Research Achievements and Peer Recognition

1. Antibody engineering and discovery: During my Ph.D. and postdoc I developed a new type of recombinant antibody fragment, called a diabody [1]. Bivalent diabodies have been widely used in molecular imaging applications since then, while bispecific diabodies have shown significant potential in the immunotherapy. More recently my group has developed an ultra-high-throughput method for antibody screening and discovery (deep screening) [2], which also forms the basis for the spinout company Sortera Bio Ltd.
2. Phage biology: Working independently, I discovered an infection pathway unique among prokaryotic viruses, but with close analogies to a wide range of eukaryotic viruses including herpes-, adeno- and lentiviruses such as HIV [3].
3. Emulsion PCR technology: During my tenure track, my group developed methods for emulsion-PCR, which enabled clonal DNA amplification within the aqueous compartments of a heat-stable water-in-oil emulsion. Based on this, we devised strategies for the directed evolution of polymerases [4]. Our methods of emulsion PCR and polymerase evolution have found wide commercial application, as part of multiple next-generation sequencing platforms (Roche; Life Technologies & Ion Torrent) and diagnostic digital PCR systems (Bio-Rad) and in polymerase discovery (New England Biolabs, Agilent).
4. Xeno-nucleic acids (XNAs): Combining nucleic acid chemistry with novel methods for in vitro evolution, my group engineered replicative DNA polymerases for the synthesis, replication and evolution of synthetic genetic polymers with entirely unnatural backbones (Xeno-Nucleic Acids (XNAs) [5]). By de novo selections directly from pools of random XNA sequences, we discovered multiple specific high-affinity binders (“XNA aptamers”) to a range of molecular targets [5] including, recently, the first ever aptamers with uncharged backbones [6]. By the same approach, we discovered catalysts (“XNAzymes”) with sequence-specific RNA endonuclease or ligase activity elaborated in four different XNA backbone chemistries [7]. This work demonstrated that three defining hallmarks of life - heredity, evolution and catalysis – are not restricted to the natural biopolymers on which life is based, but rather are emerging properties of a potentially wide range of synthetic sequence defined polymers.
5. RNA-catalyzed RNA replication: As a critical test of the RNA world hypothesis of the origin of life, my group has explored the possibility of RNA self-replication. Based on a previously described RNA polymerase ribozyme (RPR), we discovered a new RPR that could synthesize RNAs up to 206 nucleotides long [8] or synthesize another ribozyme [9]. RNA function (such as RPR activity) critically hinges on the folding of RNA strands into a defined structure, but such structures proved strong impediments to RNA replication. In order to overcome this apparent paradox at the heart of RNA self-replication, my group engineered a novel polymerase ribozyme that utilizes RNA trinucleotides (triplets) as substrates [10]. Due to their ability to bind RNA template strands with much higher affinity than mononucleotides, triplets can cooperatively invade and open up even highly stable RNA structures for “copying” and thus resolve this central incongruity of RNA-catalysed RNA replication. Triplet-based RNA replication enabled this ribozyme to copy its own sequence in segments as well as non-canonical primer-free and reverse direction (3’-5’) modes of RNA replication not seen in nature [10].
6. Eutectic ice phases: In the course of this work, we discovered that water-ice, a simple medium likely to have been widespread on the early Earth, promotes RNA stability, replication and evolution. These counterintuitive effects arise from the unique physico-chemical properties of the ice environment, including the concentration and quasi-cellular compartmentalization of solutes within an eutectic brine phase surrounding the ice crystals, which also enables in-ice evolution of RNA polymerase ribozyme activity [8].

1. Holliger P, Prospero T & Winter G. (1993) Diabodies; small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA. 90: 6444-48. (cited by 2329)*.
   (* Google Scholar)
2. Porebski BT, Balmforth M, Browne G, Riley A, Jamali K, Fürst MJLJ, Velic M, Buchanan A, Minter R, Vaughan T & Holliger P (2024) Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening. Nature Biochem Eng 8: 214–32. (cited by 63)*
3. Riechmann L & Holliger P (1997) The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli. Cell 90: 351-60 (cited by 327)*
4. Ghadessy FJ, Ong JL & Holliger P (2001) Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci USA 98: 4552-57 (cited by 608)*
5. Pinheiro VB, Taylor AI, Cozens C, Abramov M, Renders M, Zhang S, Chaput JC, Wengel J, Peak-Chew SY, McLaughlin SH, Herdewijn P & Holliger P (2012) Synthetic genetic polymers capable of heredity and evolution. Science 336: 341-4. (cited by 848)*
6. Arangundy-Franklin S, Taylor AI, Porebski BT, Genna V, Peak-Chew S, Vaisman A, Woodgate R, Orozco M & Holliger P (2019). A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids. Nature Chemistry 11: 533-42 (cited by 101)*
7. Taylor AI, Pinheiro VB, Smola MJ, Morgunov AS, Peak-Chew SY, Cozens C, Weeks KM, Herdewijn P & Holliger P (2015) Catalysts from synthetic genetic polymers. Nature 518: 427-30 (cited by 319)*
8. Attwater J, Wochner A & Holliger P (2013) In-ice evolution of RNA polymerase ribozyme activity. Nature Chemistry 5 :1011-8. (cited by 340)*
9. Wochner A, Attwater J, Coulson A & Holliger P (2011) Ribozyme-catalyzed transcription of an active ribozyme. Science 332: 209-12. (cited by 492)*
10. Attwater J, Raguram A, Morgunov AS, Gianni E & Holliger P (2018) Ribozyme-catalysed RNA synthesis using triplet building blocks. eLife. 7: e35255. (cited by 149)*