Designer bases, base pairs, and genetic sets: biochemical and biological activity

Emily M. Harcourt and Eric T. Kool

DOI: 10.1039/9781849737845-00001

A chemical approach to synthetic biology allows researchers to build new chemical monomers and evaluate their activity in a biochemical and biological context. Here we describe the work our laboratory has done in developing alternative bases for DNA, including non-hydrogen bonding base analogs, which have been used to study stacking, hydrogen bonding, and steric requirements in duplex stability and enzyme recognition; and size-expanded DNA bases for the development of a non-Watson-Crick genetic set. The designed molecules are useful in basic science, allowing a better understanding of the functions of natural DNA and RNA, and are also contributing practical new tools for biology and medicine.

1 Introduction

1.1 New bases for DNA and RNA: a bottom-up approach to synthetic biology

A conversation with the uninitiated about work on "expanded DNA" and a "new genetic system" can lead to worried looks and a voiced concern that alien life forms will soon be unleashed from the laboratory. While this is far from true at the moment, synthetic biology does involve both the generation of new functionality in living systems, and the quest to understand and recreate life from its simplest building blocks.

The field of synthetic biology is pursued along two paths: an engineering "top down" approach, which views existing biological components as modules that can be combined in new ways to obtain new functionalities, and a chemical "bottom up" approach, which aims to understand the functioning of biological components by creating new components and testing them in a biochemical or biological context. In some respects these approaches have seemingly opposite goals, as chemists first want to replicate the basic processes of living systems with designed components, while engineers are using existing components to generate new activities. However, both use synthesis as a strategy to address a "grand challenge" which will test underlying theories and knowledge of chemical principles and biological systems.

Some of the challenges taken up through the "top-down" approach of the biological engineering community include the creation of synthetic genetic circuits, metabolic engineering for drug and fuel production, and the development of the first cell with an entirely artificial genome. This work has been extensively reviewed and is discussed in other chapters of this book.

Chemists, on the other hand, have identified the molecular components of cells and attempted to replace or alter these components while retaining their functionality. Using this "bottom-up" approach, work has included exploration of alternatives to the (deoxy)ribose-phosphate backbone found in natural genetic material, attempts to design proteins, and the creation of polymer-based analogs of cell membranes. Another goal – the topic of this chapter – is the synthesis of functional nucleic acid bases for a new or expanded genetic set.

1.2 Goals of designer base pairs and genetic systems

Designing new bases and base pairs and studying them in a biochemical context allows us to both better understand the existing genetic alphabet and expand the capabilities of the genetic code. In the short term, this has already led to the development of new tools for biotechnology and new probes for biological mechanisms. For example, one of the first novel base pairs, Benner's isoG-isoC, has been used to increase the specificity of clinical detection of HIV viral loads. The non-hydrogen-bonding shape mimics developed in our own laboratory can serve as tools to separate the importance of hydrogen-bonding and solvation from steric effects in biochemistry.

A long-term goal of this field is to develop evolving, replicating, living systems with modified genetic structures. Several groups are working toward this goal, and there has been progress including new base pairs that function in PCR, the evolution of enzymes to tolerate unnatural base pairs, the establishment of function in living cells, and the incorporation of unnatural amino acids using codons with designer bases.

In our laboratory, there have been two main design strategies. First, the study of non-polar base analogs of different shapes and sizes has allowed us to tease apart effects of shape and size in base-pairing, enzymatic recognition, and helix stability. This work paved the way for the later development of non-hydrogen-bonding base pairs that are used very successfully today. Second, we are working toward a genetic set that retains natural hydrogen-bonding patterns but is of expanded size. This genetic set is different than those mentioned above in that it is not designed to function in the context of natural DNA. Advantages of this expanded genetic set include an 8-letter genetic alphabet, inherent fluorescence, and increased duplex stability; however, it pushes the limits of what natural enzymes can tolerate. This chapter will describe both of these aspects of our research in synthetic biology, providing insight into the challenges and knowledge that synthetic nucleic acids research can bring.

2 Nonpolar, non-hydrogen-bonding DNA bases and pairs

In order to make working alternatives and modifications to nature's genetic set, one needs to develop an understanding of the parameters for successful design. One crucial strategy for doing this is through perturbation. By redesigning one element of the system and then determining what difference the perturbation has made, we can find out which elements are essential and where there is room for change.

In this respect, each of the three components of nucleic acids – the phosphate backbone, the ribose or deoxyribose sugar, and the nitrogenous bases –...