Recognition of Synthetic Unnatural Base Pairs by RNA Polymerase

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Project Summary/Abstract All life forms on Earth use the same set of natural genetic alphabets: adenine (A), cytosine (C), guanine (G), thymine (T) (and uracil (U)) as the building blocks for storage and retrieval of their genetic information. Recently, a major breakthrough was made in developing the first semi-synthetic organism that is able to store and retrieve genetic information containing an unnatural base pair (UBP) in vivo. However, the molecular basis of transcription processing of UBPs is poorly understood. An important and long-standing question remains unanswered: How are these UBPs recognized by cellular transcription machinery? A lack of clear answers to this important question represents a major knowledge gap in the field. The long-term goal of this project is to tackle this important question. We hypothesize the transcription recognition of UBPs is governed by two layers of specific interactions: specific interactions between the unnatural nucleic acid template and substrates as well as their interplays with the active site of RNA polymerase. We will perform kinetic studies and compare the transcription processing of three classes of representative UBPs by different RNA polymerases, including single-subunit and multi-subunit RNA polymerases. We will determine the structural basis of transcription recognition of UBPs and gain the mechanistic insights into how transcription machineries recognize unnatural nucleotide substrate and catalyze the nucleotide addition reaction. We will utilize a combined approach that includes X-ray crystallography, cryoEM, biophysics, biochemistry, computational biology, and nucleic acid chemistry. The proposed research is significant and groundbreaking, because the novel knowledge and structures obtained from this proposed research will have a transformative impact on the fields of transcription, nucleic acid chemistry, as well as synthetic biology and vertically advance our understanding of the protein- nucleic acid interactions and how unnatural nucleic acids and nucleotides are recognized by different RNA polymerases. Ultimately, such knowledge will provide a framework for developing next generation of UBPs and would produce novel therapeutic nucleic acids and proteins containing new functional groups.