Fundamental and applied studies of nucleic acids

Fundamental and applied studies of nucleic acids For 30 years the Maher laboratory has been investigating unresolved problems in nucleic acids biology and biophysics. We will continue our collaborative efforts related to two major challenges in the field. These challenges are deliberately diverse and multidisciplinary, spanning fundamental and applied aspects of nucleic acid structure and function. This work brings unique opportunities for synergy and creates an exceptional training environment. Challenge 1: Can principles learned from studies of bacterial DNA looping be applied to artificial DNA looping for gene regulation? Hypothesis 1.1: We hypothesize that small DNA loops form more easily in vivo than in vitro because a) proteins bridge DNA loops to reduce required DNA bending, b) DNA supercoiling pre-bends DNA, and c) architectural DNA binding proteins kink DNA. Molecular biology experiments will be performed in vitro and using elements of the lac operon in living E. coli cells. Hypothesis 1.2: We hypothesize that designed sequence-specific DNA binding proteins can be used to create artificial gene regulatory loops in bacterial and eukaryotic systems. Our studies will implement novel Transcription Activator-like Effector (TALE) proteins controlled by chemically-induced heterodimerization to regulate model and endogenous genes by creating tight DNA loops that exclude RNA polymerase from gene promoters. This work will impact synthetic biology efforts. Challenge 2: Can we identify naked DNA aptamers that home to sub-cellular compartments? Hypothesis 2.1: We hypothesize that the mechanism of nucleus-homing DNA aptamers we previously identified using Ligase Proximity Selection can be understood by proteomics and this selection concept extended to discover naked DNA aptamers that exhibit tissue-specific nuclear homing in mice. Hypothesis 2.2: We hypothesize that our new Peroxidase Proximity Selection will identify homing DNA aptamers specific for different sub-cellular compartments of interest. We will study peroxidase biotinylation reward chemistry, undertake selections in live cultured cells, and explore the mechanism of discovered homing aptamers as possible delivery agents for proteins and drugs. Support for this proposal will sustain the Maher laboratory's productive research program addressing these two important and unresolved challenges. The laboratory's track record shows that this investment will trigger further innovations with impact beyond these two problems.