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NMR experiments for labeled nucleic fiala/Graphics/NucleicAcids_3D.pdf NMR Strategies for labeled Nucleic Acids Sugar 3D HCCH-COSY 3D HCCH-TOCSY 3D H(C)CH-E.COSY 3D TROSY-(H)CCH-COSY

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Text of NMR experiments for labeled nucleic fiala/Graphics/NucleicAcids_3D.pdf NMR Strategies for labeled...

  • NMR Strategies for labeled Nucleic Acids

    Sugar

    3D HCCH-COSY 3D HCCH-TOCSY

    3D H(C)CH-E.COSY 3D TROSY-(H)CCH-COSY 3D HCC-TOCSY-CCH-COSY 3D HCC-TOCSY- CCH-E.COSY

    Sequential 31P-based

    2D HP HETCOR 2D HP HSQC 2D HP HeteroTOCSY

    2D HP HSQC-NOESY 2D & 3D HP HeteroTOCSY-NOESY

    2D P(C)H 2D P(CC)H

    3D P(H)CH

    3D HCP 3D HCP-CCH-TOCSY

    2D for Base

    2D H(NC)C 2D H(NCC)H 2D H(CCN)H

    2D H(CN)C

    3D for Base

    3D H b C

    b N

    b

    3D (H)N(C)CH

    3D HC(CN)H 3D H6(CCC)NH 3D H(CC)NH-COSY 3D H(CC)NH-TOCSY 3D (H)C(C)NH-TOCSY

    2D Sugar-to-base

    2D H s (C

    s N

    b C

    b )H

    b

    2D (H s )C

    s (N

    b C

    b )H

    b

    2D (H b )C

    b (N

    b C

    s )H

    s

    2D H s (C

    s N

    b )C

    b

    2D H s (C

    s N

    b )H

    b

    2D H s (C

    s )N

    b

    3D Sugar-to-base

    3D H s C

    s N

    b

    3D H s C

    s (N

    b C

    b )H

    b

    3D H s (C

    s N

    b )C

    b H

    b

    3D H s (C

    s )N

    b (C

    b )H

    b

    3D (H b )C

    b N

    b (C

    s )H

    s

    3D (H s )C

    s N

    b (C

    b )H

    b

    3D (H s C

    s )N

    b (C

    b )C

    b H

    b

    Through-hydrogen Bond

    2D H(N)N-COSY 2D H(NN)H

    NOE Experiments

    3D NOESY-C-HSQC

    3D NOESY-N-HSQC

    NMRGuide 4.3 - TOPSPIN 3.0 Written by Teodor Parella Copyright © 1998-2009 BRUKER Biospin. All rights reserved.

    CLOSE BACK

    NMR experiments for labeled nucleic acids http://localhost:6600/guide/eNMR/3Dtrip1.html

    1 of 1 22.10.2013 14:59

  • H1' 5-6 H2' 2.3-2.9(A,G) 1.7-2.3(T,C) H2'' 2.4-3.1(A,G) 2.1-2.7(T,C) H3' 4.4-5.2 H4' 3.8-4.3 H5' 3.8-4.3 H5'' 3.8-4.3

    H1' 5-6 H2' 4.4-5.0 H3' 4.4-5.2 H4' 3.8-4.3 H5' 3.8-4.3 H5'' 3.8-4.3

    C1' 83-89 C2' 35-38 C3' 70-78 C4' 82-86 C5' 63-68

    C1' 87-94 C2' 70-78 C3' 70-78 C4' 82-86 C5' 63-68

    RNADNA

  • Adenine Guanine H2 7.5-8 C2 152-156 - - C2 156 H8 7.7-8.5 C8 137-142 H8 7.5-8.3 C8 131-138 N6H 5-6/7-8 N6 82-84 N1H 12-13.6 N1 146-149 - - - N2H 5-6/8-9 N2 72-76

    C4 149-151 C4 152-154 C5 119-121 C5 117-119 C6 157-158 C6 161 N1 220-226 N1 146-149 N3 214-216 N3 167

    N7 224-232 N7 228-238 N9 166-172 N9 166-172

    Thymidine Uridine Cytidine H6 6.9-7.9 C6 137-142 H6 6.9-7.9 C6 137-142 H6 6.9-7.9 C6 136-144 Me5 1.0-1.9 Me5 15-20 H5 5.0-6.0 C5 102-107 H5 5.0-6.0 C5 94-99 N3H 13-14 N3 156 N3H 13-14 N3 156-162 - - N3 210 - - - - - - - N4H 6.7-7.0

