The 3D HNCO experiment is specifically designed to correlate crucial sequence backbone connectivities between the ¹⁵N-¹H pair of one residue with the carbonyl (¹³CO) resonance of the preceding residue by means of the ¹J(NH) and ¹J(N,CO) coupling constants.

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 HNCO pulse sequence ( 93ANG1489 , 94JMRB203-103 , 94JMRA129-109 , 95JB11 , and 96JB315 ) is closely analog to the 3D HNCA experiment and consists of the following "out and back" steps:

1. Initial transfer from ¹HN to ¹⁵N via ¹J(NH) using an INEPT pulse sequence.
2. Fixed evolution delay to achieve antiphase ¹⁵N magnetization with respect to ¹³CO via ¹J(N,CO) and refocusing of ¹J(NH).
3. ¹³CO chemical shift evolution during the variable evolution t₁ period in an HSQC-type way followed by ¹⁵N chemical shift evolution during a constant-time evolution t₂ period with evolution of ¹J(NH) and refocusing of ¹J(N,CO).
4. Magnetization is finally transferred back to the NH protons by applying a retro-INEPT scheme and proton acquisition is recorded under ¹⁵N decoupling.

Otherwise, several versions have been also proposed incorporating the following modifications:

• The original sequence used a different pathway in which ¹⁵N chemical shift evolution takes place before the ¹³CO chemical shift evolution delay ( 90JMR496-89 ).
• Constant-time period (CT) in the F₁(¹⁵N) dimension, HSQC-like transfer in F₂(¹³CO) dimension and optional ¹H decoupling instead of 180º ¹H pulses was first proposed in 92JMR432-96 .
• Incorporation of PFGs ( 92JB395 ).
• Improved sensitivity incorporating the PEP methodology in phase-cycled ( 92JMR431-100 ) and in gradient-enhanced ( 93ANG1489 , 94JMRB203-103 , 94JMRA129-109 , 95JB11 , and 96JB315 ) versions.
• Improved sensitivity and resolution using the TROSY approach ( 99JB3 , 98PNAS13585 , 99JMR180-141 and 99JB273-14 ). Application to the measurement of scalar couplings across hydrogen bonds ( 98JACS8293 , 98PNAS14147 , 00JMR431-143 and 00JMR387-143 ) and dipolar couplings ( 98JB325 and 00JB43-17 ).
• The sensitivity of the HNCO-type experiments can be enhanced by quenching scalar coupling mediated relaxation. This is performed by applying CPD on alpha carbons, when a long 15N-13CO defocusing/refocusing period is required. This can be highly useful to detect weak, through hydrogen-bonds HN-CO dipolar couplings without resorting to perdeuteration. ( 00JB55-17 and 00JB79-17 )
• For small proteins, a related 2D H(N)CO experiment can give unambiguous ¹³CO resonance assignment