Dr Vitor Pinheiro


ISMB Lectureship in Synthetic Biology

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Research interests

Directed evolution as a tool for Synthetic Biology

Detailed and systematic characterisation is the traditional scientific tool used to understand the function and mechanism of biological systems and their components. Although undoubtedly very successful, characterisation alone may not be sufficient to give us a complete understanding of any particular system or component.

Synthetic Biology seeks an alternative approach in which well-characterised parts are assembled to reconstitute biological function. This ‘bottom-up’ approach can yield novel insights at all levels of the assembled system – i.e. at the level of individual components, the biological system being assembled and the more general principles that emerge in biology (see below).

However, the understanding required to re-engineer components for novel function is limiting and generally not available. Directed evolution allows that gap in our current understanding to be bridged.

Directed evolution, implemented through sequential rounds of sequence diversification and purifying selection, allows an original biopolymer (be it protein or nucleic acid) to be systematically modified towards the desired function. Crucially, in principle, sequence diversity can be introduced without any knowledge of the underlying component or mechanism of action – although in practice, all available information on the system is used to target diversity and minimise the number of variants to be used as input into selection.

Methodologies for protein and nucleic acid directed evolution

Selection and screening are central to all directed evolution methodologies. The goal of selection is to create a strong link between phenotype and genotype, such that isolation of functional molecules, or molecules with the desired function, results in the co-isolation of their respective genetic information.

A number of versatile selection platforms have been developed, differing in how selective pressure can be introduced and modulated. Our goal is to adapt existing and develop novel selection platforms, establishing a technological toolbox for the directed evolution of natural and synthetic biopolymers - whether in vitro, ex vivo or in vivo.


The development of synthetic genetic materials (xeno-nucleic acids or XNAs) by systematically engineering DNA polymerases [1] is a clear example of the power of directed evolution for synthetic biology, and the first step towards developing an organism based on a synthetic genetic material.

Directed evolution of DNA polymerases for XNA synthesis was achieved through selection, by compartmentalised self-tagging (CST), and high throughput screening [1]. CST is an emulsion-based selection platform developed to allow the isolation of thermophilic DNA polymerases capable of incorporating modified nucleotides. Reminiscent of a primer extension assay, active polymerase variants incorporate the modified nucleotides provided extending a biotinylated primer against their own plasmids – extension of the primer stabilises its hybridization to the plasmid used as template. Recovery of the biotinylated primers leads to recovery of stably hybridised plasmids, and thus the recovery of the genotype of active polymerase variants.

Two rounds of selection were sufficient to allow the isolation of DNA polymerase variants capable of synthesising a number of XNAs, including hexitol nucleic acids (HNA), ‘locked’ nucleic acids (LNA), fluoroarabino nucleic acids (FANA) as well as a number of 2’-modifications (e.g. RNA, 2’-fluoro-DNA, 2’-azido-DNA) [1,2]. Together with a rationally designed polymerase variant capable of synthesising DNA from an XNA template, a total of eight synthetic genetic systems were established, with potential application in diagnostics and therapeutics through aptamer selection and nanotechnology [4].

This approach not only enabled the development of the first synthetic genetic materials, but also identified a novel region in the DNA polymerase involved in substrate recognition and discrimination [2]. In addition, development of synthetic genetic systems allows exploration of the boundary conditions of chemical information storage [3]. Although the engineered polymerases were capable of synthesising XNAs in a test tube environment, considerable work is still required before XNA polymerases will be capable of functioning within a true biological system.

Our goal is to further engineer biological parts involved in the storage, maintenance and interpretation of chemical information with a view towards learning how these systems emerged (or could emerge) and towards assembling synthetic components orthogonal to (i.e. independent and unable to interact with) biology.


Key publications

[1] Pinheiro, V.B., Taylor A.I., Cozens, C., Abramov, M., Renders, M., Zhang, S., Chaput, J., Wengel, J., Peak-Chew, S-Y., McLaughlin, S.H., Herdewijn, P. and Holliger, P. (2012) Synthetic genetic polymers capable of heredity and evolution. Science, 336, 341-4.

[2] Cozens, C., Pinheiro, V.B., Vaisman, A., Woodgate, R. and Holliger, P. (2012) A short adaptive path from DNA to RNA polymerases. PNAS, 109, 8067-8072

[3] Pinheiro, V.B.*, Loakes, D. and Holliger, P. Synthetic polymers and their potential as genetic materials. (2013) Bioessays, (DOI: 10.1002/bies.201200135.R1). *corresponding author.

[4] Pinheiro, V.B. and Holliger, P. (2012) The XNA world: Progress towards replication and evolution of synthetic genetic polymers. Current Opinion in Chemical Biology, 16, 245-252.









Vitor PinheiroDr Vitor Pinheiro



Institute of Structural and Molecular Biology, University of London

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