Project Summary The 3D structure of genome has long been recognized as an important realm of cellular regulation. Molecular analysis of the 3D genome structure is becoming a reality due to new technology development. Preliminary studies suggest that direct physical models of the genome can be generated from genome-wide mapping of DNA-DNA proximities and population-based modeling, and that the resulting models can yield insights about genomic functions via statistical analyses. While these studies provide a glimpse of the potential of understanding cellular functions from the molecular structures of the genome, it remains a major challenge to develop an accurate physical model of the genome in space and time and relate the model structures to cellular functions. To meet this challenge, the proposed studies will focus on the three major technical barriers faced by all current mapping technologies: (i) inefficient and potentially biased data acquisition; (ii) lack of temporal resolution; and (iii) missing higher-order contact information. The proposed studies have two specific aims. One is to adapt current mapping technologies to a new structure capturing technique, namely flash freezing and cryomilling, for instant capturing and efficient processing of the nucleus thereby enabling extensive and unbiased mapping of maximum amount of chromatin contacts from fewer cells including single cells. The second aim is to develop new chemical approaches for DNA end joining, a critical and universal step in current mapping technologies. The new chemical approaches will not only improve data acquisition efficiency but also enable analyses and mapping of higher-order DNA-DNA contacts in single cells as well as ensemble of cells. These studies have the potential to shift the current focus of genome conformation analysis from 2D maps to 3D structures, thereby enhancing our ability to study the link between structure and function in the genome.