Biology subjects

Sieskind, R.

Publications and source records attributed to Sieskind, R..

5 recordsLinked to original sources

bioRxiv2026

Directed evolution of a thermostable family-A DNA polymerase into a reverse transcriptase for 2'-modified RNA

RNA-based therapeutics increasingly rely on nuclease-resistant xeno-nucleic acids (XNAs), but engineering polymerases capable of reading and amplifying these non-canonical substrates remains a major challenge. Natural DNA polymerases cannot process C2' sugar modifications, and the complete structural determinants governing this limitation remain poorly understood, limiting rational design strategies. In this study, we apply directed evolution methods to Thermus thermophilus DNA polymerase using a microfluidic implementation of the Compartmentalized Self-Replication (CSR). We identify an engineered variant, termed E5, capable of reverse transcribing C2'-modified RNA, a previously inaccessible activity for this enzyme. Next-generation sequencing allow us to track evolutionary trajectories across nine rounds of evolution. Through an integrated analysis combining statistical methods, protein language models, and crystallographic data, we reveal unknown epistatic networks controlling polymerase function. This large-scale experimental dataset (>196,000 thermostable enzyme variants) provides a robust basis for predicting sequence-to-function relationships, accelerating development of enzymes for aptamer discovery and synthetic biology applications.

synthetic biology↗

bioRxiv2026

Polymerase Evolution Enables Access to Glycosylated Xeno-Nucleic Acids with Expanded Chemical Functionality

Chemical modifications expand the functional repertoire of nucleic acids, but the extent to which complex glycans can be integrated into genetic polymers remains largely unexplored. Glycosylated nucleic acids, including glycoRNAs and bacteriophage genomes, are emerging as key mediators of host-pathogen interactions, yet their synthetic accessibility is severely limited by the inability of polymerases to process substrates bearing simultaneous base and backbone modifications. Here we report the directed evolution of a family B DNA polymerase that enables the synthesis of glycosylated xeno-nucleic acids (XNAs). Using fluorescence-activated droplet sorting, we identified an engineered polymerase, termed G2, that efficiently incorporates nucleotides bearing mono- to trisaccharide base-appendages together with C2 substitutions, yielding XNAs containing up to 30% glycosylated bases. Deep sequencing of >93,000 chimeras reveals that this activity arises from epistatic networks concentrated within catalytic domains. These results establish a route to glycan-encoded nucleic acids and advance polymerase engineering for chemically expanded therapeutics.

synthetic biology↗

bioRxiv2025

Functional selection in a population of synthetic cells with a minimal metabolism

Various synthetic microcompartment systems have been developed to mimic key features of living cells. Here, we focus on artificial cells that capture their capacity to serve as vessels for Darwinian evolution. We assemble micro-compartmentalized In Vitro Transcription-Translation-Replication systems containing a minimal genome, a basic metabolic pathway, a reconstituted protein expression machinery, and a simple DNA replication module, wired in a positive feedback loop. The minimal genome encodes the enzyme deoxyribonucleoside kinase (DNK) whose expression, and then metabolic activity, is required for the genomes replication. We show that these compartments act as minimal Darwinian elements by filtering out non-functional genotypes. We track individual replicators from a library of 42 genetic variants to reveal the systems dynamics at both the population and the single replicator levels. At the population level, we extract the fitness function, which links a genomes metabolic efficiency to its selective success, considering co-encapsulation and hitch-hiking effects. At the individual replicator level, we observe a bimodal distribution of replication yields and propose a mixed model with an inter-droplet heterogeneity with presence or absence of a metabolic feedback loop on the replicator. In addition, we leverage this autonomous self-selection loop to generate a high-resolution mutational map of the DNK enzyme.

synthetic biology↗

bioRxiv2025

Structural Basis for DNA Replication and Uracil Repair in Phage A-Family DNA Polymerases

Replicative DNA polymerases (DNAP) play a critical role in genome duplication, ensuring the accurate transmission of genetic information in all kingdoms of life. This process is essential also for DNA-dependent viruses, including bacteriophages. In many phage genomes, a uracil-DNA glycosylase (UDG) is encoded in trans. In this paper, we identify a new subfamily of A-family DNAP in phages that are fused to an active (UDG) domain. Two members of this subfamily, B. subtilis phage SP-15 and YerA41 are known to be hypermodified on their thymidines. Here, we present cryo-EM structures at high resolution for two of its members in various functional and conformational states, from YerA41 and phiLo phages. The structures explain how these DNAPs can have an activity, distinct from copying genetic information, which reads dU bases ahead of the replication fork and creates abasic sites that are efficiently bypassed by the DNAP. Additionally, we report the co-occurrence of both a X-family DNAP and a DNA ligase in the corresponding phage genomes, and show that both enzymes are capable of repairing the abasic sites. The former makes a stable complex with the replicative DNAP, which thus appears as a platform for recruitment of the enzymatic activities necessary for the repair of dU bases during replication, that result from the incorporation of residual dUTP in the pool of nucleotides. Strikingly, the location of the UDG domain is the same as in the structure of Mpox virus replicative complex involving a B-family DNAP.

molecular biology↗

bioRxiv2021

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

High-throughput directed evolution, implemented in well-controlled in vitro conditions, provides a powerful route for enzyme engineering. Most existing technologies are based on activity screening and require the sequential observation and sorting of each individual variant. By contrast, approaches based on autonomous feedback loops, linking phenotype to genotype replication, enable autonomous selection without screening. However, these approaches are only possible in vivo, or applicable to very specific activities, such as polymerases or ligases. Here, we leverage synthetic molecular networks to create a programmable in vitro feedback loop linking a target enzymatic activity to gene amplification. After encapsulation and lysis of up to 107 transformed variants, the genes present in each droplet are amplified according to the activity of the encoded enzyme, resulting in the autonomous enrichment of interesting sequences. Applied to a nicking enzyme with thermal or kinetic selection pressures, this method reveals detailed mutational landscapes and provides improved variants.

bioengineering↗