A NOVEL COUPLING-REACTION APPROACH TO MULTIPLEX IMAGING DNA REPLICATION DYNAMICS BY FLUORESCENCE

ASSESSMENT OF CELL PROLIFERATION IS OF CRUCIAL IMPORTANCE IN VARIOUS AREAS OF BIOMEDICAL RESEARCH SUCH AS CANCER OR DRUG DISCOVERY, AND IN MANY BASIC RESEARCH PROJECTS AS WELL. ONE OF THE MOST WIDELY USED TECHNOLOGIES IS TO MEASURE THE INCORPORATION OF ONE OR MORE NUCLEOTIDE ANALOGS IN NEWLY SYNTHESIZED DNA. TO DATE THIS IS ACHIEVED USING IMMUNOLOGICAL PROCEDURES THAT PRESENT SEVERAL DRAWBACKS: (I) NEED OF HARSH TREATMENTS TO DENATURE THE DNA, AFFECTING THE MORPHOLOGY OF CELLS AND TISSUES, AND (II) THE PROCEDURE IS NOT COMPATIBLE WITH THE USE OF OTHER ANTIBODIES, E.G. FOR DETECTING MODIFICATIONS OF CHROMATIN STRUCTURE. THEREFORE, DEVELOPMENT OF NOVEL PROCEDURES IS A MUST IF WE WANT TO INCREASE OUR PORTFOLIO OF TECHNIQUES TO EVALUATE CELL PROLIFERATION IN WHOLE TISSUES AND ORGANS. THE REPLICOLORS PROPOSAL AIMS AT SOLVING THIS PROBLEM BY GENERATING NOVEL AND HIGH VALUE TOOLS USEFUL FOR BASIC EXPERIMENTAL AND BIOTECHNOLOGICAL PURPOSES. IN PARTICULAR, WE PROPOSE TO DEVELOP NEW CHEMICAL REACTIONS TO DETECT MODIFIED NUCLEOTIDES IN NATIVE DNA THAT ARE COMPATIBLE WITH THE CLICK CHEMISTRY, SPECIFICALLY, THE CU(I)-CATALYZED AZIDE-ALKYNE HUISGEN CYCLOADDITION, ALREADY USED IN THIS FIELD. THIS WOULD ALLOW MULTIPLE LABELING OF THE NEWLY SYNTHESIZED DNA AVOIDING THE USE OF HARSH IMMUNOLOGICAL DETECTION AND PREVENTING CELL, TISSUE AND ORGAN STRUCTURE. SUCCESSFUL ACHIEVEMENT OF THIS GENERAL OBJECTIVE NEEDS TO BRING TOGETHER EXPERTISE IN CHEMISTRY AND BIOLOGY FROM TWO DIFFERENT RESEARCH TEAMS. FURTHERMORE, THE MAIN OBJECTIVE IS NOT PART OF OUR CURRENT FUNDED PROJECTS. MORE IMPORTANTLY, THIS PROPOSAL WILL NOT AND CANNOT BE TACKLED IN THE INDIVIDUAL LABORATORIES SEPARATELY UNLESS THEY ARE COORDINATED TOWARDS SETTING UP NEW CHEMICAL REACTIONS AND ADAPTING THEM PROPERLY TO BIOLOGICAL MATERIAL. IT IS RISKY, BUT VERY ATTRACTIVE AND OF HIGH GAIN, AND BASED ON THE EXPERTISE OF THE TWO TEAMS INVOLVED, THERE ARE REASONABLE CHANCES OF SUCCESS.