Nucleic acids can assume numerous structures; B-DNA, Z-DNA and G4-quadruplex DNA. These DNAs have different conformations and functions in cells. The 3-D organization of the nucleus is very dynamic. This results in the complex regulation of gene expression. We wanted to demonstrate, for the first time, the spatial genomic organization (SGO) of three different DNAs simultaneously in HeLa cells, and tissue sections. This was accomplished by employing anti-B-DNA, anti-Z-DNA and anti-G4 quadruplex DNA antibodies. No one is examining the association between SGO, and structures of DNAs. We believe that B-DNA, Z-DNA and G4-quadruplex DNA exist in the genome and are simultaneously expressed in different regions. Samples were processed in different fixatives, and AR was employed to improve immunohistochemical (IHC) colorimetric staining. IHC protocols (e.g., enzyme: HRP or AP) were done manually and by autostainer. Images were obtained by a brightfield light microscope. This was accomplished by using enzymes conjugated to secondary antibodies and color developed by chromogenic substrates [e.g., B-DNA (Blue) AP Substrate, Z-DNA (Red/Magenta) AP Substrate, and G4-quadruplex DNA (Brown), DAB Peroxidase (HRP) Substrate]. The order of the primary antibodies and the substrates is critical. Negative and positive controls, isotype controls, and blocking background were considered. Advanced image analysis unmixing software (HALO), allowed us to characterize (e.g., intensity) stained DNA; since multiplex color reactions cannot be viewed by the naked eye. The largest amount of DNA was in the form of B-DNA, followed by the other two DNA structures. Counterstains revealed non-nuclear cellular components. Genomics is divided into two basic areas: structural genomics, and functional genomics. The novel method signifies a new subfield/discipline of structural and functional genomics, which has been named SGO of DNA Structures, i.e., Genomesorganizomics. This multiplex method will allow for the characterization of concurrent expression of various DNAs that control the molecular biology of both normal and pathological cells. This approach will deliver valuable data on transcription, based on the structures of different DNA molecules in genes and telomeres. This new genomics approach will allow for a new way of characterizing nuclear genomic architecture, expand the range of metrics, and produce highly quantified data.

Spatial genomic organization of DNA structures, i.e., genomesorganizomics: Multiplex high-dimensional immunohistochemical colorimetric demonstration of B-DNA, Z-DNA and G4-quadruplex DNA in human cells and tissues

ACS Spring 2022

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