairs is given by Leontis and Westhof (RNA 2001 7, 499). Briefly, three

base pairing faces can be identified for the purines (Watson-Creek, Hoogsteen, and sugar edge), three for the

pyrimidines (Watson-Creek, C-H and sugar edge) and two glycosidic bond orientations (cis if the sugars are on

the same side of an ideal line which runs parallel to the hydrogen bond directions; trans if the sugars are on

opposite sides with respect to this line) (Fig. 15).

Leontis and Westhof (RNA 2001 7, 499)

Fig. 15

This gives a total of 12 orientations, that can be classified according to the following table:

Leontis and Westhof (RNA 2001 7, 499)

RNA structure

RNA can assume many different secondary structures (Fig. 16). Loops, buldges, junctions, pseudoknots

interrupt the regular double helix form. Tertiary interactions among the secondary structural elements stabilize

a variety of structural motifs in RNA (kissing loops, peseudoknots, etc). This structural variability allows RNA

to interact with other molecules (RNA, proteins, ions, small molecules) very efficiently and is at the basis of the

multifunctionality of RNA.

In Fig. 17 the structures of three stable tetraloops is shown, where non canonical hydrogen bond patterns and

stacking interactions between bases stabilize the structure. Pseudoknots structures are also stabilized by non

canonical triple base pairing (Fig. 18).

When the regular double helix structure is interrupted by buldges, it becomes a critical point for he interaction

with ions or other molecules in general. In the simplest case a buldge consists of only one nucleotide (Fig. 19).

The buldged nucleotide can be accommodated between two bases of the helix via stacking interactions, can be

buldged out in solution, can be accommodated in the shallow groove, or can flap over a ligand closing the

binding site (Fig. 19). These structures are examples to understand the variability of the RNA structure and

Fig. 16

Fig. 17

Fig. 18

Fig. 19

therefore the necessity of having tools for the accurate structure determination of RNA. NMR is a particularly

suitable technique for this purpose, due to the flexibility of RNA molecules, that renders crystallization

difficult.

Structure determination of RNA by NMR

The first step for a structure determination of RNA by NMR is the preparation of the sample. This is obtained

by in vitro transcription out of a DNA template with the help of the T7 polymerase (Fig. 20). Reaction

conditions, like magnesium, NTP (nucleotidetriphosphate), template and enzyme concentrations are optimised

for optimal yields. A 15N-13C labelled sample is produced by usage of 15N-13C labelled NTPs.

Fig. 20

The general procedure to determine RNA structure by NMR is quite similar to that for determining protein

structure by NMR. First the 1H,15N,13C resonances have to be assigned, then structural restraints like NOEs, J-

coupling, Cross-Correlated relaxation rates, dipolar couplings and H-bonds have to collected (Fig. 21). This

parameters are then used as restraint for molecular dynamics calculations, that starting from a random structure

look for the structure that best satisfy all the experimental constraints (Fig. 22).

Fig. 21

Fig. 22

The 1D proton spectrum of a RNA is shown in Fig. 23. The sugar resonances (H2’, H3’, H4’, H5’ and H5’’) are

very overlapped in the region 4.8-4.0 ppm. The H1’ of the ribose are usually quite well resolved arount 5.6

ppm. Well resolved are also the protons of the bases around 5.5 ppm (H5 of C and U) and around 7.8 ppm (H8

of G and A and H6 of C and U). The imino (NH) protons of G and U are only observable if involved in

hydrogen bonding and have a characeristic chemical shift around 13.0 ppm. The NH2 protons of G and A are

usually very broad due to exchange with water and rotation around the C-N bond; those of C are sharper and

deliver two distinct signal per NH2 group due to the partial double bond nature of the C-N bond.

Fig. 23

The imino region of a RNA can be considered as the fingerprint region in a 1D spectrum. In Fig. 24 the imino

region of the TAR RNA, a 30mer RNA that binds to an arginine rich region of the Tat protein, is shown. All the

imino resonances involved in H-bonds are visible apart from that of the 5’ terminus G and that of the U40

preceding the buldge region. The 2D NOESY spectrum in the imino region shows weak NOE peaks between

the imino protons that allow to assign sequential base pairs (i.e. G26-U38-G28-G38 in the upper stem). Such a

correlation is fundamental to prove the existence of a double helical region. Another fundamental spectrum,

that is very useful for the assignment of the base protons as well as for checking the integrity of the RNA, is the

COSY correlation between the H6 and H5 protons of C and U (Fig. 26). For the assignment of the overlapped

sugar resonances in non-labelled RNA molecules, it is convenient to start from the well resolved H1’ region

and to assign the H2’ resonances through a H1’-H2’ COSY sorrelation, as shown in Fig. 27. This spectrum is

also useful to get an estimation of the J(H1’,H2’), that is indicative of the sugar pucker (large J for C2’ endo,

small J for C3’ endo).

