The collagen IV scaffold serves as a fundamental structural unit of the basement membrane (BM). Understanding its structure, assembly, and function is essential for tissue engineering, the design of organoid models, and developing therapies for diseases such as Alport syndrome, Gould syndrome, psoriasis, eye abnormalities, hearing loss, and others, where collagen IV is required for structural integrity and functionality of the BM. The collagen IV molecule is a 400 nm long heterotrimer, comprising non-collagenous 1 (NC1), collagenous, and 7S domains. The assembly of the collagen IV scaffold involves oligomerization of the C-terminal NC1 and the N-terminal 7S domains, along with lateral associations within the collagenous domain. However, the detailed architecture and assembly mechanisms of the collagen IV scaffold remain unclear. Here, we investigated the potency and mechanism of recombinant single-chain NC1 trimer incorporation into the collagen IV scaffold. We discovered that the NC1 trimer influences the overall assembly of the basement membrane by affecting the quality of the developing collagen IV scaffold in a dose-dependent manner, without impacting already established scaffolds. This interference occurs through the hexamerization of supplemented NC1 trimers with endogenous NC1 domains, as the NC1 trimer becomes sulfilimine crosslinked with the existing chains. Overall, the single-chain NC1 trimer of collagen IV is crucial for developing novel extracellular therapies in two main ways: (1) facilitating the delivery and incorporation of functional replacements like collagen IV fragments and (2) inhibiting the formation of new basement membranes in conditions such as tumor growth and detrimental vascularization.