Class 2017
PhD Grant: PD/BD/135138/2017 | COVID/BD/151841/2021
PhD thesis
Biomanufacturing of DNA-origami nanostructures for bio-imaging
Supervisor
• Miguel F. Prazeres, Professor @ IST/ULisboa, PT
• Pedro Paulo, Research Scientist @ IST/ULisboa, PT
Collaborators
• Hendrik Ditz, Professor @ TUMünchen, Munique, D
PhD Degree complete: Discussion on March 1, 2023
THESIS ABSTRACT
DNA-origami nanostructures are emerging as components of nanomachines, nanopores, drug delivery systems and biosensors. They rely on the self-assembly of a long (~103–104 bases) single stranded (ssDNA) DNA molecule (the scaffold) into a target nanostructure with the assistance of short oligonucleotides (the staples). Scaffolds are usually purified by (i) extraction of phage genomic DNA, produced in E. coli, which has sequence limitations and raises safety concerns regarding contamination of following cultures, preventing the scalability potential of the technique and its applications, or (ii) by agarose gel extraction., which is a laborious, time consuming, limited and not scalable technique that presents low recovery, delivers low-quality products, and requires specific equipment, making it not suitable for pharmaceutical or analytical applications.
The main goal of this work is to improve the recovery and quality of ssDNA scaffolds. Firstly, a phage-free method, in E. coli, called reverse infection was used to produce ssDNA scaffolds containing random sequences based on the use of a helper plasmid and phagemid particles. This method allowed the generation of high titers of phagemid particles not capable of self-replicating, overcoming the safety concerns related with phage production. On the other hand, production of ssDNA scaffolds using asymmetric PCR (aPCR) was also explored. Besides generating the target ssDNA molecules, contaminants of aPCR comprise double stranded DNA, dNTPs, excess of primers and enzyme. To purify these mixtures two approaches were tested: (i) affinity between functionalized magnetic beads and the target ssDNA and (ii) anion-exchange and multimodal chromatography as a scalable platform for the purification of ten aPCR mixtures. An analytical chromatography tool based on ion-pair reverse phase high performance liquid chromatography was also developed for the quantification of ssDNA scaffolds. Finally, the applicability of DNA-origami tetrahedrons for bio-imaging and as a drug delivery model was evaluated by photochemical techniques.
RESUMO DA TESE
As nanoestruturas de DNA-origami assentam na auto-organização de uma molécula de DNA de cadeia simples (ssDNA) longa (~ 103-104 bases) (scaffold) numa nanoestrutura alvo com a ajuda de oligonucleótidos curtos (agrafos). Os scaffolds são geralmente purificados por (i) extração do DNA genómico de fago produzido em E. coli, que tem limitações na sequência e levanta questões de segurança quanto à contaminação das culturas seguintes, limitando o potencial de escalabilidade e aplicações desta técnica, ou (ii) por extração de gel de agarose, que é uma técnica trabalhosa, demorada, limitada, não escalável, apresenta baixa recuperação com baixa qualidade e requer equipamentos específicos, tornando-a inadequada para aplicações farmacêuticas ou analíticas.
O principal objetivo deste trabalho é melhorar a recuperação e a qualidade dos scaffolds. Primeiramente, um método livre de fagos, infeção reversa em E. coli, foi usado para produzir scaffolds contendo sequências aleatórias recorrendo a um plasmídeo ajudante e partículas de fagemídeo. Este método permitiu a produção de altos títulos de partículas de fagemídeoincapazes de auto-replicação, ultrapassando as preocupações de segurança relacionadas com a produção de fagos. Por outro lado, a produção de scaffolds usando PCR assimétrico (aPCR) também foi explorada. Além de gerar as moléculas ssDNA alvo, a reação de aPCR contémDNA de cadeia dupla, dNTPs, excesso de primers e enzimas como contaminantes. Para purificar essas misturas foram testadas duas abordagens: (i) afinidade entre esferas magnéticas funcionalizadas e o ssDNA alvo e (ii) cromatografia de troca aniónica e multimodal como plataforma escalável para a purificação de dez misturas de aPCR. Uma ferramenta de cromatografia analítica baseada em cromatografia líquida de alta eficiência de fase reversa de pares de iões também foi desenvolvida para a quantificação de scaffolds de ssDNA.Finalmente, a aplicabilidade de tetraedros de DNA-origami para bioimagem e como modelo de entrega de drogas foi avaliada por técnicas fotoquímicas.
PUBLICATIONS
Papers
Silva-Santos AR, Paulo PMR, Prazeres, DMF, Scalable purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures: Exploring anion-exchange and multimodal chromatography, Separation and Purification Technology, 298, 121623, 2022.
Behler KL, Honemann MN, Silva‐Santos AR, Dietz H, Weuster‐Botz D, Phage‐free production of artificial ssDNA with Escherichia coli, Biotechnology and Bioengineering, 119(10), 2878-2889, 2022.
Silva-Santos AR, Oliveira-Silva R, Rosa SS, Paulo PMR, Prazeres DMF, Affinity-Based Magnetic Particles for the Purification of Single-Stranded DNA Scaffolds for Biomanufacturing DNA-Origami Nanostructures, ACS Applied Nano Materials, 4 (12) 14169–14177, 2021
Silva-Santos AR, Alves CPA, Monteiro M, Azevedo AM, Prazeres DMF, Multimodal chromatography of supercoiled minicircles: A closer look into DNA-ligand interactions, Separation and Purification Technology, 212, 161-170, 2019.