Fig. 24

Fig. 25

Fig. 26

Fig. 27

Fig. 28

For large RNAs (MW>7 KDa) heteronuclear multidimensional experiments on 15N-13C labelled samples

become essential. In Fig. 28, the 13C-1H HSQC correlation of the ribose of the TAR RNA shows that, even after

13C labelling, the H2’, H3’, H4’, H5’ and H5’’ resonances are severely overlapped. The lack of chemical shift

dispersion constitutes the difficulty of RNA spectroscopy. For very large RNAs this extensive overlap can be

solved only with selective deuteration of the sugar ring.

The 15N-1H HSQC of the imino region is much better resolved and allows to distinguish among of G and U

imino resonances thanks to the different nitrogen chemical shift (Fig. 29).

Fig. 29

The assignment of the spin system of a nucleotide takes place through multidimensional experiments (Fig. 30).

The resonances of the sugar moiety can be assigned in HCCH-TOCSY and HCCH-COSY experiments. The

linkage between the sugar moiety and the base takes place through HCN correlations: the H1’-C1’ and H8/H6-

C8/C6 resonances are linked in a HCN spectrum to the same nitrogen, allowing to correlate each ribose spin

system with a base spin system (Hu et al. 2001 JBNMR 167,167; Riek et al. 2001 JACS 123, 658; Fiala et al.

2000 JBNMR 16, 291). Alternatively magnetization can be transferred from the H1’/H8/H6-C1’/C8/C6 to the

nitrogen and back to the H1’/H8/H6-C1’/C8/C6 in a (H)CN(C)H experiment (Fig. 31) (Fiala et al. 2000

JBNMR 16, 291). The correlation between the non exchangeable protons in the base takes place through a

HCCH-COSY for the pyrimidines and through a HCCH-TOCSY (Legault et al. 1994 JACS 116, 2203) or a

relayed HCCH COSY (Simon et al. 2001, JBNMR 20, 173) for the adenine. Exchangeable and non-

exchangeable protons in the bases are correlated through HCCNH-TOCSy experiments (Fiala et al. 1996 JACS

118, 689; Sklenar et al. 1996 JBNMR 7, 83). An interesting experiment has recently appeared in the literature

for the assignment of the sugar proton and carbon resonances, that combines a HCCH-COSY step and an

HCCH-TOCSY step in a single experiment (Hu et al. 1998 JBNMR 12, 559). The analysis of the pulse

sequence (Fig. 32) is left to the student as an exercise. Briefly, in a Ha-Ca-Cb-Hb-Cc-Hc-Cd-Hd moiety, the

magnetization generates from the proton Ha (F1), is transferred to its directly bound carbon Ca and from there

to the carbon Cb in a HCCH-COSY step. The chemical shift of Cb is then recorded in F2. Form Cb the

magnetization is transferred through a carbon TOCSY step to all carbons of the spin system. A final INEPT

step transfer magnetization from the carbons to the protons Ha, Hb, Hc and Hd, the chemical shifts of which are

then recorded in F3. The resulting spectrum is shown in Fig. 33 and allows assignment of the all ribose spin

systems in one experiment.

Adenine

H8-H1’ correlation, HCNH8-H1’ correlation, HCN

H1’,H2’, H3’ H4’H5’H5” correlations, HCCH-TOCSY

H1’,H2’, H3’ H4’H5’H5” correlations, HCCH-TOCSY

H2-H8 or H5-H6correlation

H2-H8 or H5-H6correlation

Fig. 30

Fig. 31

Fig. 32

F2 plane F3 plane

Fig. 33

The spin systems belonging to two different nucleotides can be linked through HCP or PCCH experiments. The

pathway of magnetization transfer is shown in Fig. 34 and 35. In a HCP experiment the H3’(n)-C3(’n) and P(n)

or H5’/H5’’(n+1)-C5’(n+1) and P(n) resonances are linked together in a 3D correlation, exploiting the J(PH3’)

and J(PH5’/H5’’) scalar couplings (Varani et al. 1995 JBNMR 5, 315); in a PCCH spectrum (Wijmenga et al.

1995 JBNMR 5, 82), the H4’(n)-C4’(n) and H4’(n+1)-C4’(n+1) resonances are linked to the P(n) resonance

exploiting the J(PC4’) scalar coupling. HCP correlations do not work for very large RNAs due to the fast

relaxation of the phosphorous and to the small J couplings used for the transfer. Inthis case the sequential

assignment is achieved through NOESY spectra, as shown in Fig. 36. The most important connectivity is that of

the H2’(n) with the H6/H8(n+1). A schematic representation of the NOEs observable in A form helices is given

in Fig. 37 and 38.

