Immunofluorescent and Immunohistochemical (Colorimetric) Characterization of B‐DNA, Z‐DNA and G4‐Quadruplex DNA in Human Tissues Demonstrating the Spatial Genomic Organization of Different DNA Structures: Genomesorganizomics

Abstract

Spatial genomic organization, relative to DNA and RNA structures, has not been examined. DNA can adopt a variety of different shapes (helical conformations), i.e., canonical right‐handed double‐stranded (ds‐) B‐DNA, alternative left‐handed ds‐Z‐DNA and multi‐stranded G4‐quadruplex DNA. Each of these DNAs has a unique structure and plays a different role in cells. The structure of the nucleus is complex, and has a major role in gene expression. For the first time, our group has demonstrated the spatial genomic distribution of three totally different DNAs concurrently in HeLa cells, paraffin‐embedded tissue sections, and frozen sections. This was achieved by means of anti‐B‐DNA, anti‐Z‐DNA and anti‐G4 quadruplex DNA antibodies, via immunofluorescence (IF), and immunohistochemistry (IHC) (colorimetric). No one is investigating the relationship between spatial genomic organization and structures of different nucleic acids. All three types of DNA exist in the human genome and perform diverse cellular genomic functions. Tissues were processed in 10% NBF, and staining protocols were done both manually and by autostainer. Antigen retrieval enhanced all DNA‐based immunostaining procedures. IF was performed using Alexa Fluor conjugated secondary antibodies [i.e., Alexa Fluor 405 (blue), Alexa Fluor 488 (green) and Alexa Fluor 555 (red)], and wheat germ agglutinin conjugated with Alexa Fluor 647 (white) (counterstain). IHC protocols (i.e., enzyme: HRP or AP) were accomplished by using enzymes conjugated to secondary antibodies, and color developed by chromogens [i.e., B‐DNA (Blue) AP Substrate, Z‐DNA (Red/Magenta) AP Substrate, and G4‐quadruplex DNA (Brown), DAB Peroxidase (HRP) Substrate]. Negative and positive controls, isotype controls, and blocking background were performed in triplicate. The order of primary antibodies (and substrates: IHC) is critical for obtaining accurate data. Images were obtained by confocal and light microscopes (i.e., 200x, 600x). Advanced computer‐based image analysis (e.g., HALO, Image Pro Plus) allowed for qualitative and quantitative characterization of immunostained DNAs. The largest percentage of DNA was in the form of B‐DNA, followed by the other two remaining structures of DNA. The novel method signifies a new subfield of structural and functional genomics, which we have named Spatial Genomic Organization of DNA Structures, i.e., Genomesorganizomics. It will aid spatial transcriptomics in the analysis of gene expression and provide information on transcriptional activity based upon the conformation of DNA in genes and telomeres. An in‐depth comparative analysis between IF and IHC will yield information about specific advantages and disadvantages of these immuno‐based tissue staining systems (e.g., sensitivity, dynamic range, easier multiplexing, photobleaching, better target colocalization). This genomics approach is both sensitive and reliable, and will allow for the characterization of nuclear genomic architecture. Future projects are examining both DNA and RNA structures of four or more nucleic acids.