    /8.1-8.8 N4 94-98

    C2 154 C2 154 C2 159 C4 169 C4 169 C4 166-168 C5 95-112 C5 102-107 C5 94-99 N1 144 N1 142-146 N1 150-156

  • C

    N

    N

    C

    O

    O

    H

    H

    R

    H

    Adenosine Guanosine

    Cytidine

    a HH

    C

    C

    C

    N

    N

    N

    N

    N

    H

    H

    R H

    H

    C

    C

    C

    N

    N

    C N

    N

    N

    O

    H

    R

    H

    HH

    N

    C

    C

    N

    N

    O

    H

    H

    R

    8888

    21

    75

    ~0.7 65 203

    0.2

    0.3

    11

    20

    216

    10.8

    90 7.5

    15

    2391

    91

    88

    63 7.6

    8.5

    216

    20

    11

    11

    91 10.7 65 179

    67 185

    13

    12 19

    19 176

    68 184

    13

    12

    ~12

    ~8

    6.5

    20

    86 86

    55

    Uridine

  • C

    N

    N

    C

    O

    O

    H

    H

    R

    H

    Adenosine Guanosine

    UridineCytidine

    HH

    C

    C

    C

    N

    N

    N

    N

    N

    H

    H

    R H

    H

    C

    C

    C

    N

    N

    C N

    N

    N

    O

    H

    R

    H

    HH

    N

    C

    C

    N

    N

    O

    H

    H

    R

    15

    5.2

    11

    7.8

    8.9 8.5

    6.0 3.7

    11

    7.9

    ~9.5 ~8 ~8

    5.7 4.6 4.4

    ~6

    15

  • C

    C

    C

    N

    N

    N

    N

    N

    H

    H

    R

    H H

    C

    C

    C

    N

    N

    C N

    N

    N

    O

    H

    R

    H

    H

    H

    N

    C

    C

    N

    N

    O

    H

    H

    R

    HH

    C

    N

    N

    C

    O

    O

    H

    H

    R

    H

    11

    ~8.511

    12 5.2

    11

    5.1

    ~9

    11

    10.7

    8.36.2

    8.9

    Adenosine Guanosine

    UridineCytidine

  • Go to Tutorial

    3D HCCH-COSY

    DESCRIPTION

    The 3D HCCH-COSY experiment is specifically designed to correlate side-chain aliphatic proton resonances with their attached 13C resonances via 1J(CH) and 1J(CC) coupling constants. The experiment provides nearly complete assignments of all aliphatic 1H and 13C resonances, with the exception of some resonances of the long aliphatic side chains (as Lys or Arg) for which substantial overlap remains.

    REQUIREMENTS

    Implementation on AVANCE spectrometers is feasible. Improved versions using pulsed field gradients (PFGs) are also available

    and, therefore, in such cases gradient technology is required. The experiment is applied on 13C-labeled proteins. Since NH protons are not involved, this experiment is performed in D2O.

    VERSIONS

    The original HCCH-COSY pulse sequence ( 90JACS888 ) consisted of the following steps:

    After the initial 90º 1H pulse, 1H chemical shift evolution during the variable evolution t1 period takes place.1.

    Fixed evolution delay to achieve antiphase 1H magnetization with respect to 13C via 1J(CH).2.

    Magnetization transfer to 13C by applying simultaneous 90º 1H and 13C pulses.3. 13C chemical shift evolution during the variable evolution t2 period. followed by a fixed period to achieve antiphase

    13C

    magnetization with respect to its 13C neighbors via 1J(CC).

    4.

    A 90º 13C pulse transfers magnetization to its coupling partner.5. 13C magnetization is transferred back to the protons by reversing the transfer steps described in points 4 and 2, respectively.6.

    Proton acquisition under 13C decoupling.7.

    Several improved versions have been proposed incorporating the following modifications:

    An improved version reduces indesirable artifacts and noise-like features by using selective 180 pulses and a modified phase-cycle ( 90JMR620-87 ).

    Incorporation of a constant-time 13C evolution period ( 91JB299 ). HCCH-E.COSY experiments to measure J(HH) ( 93JMRA321-105 and 98JMR216-135 ) and J(CC) ( 93JACS7878 ) coupling constants .

    Use of pulsed-field gradients and PEP methodology ( 94JACS2203 and 98JMR185-135 ).

    3D HCCH-COSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchco3d.html

    1 of 2 22.10.2013 15:40

  • A related 2D experiment has been proposed to trace out heteronuclear connectivites at natural abundance (see 2D ADEQUATE experiment) Specific 2D and 3D HCCH-COSY experiments have been reported to assign deoxyribose spin systems in doubly labeled DNA ( 98JB25 ). A 3D H(C)CH-COSY pulse sequence with two carbon-dimensions has been recently reported ( 98JMR185-135 ). A modified version has been used to measure J(CP) coupling constants in RNA ( 98JMR236-133 ).

    Use of an isotropic 13C mixing period instead of the 90º 13C pulse (see 3D HCCH-TOCSY experiment).

    Use of the TROSY approach ( 98JACS6394 , 99JMR447-139 ) to be applied on aromatic 1H-13C spin systems. Editing and Suppression of diagonal peaks in such experiments has been also reported ( 00JMR171-144 and 01JB69-19 ). TROSY-relayed HCCH-COSYfor correlation H2/H8 resonances in 13C-labeled RNA ( 01JB173-20 ) A forward-directed quantitative HCCH experiment has been proposed for the measurement of the sugar conformation in RNA oligonucleotides from CH-CH dipole-dipole cross-correlated correlation ( 99JACS1956 ). A HCC-TOCSY-CCH-E.COSY experiment to measure J(HH) for ribose protons in uniformly 13C-labeled RNA ( 95JACS7251 ).

    EXPERIMENTAL DETAILS

    The 3D HCCH-COSY experiment can be recorded in automation mode. More details on practical implementation of the 3D HCCH-COSY experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HCCH-COSY experiment

    SPECTRA

    The HCCH-COSY experiment affords a 3D spectrum in which 1H, 13C and 1H chemical shifts are displayed in three independent

    dimensions. Cross-peaks are due to 1H-13C-(13C)-1H spins systems.

    RELATED TOPICS

    Analogs 2D HCCH-COSY experiments are also possible, yielding a COSY-type spectra. However, for larger proteins such 2D spectra show very severe overlap.

    NMRGuide 4.0 - TOPSPIN 1.3 Written by Teodor Parella Copyright © 1998-2004 BRUKER Biospin. All rights reserved.

    NMRGuide 4.0 - TOPSPIN 1.3 Written by Teodor Parella Copyright © 1998-200