Adenine

OH

Guanine

H-C-Pexperiment

H3’-C3’-PResidue nH3’-C3’-PResidue n

H5’,H5’’(n+1)-C5’(n+1)-P(n)H5’,H5’’(n+1)-C5’(n+1)-P(n)

2 spin systems can be linked

Fig. 34

OH

H4‘(n)-C4‘(n)-P(n)H4‘(n+1)-C4‘(n+1)-P(n)

Fig. 35

Fig. 36

Fig. 37

A H2‘ to H1‘: two residues base pair above and belowG NH to H1‘: two residues base pair above and below

Fig. 38

The structural information that has to be collected to determine the structure of a RNA is: NOE: used to derive interproton distances

J-coupling + cross-correlated relaxation: used to derive the dihedral angles in the backbone and the

conformation of the sugar

Dipolar couplings: used for structural refinement and for the definition of the relative orientation of secondary

structure elements, namely of the tertiary folding

H-bonds: used for the definition of base pairing

Chemical shifts: used for structure refinement and to obtain local structural information

The following table summarize the J-couplings that needs to be measured to access the backbone and ribose

dihedral angles:

Coupling constants for RNA conformation

Angle 3J-couplings 3J-couplingsAngle

A generalized Karplus equation can be given for the J(H;H) according to the following formula:

i=1

4

in a HaS1 (S11 ,S12 ,S13 )S3 (S31 ,S32 ,S33 )-C1-C2-S2 (S21 ,S22 ,S23 )S4 (S41 ,S42 ,S43 )moiety

α β β β α β β βα β β β α β β β

ii

i

All the J(H,H) of the ribose can be calculated from this equation.

The Karplus dependence for the J(HP) and J(CP) couplings are:

3JHCOP = 15.3cos2φ - 6.2cosφ + 1.5

3JCCOP = 8.0cos2φ - 3.4cosφ + 0.5

Fig. 39

Fig. 40

The torsion angle γ can be determined for unlabelled RNA by a combination of J(H,H) couplings and

interproton distances, as shown in Fig. 41

Fig. 41

The glycosidic torsion angle can be determined by means of heteronuclear J(H,C) couplings, as shown in Fig.

42.

The sugar pucker can be determined by means of J(H,H) couplings (Fig. 43). A large J(H1’,H2’) coupling and a

small J(H3’,H4’) coupling are indicative of the C2’ endo conformation; a large J(H3’,H4’) coupling and a small

J(H1’,H2’) coupling are indicative of the C3’ endo conformation. Also cross-correlated relaxation rates can be

used to derive the conformation of the ribose ring. Γ(C1’H1’,C2’H2’) and Γ(C3’H3’,C4’H4’) depend on the

sugar pucker in a similar way as J(H1’,H2’) and J(H3’,H4’) (Fig. 44). A large and positive Γ(C1’H1’,C2’H2’)

and a small and negative Γ(C3’H3’,C4’H4’) are indicative of the C2’ endo conformation, while a small and

negative Γ(C1’H1’,C2’H2’) and a large and positive Γ(C3’H3’,C4’H4’) are indicative of the C3’ endo

conformation. Cross-correlated relaxation rates are particularly useful for large RNAs, where the measurement

of the coupling constants might be impeded by large linewidths. The cross correlated relaxation rates can be

obtained with the G-quantitative method by the ratio of two corresponding peaks in a cross and reference

experiment (Fig. 45). The sign and the intensity of the peaks in the cross experiment are proportional to the

Fig. 42

Fig. 43

North South5.0

0.0

P [°]300.0200.0100.00.0

Γ34

H1'

H2'

H4'

H3'

H1'

H2'H3'

H4'

Γ23Γ12

Fig. 44

cross-correlated relaxation rate: from Fig. 45 it can be concluded that U4 is in the C3’ endo conformation while

U5, U6 and U7 are in the C2’ endo conformation.

δ1H (ppm)

δ13C

(ppm

)

δ1H (ppm)

δ13C

(ppm

)

U5

U6

U4

U7

a) b)

H2‘-C1‘ region: measurement of the Γ(C1‘H1‘,C2‘H2‘) cross-correlated relaxation

cross reference

Fig. 45

The conformation of the backbone torsion angles α and ζ cannot be obtained from J-coupling measurements,

due to the fact that the oxygen nucleus is magnetically silent. However, cross correlated relaxation rates

between the phosphorous CSA tensor (Fig. 46) and the C3’H3’ or C2’H2’ vectors, Γ(P,C3’H3’) and

Γ(P,C2’H2’) depend on the torsion angles ε and ζ, while the cross correlated relaxation rates between the

phosphorous CSA and the C5’H5’ or C5’H5’’ vectors, Γ(P,C5’H5’) and Γ(P,C5’H5’’) depend on the torsion

angle α and β. In a recent work (Richter et al. 2000 JACS 122, 12728) these CCR rates are used to determine

the two torsion angles α and ζ (Fig. 47).