Functional DNA-based assemblies

Lead Research Organisation: University of Birmingham
Department Name: School of Chemistry

Abstract

Deoxyribonucleic acid, or DNA, is a long polymer made up of repeating units called nucleotides. Each nucleotide consists of three components: sugar, phosphate and base. There are four possible bases that can be attached to the sugar-phosphate backbone, thymine (T), adenine (A), guanine (G) and cytosine (C). A specific base sequence along the chain enables DNA to encode a set of unique instructions for the synthesis of various biological components of the cell, for example a particular protein. DNA can adopt different conformations and structures in solution but the most common one is called B-DNA, where two polymer strands combine (hybridise) through base-pairing to form a right-handed double helix (or duplex).Due to its crucial role in initiating cell division (i.e. growth of an organism), DNA has long been a target for drugs, in particular anti-cancer compounds. However more recently, chemists have become interested in ways of chemically modifying DNA by attaching other groups (or tags) to the polymer chain. Part of this interest has stemmed from a need to sense DNA, in particular specific base sequences that could signify a genetic disease. We have shown that by attaching fluorescent groups to a DNA probe strand, a selective sensor can be designed that enables two target strands, 15 bases long, that differ only in the identity of one of their bases (G instead of A), to be told apart. This is a result of the two duplexes (each formed from the target strand binding to the probe strand) giving different different emission profiles (colour intensities) upon hybridisation. In this fellowship, we wish to explore and rationalise these findings in more detail and work with end users so that the viability of this new approach to detecting these so-called single nucleotide polymorphisms (or SNPs, pronounced 'snips') in DNA can be assessed.We also wish to extend the chemical modification of DNA further by introducing groups that give DNA even more functionality. One particular aspect concerns so-called photochromic groups that undergo a reversible structural change upon their exposure to light. If these groups are attached to DNA, then the structure of DNA should also change upon photo-irradation, which in turn should control its biological function.Using a similar approach, we will also attach groups to DNA that respond to an oxidising potential rather than light. These so-called redox-active groups can be oxidised which, if attached to DNA, allow DNA to be sensed electrochemically through the flow of current. We wish to use redox-active groups that are not only tagged to DNA but also interact with the structure itself through a process called intercalation, where a group inserts itself between the base pairs of duplex DNA. Through this approach, we expect that electrochemical DNA sensing can be made more effective and sensitive. Finally, we wish to incorporate the redox-active group ferrocene into the actual backbone of DNA through its replacement of a sugar-phosphate-sugar motif to create synthetic mimics of DNA. If such a process is successful, then oxidation of the ferrocene groups could change the stability of the DNA duplex in an unprecedented manner, allowing redox processes to control various DNA functions.

Publications

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Carr-Smith J (2015) Polymerase Chain Reaction on a Viral Nanoparticle. in ACS synthetic biology

 
Description We have developed a new metal-containing form of a nucleic acid, called ferrocene nucleic acid (FcNA). FcNA is a structural analogue of DNA (the most common natural form of a nucleic acid). The monomeric components of FcNA show promising anticancer and antiviral activity, themselves being structural analogues of the monomeric components of DNA. We have also developed a new form of metal-containing DNA that consists of a macrocyclic complex tag containing copper of nickel. These probes act as electrochemical sensors for nucleobase changes in target DNA.

We have also developed photo-active DNA molecules that act as novel nucleic acid sensors (for both nucleobase changes and modifications in DNA) and as light-controllable DNA binders.

Finally we have developed a new way of sensing long stretches of DNA using a PCR-based linear dichroism (LD) spectroscopy assay involving the M13 bacteriophage.
Exploitation Route The work may be useful for scientists and researchers working in the following areas:
(1) the pharmaceutical sector, e.g. in anticancer drug discovery (2) the healthcare diagnostics sector e.g. for DNA sensing applications.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.birmingham.ac.uk/staff/profiles/chemistry/tucker-james.aspx
 
Description In two patents, one on drug discovery and the other on sensing.
First Year Of Impact 2014
Sector Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC Synthetic Biology
Amount £2,219,000 (GBP)
Funding ID BB/M025624/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2015 
End 04/2016
 
Description EPSRC Follow on funding
Amount £60,601 (GBP)
Funding ID UOBFOF54 
Organisation University of Birmingham 
Department University of Birmingham EPSRC Follow On Fund
Sector Academic/University
Country United Kingdom
Start 12/2014 
End 09/2015
 
Description Leverhulme award
Amount £118,436 (GBP)
Funding ID RPG-2013-100 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 09/2015
 
Description MRC Confidence in Concept Award
Amount £59,762 (GBP)
Organisation Medical Research Council (MRC) 
Department MRC Confidence in Concept Scheme
Sector Academic/University
Country United Kingdom
Start 09/2014 
End 03/2015
 
Description Collaboration with QUB on DNA synthesis 
Organisation Queen's University Belfast
Department School of Chemistry and Chemical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution One student was sent over the Belfast to learn DNA synthesis
Collaborator Contribution QUB provided intellectual input on DNA research during fellowship
Impact Publications under the author name Vyle and Tucker; multidisciplinary
 