Pre-prints:
Ana Rita Silva-Santos, Pedro Paulo, Miguel Prazeres. Scalable purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures: exploring anion-exchange and multimodal chromatography. Authorea. March 31, 2022. (10.22541/au.164870304.41072457/v1)
Book chapters:
Wagner A, Silva-Santos AR, Rosa SS, Gierak S, Azevedo AM, Prazeres DMF, Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation, DNA Vaccines, 2021, 151-165, Humana, New York, NY
Silva-Santos AR, Rosa SS, Prazeres DMF, Azevedo AM, Purification of Plasmid DNA by Multimodal Chromatography, DNA Vaccines, 2021, 193-205, Humana, New York, NY
Oral Communications
Silva-Santos AR, Prazeres DMF, Quantification of ssDNA scaffold production by ion-pair reverse phase chromatography, BPP 2022 – Biopartitioning & Purification conference, September 2022, Aveiro, Portugal.
Silva-Santos AR, Prazeres DMF, DNA-origami nanostructures: exploring chromatography for the purification of single stranded DNA scaffolds, 40th ISPPP – International Symposium on the Separation of Proteins, Peptides and Nucleotides, November 2021, Porto, Portugal
Silva-Santos AR, Oliveira-Silva R, Paulo PMR, Prazeres DMF, Magnetic particles as affinity-based ligands: purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures, 6th ECAB – European Congress of Applied Biotechnology, September 2021, virtual event
Silva-Santos AR, Prazeres DMF, Chromatographic isolation of single stranded DNA scaffolds for biomanufacturing scale-up of DNA-origami nanostructures, 6th ECAB – European Congress of Applied Biotechnology, September 2021, virtual event
Silva-Santos AR, Oliveira-Silva R, Paulo PMR, Prazeres DMF, Magnetic particles as affinity-based ligands: purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures, Affinity 2021, June 2021, virtual event
Silva-Santos AR, Oliveira-Silva R, Paulo PMR, Prazeres DMF, Magnetic particles as affinity-based ligands for the purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures, 13thESBES – European Symposium on Biochemical Engineering Sciences 2021, May 2021, virtual event
Silva-Santos AR, Prazeres DMF, Chromatographic isolation of single stranded DNA scaffolds for biomanufacturing of DNA-origami nanostructures, 15th SBCN – International Symposium on Biochromatography and Nanoseparations, May 2021, virtual event (Bronze VIJI award)
Silva-Santos AR, Prazeres DMF, “Chromatographic purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures”, MicroBiotec2019, December 5-7, 2019, Coimbra, Portugal
Capela EV, Silva-Santos AR, Campos-Pinto I, Rodríguez MA, Gavara PR, Fernandez-Lahore M, Aires-Barros MR, Azevedo AM, “LYTAG-driven purification strategies as a key to integrate and intensify the downstream processing of monoclonal antibodies”, MicroBiotec2017, December 7-8, 2017, Porto, Portugal
Poster Communications
Silva-Santos AR, Paulo, PMR, Prazeres DMF, Purification of ssDNA scaffolds for DNA-origami nanostructures biomanufacturing, 2nd Chem & Biochem Students Meeting, July 2022, Lisboa, Portugal
Silva-Santos AR, Oliveira-Silva R, Paulo PMR, Prazeres DMF, Single-step purification of ssDNA scaffolds using affinity magnetic beads for DNA-origami biomanufacturing, 40th ISPPP – International Symposium on the Separation of Proteins, Peptides and Nucleotides, November 2021, Porto, Portugal (Best Poster Prize)
Silva-Santos AR, Prazeres DMF, Chromatographic purification of single stranded DNA scaffolds for DNA-origami nanostructures biomanufacturing, 13th ESBES – European Symposium on Biochemical Engineering Sciences 2021, May 2021, virtual event
Silva-Santos AR, Prazeres DMF, “Biomanufacturing of DNA-origami nanostructures for bio-imaging”, PhD open days 2018Instituto Superior Técnico, Lisboa, Portugal
A.Rita Silva-Santos, Cláudia P.A. Alves, Michaela Simčíková, Gabriel Monteiro, Ana M Azevedo, Duarte Miguel F. Prazeres, “Multimodal chromatography of supercoiled minicircles: a closer look into DNA-ligand interactions, 12th ESBES – European Symposium on Biochemical Engineering Sciences, September 2018, Lisboa, Portugal
Silva-Santos AR, Alves CPA, Šimčiková M, Monteiro G, Azevedo AM, Prazeres DMF, “Multimodal chromatography: a closer look to supercoiled minicircle DNA interactions”, MicroBiotec2017 December 7-8, 2017, Porto, Portugal
DOCTORAL PROGRAM (36 ECTS)
Curricular units:
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Nanobiotechnology (6 ECTS)
• Advanced Topics in Bioengineering and Biosciences (6 ECTS)
Advanced courses:
• ABC2 – Anything but conventional chromatography workshop, Lisbon, Portugal, November 22-24, 2017
• BIOPRO World Talent Campus, organized by DTU and sponsored by NovoNordisk Foundation, Copenhagen, Denmark, August, 2018
• Cell Culture-based Viral Vaccines course – 3rd edition, organized by the European Society for Animal Cell Technology in Llafranc, Girona, Spain, September, 2018
• Drug Development course – 3rd edition, organized by the European Society for Animal Cell Technology in Llafranc, Girona, Spain, September, 2018
Current position
Research Scientist at IBB, Instituto Superior Técnico, PT