The 3D HN(CA)CO experiment is specifically designed to correlate inter- and intra-residue backbone connectivities between the amide ¹⁵N-¹H pair and the carbonyl ¹³CO resonance via the intervening ¹³CA nucleus by means of the ¹J(NH), ^1,2J(N,CA) and ¹J(CA,CO) coupling constants. On the other hand, inter-residues correlations can be exclusively obtained from a 3D HNCO experiment.

Implementation on AVANCE spectrometers equipped with a third channel. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required.

The experiment is applied on ¹⁵N,¹³C-labeled proteins. Because the amide (NH) protons are involved, the HNCO experiment must be recorded in H₂O. For this reason, a small isotope shift about 0.08ppm must be taken in account when comparison of absolute carbonyl chemical shifts are made in D₂O and H₂O.

The original HN(CA)CO pulse sequence ( 92JMR213-97 ) consisted of the following steps:

1. Initial transfer from ¹H to ¹⁵N via ¹J(NH) using an INEPT pulse sequence.
2. ¹⁵N chemical shift evolution during the constant-time evolution t₁ period.
3. Evolution delay to achieve antiphase ¹⁵N magnetization with respect to ¹³CA via ^1,2J(N,CA) followed by simultaneous 90º ¹⁵N and ¹³C, pulses for magnetization transfer to ¹³CA.
4. Evolution delay to achieve antiphase ¹³CA magnetization with respect to ¹³CO via ¹J(CA,CO) followed by simultaneous 90º ¹³CA and ¹³CO pulses for magnetization transfer to ¹³CO.
5. ¹³CO chemical shift evolution during the variable evolution t₂ period in an HSQC-type way.
6. Magnetization is transferred back to the NH protons by reversing the transfer steps described in points 4, 3 and 1, respectively.
7. Proton acquisition under ¹⁵N decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Incorporation of PFGs ( 95JMRB318-109 ).
• Improved sensitivity incorporating the PEP methodology in gradient-enhanced ( 96JMRB112-110 ) versions.
• Improved sensitivity by ²H decoupling in  ¹⁵N,¹³C,²H-labeled proteins ( 96JMRB112-110 and 94JACS11655 ).
• ¹³CO chemical shift evolution during the variable evolution t₂ period in an HMQC-type way  ( 95JMRB318-109 ).
• Improved sensitivity and resolution using the TROSY approach ( 99JACS844 ).
• Other mechanism starting from ¹HA-¹³CA magnetization has been proposed in the (HCA)CONH experiment ( 95JB189 , 96JMRB65-110 , 00JB229 ) to improve the sensitivity.
• In overlapped spectra it could be advisable to record a slightly modified 3D H(N)CACO experiment.
• Intraresidue correlation can be achieved from a 3D intra-HN(CA)CO experiment ( 04JB131-28 ).

The 3D HN(CA)CO  experiment can be recorded in automation mode. More details on practical implementation of the 3D HN(CA)CO  experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HN(CA)CO experiment

The HN(CA)CO experiment affords a 3D spectrum in which ¹H, ¹⁵N and ¹³CO chemical shifts are displayed in three independent dimensions. Two different connectivities are present:

• Intra-residue correlations due to ¹J(NH) +  ¹J(N,CA) + ¹J(CA,CO).
• Sequential correlations due to ¹J(NH) +  ²J(N,CA) + ¹J(CA,CO).

See list of 3D triple-resonance NMR experiments for doubly-labeled proteins.