Description Collaboration with the University of Bordeaux 
Organisation University of Bordeaux
Department Institute of Molecular Sciences (ISM)
Country France 
Sector Academic/University 
PI Contribution We supplied samples to the research team in Bordeaux for photophysical measurements. The collaborators are Dario Bassani and Nathan McClenaghan.
Collaborator Contribution The research team in Bordeaux carried out photo-physical measurements on anthracene-containing samples. These have included supramolecular receptors and also DNA systems (for the EPSRC Leadership Fellowship).
Impact 1. Photocontrolled Binding and Binding-Controlled Photochromism within Anthracene-Modified DNA PIs: Bassani, DM; Tucker, JHR, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2012, 134, 10791-10794, 10.1021/ja304205m 2. Detection of DNA base variation and cytosine methylation at a single nucleotide site using a highly sensitive fluorescent probe PIs: Bassani, DM; Tucker, JHR, CHEMICAL COMMUNICATIONS, 2011, 47, 6629-6631, 10.1039/c1cc11205h 3. Anthracene-modified oligonucleotides as fluorescent DNA mismatch sensors: discrimination between various base-pair mismatches PIs: Bassani, DM; Tucker, JHR, SUPRAMOLECULAR CHEMISTRY, 2011, 23, 273-277, 10.1080/10610278.2010.523117 4 Single Site Discrimination of Cytosine, 5-Methylcytosine, and 5-Hydroxymethylcytosine in Target DNA Using Anthracene-Tagged Fluorescent Probes PIs: Bassani, DM; Tucker, JHR, ACS CHEM BIOL, 2016, in press, 10.1021/acschembio.5b00796 Other recent pieces of work not linked to work on EPSRC grant EP/G007578/1 are as follows: Photorelease of an organic molecule in solution: Light-triggered blockage of a hydrogen-bonding receptor site PIs: Bassani, DM; Tucker, JHR, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2005, 44, 1072-1075 Structural effects on the ground and excited-state properties of photoswitchable hydrogen-bonding receptors PIs: Bassani, DM; Tucker, JHR, JOURNAL OF ORGANIC CHEMISTRY, 2006, 71, 8523-8531 Detection of a single DNA base-pair mismatch using an anthracene-tagged fluorescent probe PIs: Bassani, DM; Tucker, JHR, CHEMICAL COMMUNICATIONS, 2006, 5003-5005 Shining light on supramolecular assemblies PIs: Tucker, JHR; Bassani, DM, PURE AND APPLIED CHEMISTRY, 2009, 81, 1677-1685 Reversible Control over Molecular Recognition in Surface-Bound Photoswitchable Hydrogen-Bonding Receptors: Towards Read-Write-Erase Molecular Printboards PIs: Tucker, JHR; Bassani, DM, CHEMISTRY-A EUROPEAN JOURNAL, 2013, 19, 12748-12758, 10.1002/chem.201301613 Formation of a Hydrogen-Bonded Barbiturate [2]-Rotaxane PIs: Tucker, JHR; McClenaghan, ND, ORGANIC LETTERS, 2014, 16, 1358-1361 Others with McClenaghan, ND: 10.1021/jo502452t 10.1039/c4cc09472g 10.1002/ajoc.201402243
Start Year 2006
 
Description Collaboration with the University of Copenhagen 
Organisation University of Copenhagen
Department Department of Cellular and Molecular Medicine
Country Denmark 
Sector Academic/University 
PI Contribution We supplied ferrocene-containing DNA strands for measurement of duplex stabilities with PNA strands
Collaborator Contribution The Denmark team made PNA strands to be used for the duplex binding studies with the ferrocene-containing DNA strands
Impact No outputs yet but a paper is being written for publication in 2016 or 2017.
Start Year 2012
 
Description Collaboration with the University of Leuven 
Organisation University of Leuven
Department Riga Institute
Country Belgium 
Sector Academic/University 
PI Contribution Samples for cell line testing to assess anticancer and antiviral activity have been sent to the group of Prof Jan Balzarini
Collaborator Contribution The group have provided cell toxicity data that has proved invaluable for the progression of the research. As of 2014, one paper and a patent has resulted from this work so far.
Impact Organometallic Nucleoside Analogues with Ferrocenyl Linker Groups: Synthesis and Cancer Cell Line Studies PIs: Balzarini, J; Tucker, JHR, JOURNAL OF MEDICINAL CHEMISTRY, 2014, 57, 5817-5822
Start Year 2011
 
Title Ferronucleobases 
Description Biological activity of ferrocene-containing nucleobase derivatives 
IP Reference WO2015092432 
Protection Patent granted
Year Protection Granted 2013
Licensed No
Impact Subject of current research activity in collaboration with the medical school and Biosciences at Birmingham
 
Title Sensor 
Description This invention relates to a genetic probe for the detection of a single nucleotide polymorphism (SNP) or single nucleotide modification of a target nucleic acid, and 5 methods of determining the percentage of single nucleotide polymorphisms (SNPs) or single nucleotide modifications of a target nucleic acid in a pool of the target nucleic acid. 
IP Reference GB1714068.2 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact Still at early stages
 
Title Diagnostic assay for SNPs in nucleic acids 
Description The assay concerns a new method to determine SNPs in nucleic acids involving a fluorescent read-out method. It was supported by an MRC Confidence in Concept award in 2014-2015. 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Initial development
Year Development Stage Completed 2015
Development Status On hold
Impact Achieved follow-on funding (MRC confidence in concept) on the back of our initial results, leading to the initial development stage. 
 
Title Ferrocene-containing drug compounds 
Description The ferrocene compounds show anti-cancer activity and further studies are being undertaken into 2016 on the back of an EPSRC follow-on fund grant that started in 2015. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2016
Development Status Under active development/distribution
Impact n/a 
 
Description Work with ChemBam 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Activities related to DNA research are described on a School of Chemistry website called https://chembam.com. One section describes the DNA research undertaken as part of this Leadership Fellowship.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
URL https://chembam.com/meet-the-scientists/jim-tucker/