Main publications (last 5 years)
Papers on XNAzymes
Taylor, A. I., Wan, C. J. K., Donde, M. J., Peak-Chew, S. Y. and Holliger, P. (2022). A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing. Nat Chem 14, 1295–1305.
Taylor, A. I. and Holliger, P. (2022). On gene silencing by the X10-23 DNAzyme. Nat Chem 14, 855–858.
Here (and in Freund et al (2023) (see below)) we describe the in vitro evolution and characterization of RNA endonuclease XNAzymes that are active at physiological Mg concentrations and are able to cleave target RNAs with single nucleotide specificity. We show that one such XNAzyme is active in vivo and is able to specifically knock down the mutant G12D KRAS-mRNA in heterozygous adenocarcinoma cells. In the accompanying “Matters arising” article, we discuss the commonly ignored pitfalls of XNAzyme analysis such as RNAseH activation, background cleavage and RT-PCR artefacts.
Papers on XNA polymerase and reverse transcriptase engineering
Houlihan, G., Arangundy-Franklin, S., Porebski, B. T., Subramanian, N., Taylor, A. I. and Holliger, P. (2020). Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity. Nat Chem 12, 683–690.
Freund, N., Taylor, A. I., Arangundy-Franklin, S., Subramanian, N., Peak-Chew, S. Y., Whitaker, A. M., Freudenthal, B. D., Abramov, M., Herdewijn, P. and Holliger, P. (2023). A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA. Nat Chem 15, 91–100.
We describe two different approaches to the engineering polymerases for the XNA reverse transcription and synthesis with a special focus on 2’OMe-RNA, a chemistry with a range of favourable physicochemical, pharmacological and immunological properties that occurs naturally in human mRNA, tRNA and rRNA and has been validated for clinical utility in the FDA-approved siRNA drug Patisiran. We furthermore describe discovery of the first 2’OMe-RNAzymes and the synthesis and characterization of 2'-O-(2-methoxyethyl)-(MOE) RNA oligomers and mixed 2’OMe- / MOE-RNA aptamers.
Deep screening paper
Porebski, B. T., Balmforth, M., Browne, G., Riley, A., Jamali, K., Furst, M., Velic, M., Buchanan, A., Minter, R., Vaughan, T. and Holliger, P. (2024). Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening. Nat Biomed Eng 8, 214–232.
We introduce deep screening, an ultra-high-throughput approach leveraging the Illumina HiSeq platform for massively parallel sequencing, display, and rapid affinity screening at the level of >10e8 individual antibody-antigen interactions. Deep screening enabled the discovery of mid- to high-picomolar single-chain Fv (scFv) antibody leads directly from unselected, synthetic scFv repertoires augmented by machine learning.
Papers on RNA-catalyzed RNA replication
Attwater, J., Augustin, T. L., Curran, J. F., Kwok, S. L. Y., Ohlendorf, L., Gianni, E. and Holliger, P. (2025). Trinucleotide substrates under pH-freeze-thaw cycles enable open-ended exponential RNA replication by a polymerase ribozyme. Nat Chem 17, 1129–1137.
We describe progress in solving the strand-separation problem of RNA replication, where RNA duplex strands serve as dead-end products due to their very high stability. We describe how coupled pH and freeze-thaw cycles in conjunction with trinucleotide substrates unlock open-ended, exponential RNA replication over many cycles. Primer-free and / or RNA replication of random RNA template showed emergence of partial ribozyme self-replication together with de novo generation of new RNA sequences with a biased triplet usage largely matching family-box codons of the genetic code.
Gianni, E., Kwok, S. L. Y., Wan, C. J. K., Goeij, K., Clifton, B. E., Attwater, J. and Holliger, P. (2026) A small polymerase ribozyme that can synthesize itself and its complementary strand. Science 391 :1022-1028. doi: 10.1126/science.adt2760.
We describe the discovery and characterization of an unprecedently small 45-nucleotide polymerase ribozyme, discovered from random sequence pools, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate (triplet) substrates. QT45 is the first ribozyme that can synthesize copies of both its complementary strand itself. The discovery of polymerase activity in a small RNA motif suggests that polymerase ribozymes are more abundant in RNA sequence space than previously thought, facilitating the emergence of self-replication.
McRae, E. K. S., Wan, C. J. K., Kristoffersen, E. L., Hansen, K., Gianni, E., Gallego, I., Curran, J. F., Attwater, J., Holliger, P. and Andersen, E. S. (2024). Cryo-EM structure and functional landscape of an RNA polymerase ribozyme. Proc Natl Acad Sci USA 121, e2313332121.
We describe the first structure of a RNA polymerase ribozyme, solved to 5-Å resolution by Cryo-EM. The structure reveals the triplet polymerase ribozyme (TPR) apoenzyme an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit held together two distinct kissing-loop interactions that are essential for polymerase function. We furthermore describe high-throughput adaptive landscape analysis, mapping functionally important residues to the structure and suggest a model for templated RNA synthesis by the TPR holoenzyme consistent with all the data.
Kristoffersen, E. L., Burman, M., Noy, A. and Holliger, P. (2022). Rolling circle RNA synthesis catalyzed by RNA. Elife 11, e75186.
Kristoffersen, E. L., McRae, E. K. S., Sorensen, N. R., Holliger, P. and Andersen, E. S. (2025). Roles of dimeric intermediates in RNA-catalyzed rolling circle synthesis. Nucleic Acids Res 53, gkaf057.
We describe the establishment and characterization of Rolling circle RNA synthesis (RCS) by the the triplet polymerase ribozyme (TPR) over multiple cycles. We explore potential RCS mechanisms by MD simulations and - in a second publication - by structural analysis of putative RCS replication intermediates by CryoEM. These indicate a progressive build-up of conformational strain within the circular RNA templates, which is relieved by dimer formation.
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