Targeted protein degradation is a powerful therapeutic approach: expanding the druggable proteome, providing enhanced selectivity, and having the ability to overcome conventional resistance mechanisms. A major class of such molecules is proteolysis-targeting chimeras (PROTACs). PROTACs are catalytic heterobifunctional small molecules that simultaneously bind a protein of interest (POI) and an E3 ligase. And thus, PROTACs induce a proximity-dependent ubiquitination of the POI and its subsequent degradation by the ubiquitin-proteasome system. While PROTACs have successfully transitioned from academia to industry, increasing awareness of off-target effects and related toxicities highlights the urgent need for precise control mechanisms over activity. Achieving this level of control, however, remains challenging, with traditional chemistries. DNA nanotechnology, with its unparalleled programmability and structural versatility, presents a powerful tool for achieving such control. Here, we report the design and characterization of oligonucleotide-linked PROTACs (OligoPROTACs). These constructs comprise PROTAC warheads covalently linked to separate complementary DNA strands, brought together in space via DNA hybridization. OligoPROTACs are able to degrade the POI in a distance-dependent manner. Furthermore, we demonstrate the first instance of a dynamic off-switch mechanism for PROTAC activity, enabled by toehold-mediated strand displacement using a third DNA strand. This work highlights the potential of DNA nanotechnology combined with the clinical emergence of nucleic acid therapeutics to enhance the safety and functionality of PROTAC systems, paving the way for more refined and translatable therapeutic strategies.

“Where we hope this work can go next is toward applications such as drug release.”Jokūbas Leikauskas, Editor, BioInsights, speaks to Michael Booth, Associate Professor of Chemical Biology and Organic Chemistry, UCL, about how nucleic acid conjugation can expand the functional capabilities of DNA and RNA, enabling precise control over their activity, localization, and delivery in therapeutic contexts. They also discuss how these strategies are being applied in synthetic cells and cell-free systems to remotely control biological processes and enable communication between synthetic and living systems.

Cell signaling and communication are fundamental to living cellular communities. Over the past two decades, there has been a continuous development of bottom-up engineered synthetic cells, which have become increasingly similar to their natural counterparts. However, we are only scratching the surface with the development of synthetic cellular communities and their integration into natural tissues. Here we review different intercellular communication mechanisms engineered for synthetic cells and classify them based on their resemblance to natural cell signaling mechanisms-autocrine, paracrine and juxtacrine. In particular, we highlight recent advances in molecular tools for intercellular communication strategies and discuss potential applications of engineering synthetic cellular communities and synthetic cell-natural cell communication. With further advances in this area, synthetic cellular communities will be powerful tools for understanding and manipulating cellular functions, thus unlocking potential applications in biosensing, cellular reprogramming and sustainability.

Antisense oligonucleotides (ASOs) are a promising class of therapeutics designed to modulate gene expression. Both key mechanisms of action for ASOs operate in the nucleus: splice-switching ASOs modify pre-mRNA, processed in the nucleus, and mRNA-degrading ASOs require RNase H, an enzyme predominantly active in the nucleus. Therefore, to achieve maximal therapeutic efficacy, ASOs require efficient nuclear delivery. In this work, we have synthesized ASO conjugates for active nuclear import, by covalent conjugation with a potent and proven small-molecule nuclear importer, (+)-JQ1. (+)-JQ1 is a well-characterized high-affinity binder for members of the BET bromodomain family of proteins and was recently shown to transport cytoplasmic proteins into the nucleus. Our (+)-JQ1-ASO conjugates outperformed their unmodified counterparts for both splice-switching and mRNA knockdown in the nucleus, across different molecular targets, backbone chemistries, and cell lines. In addition, we show that the improvement in on-target efficacy correlates with increased nuclear localization of the (+)-JQ1-modified ASOs by subcellular fractionation and immunocytochemistry. Notably, we improved the performance of Oblimersen, a BCL-2 ASO drug that failed in phase-III clinical trials. (+)-JQ1-Oblimersen showed increased effectiveness in an acute myeloid leukemia cell model, showing that this therapeutic may merit re-evaluation. This work demonstrates that the covalent modification of ASOs with a small-molecule nuclear importer can significantly improve target engagement and pave the way for more effective therapeutics.

Antisense oligonucleotides (ASOs) are a promising class of therapeutics designed to modulate gene expression. Both key mechanisms of action for ASOs operate in the nucleus: splice-switching ASOs modify pre-mRNA, processed in the nucleus, and mRNA-degrading ASOs require RNase H, an enzyme predominantly active in the nucleus. Therefore, to achieve maximal efficacy, ASOs require efficient nuclear delivery. Current ASO therapeutics shuttle in and out of the nucleus inefficiently. In this work, we have synthesised ASO conjugates for active nuclear import, by covalent conjugation with a potent small-molecule nuclear importer, (+)-JQ1. (+)-JQ1 is a well-characterised high-affinity binder for members of the BET bromodomain family of proteins and was recently shown to transport cytoplasmic proteins into the nucleus. Our (+)-JQ1-ASO conjugates outperformed their unmodified counterparts for both splice-switching and mRNA knockdown in the nucleus. In particular, we improved the performance of Oblimersen, a BCL-2 ASO drug that failed phase-III clinical trials, showing that this therapeutic may merit re-evalutaion. This work shows that the covalent modification of ASOs with a small-molecule nuclear importer can significantly improve target engagement and pave the way for more effective therapeutics.

Targeted protein degradation is a powerful therapeutic approach: expanding the druggable proteome, providing enhanced selectivity, and having the ability to overcome conventional resistance mechanisms. A major class of such molecules are proteolysis-targeting chimeras (PROTACs). PROTACs are catalytic heterobifunctional small molecules that simultaneously bind a protein of interest (POI) and an E3 ligase. These PROTACs induce a proximity-dependant ubiquitination of the POI, which causes its subsequent degradation by the ubiquitin–proteasome system. While PROTACs have successfully transitioned from academia to industry, increasing awareness of off-target effects and related toxicities highlight the urgent need for precise control mechanisms over their activity. Achieving this level of control, however, remains challenging with traditional chemistries. DNA nanotechnology, with its unparalleled programmability and structural versatility, presents a powerful tool for achieving such control. Here, we report the design and characterization of oligonucleotide-linked PROTACs (OligoPROTACs). These constructs comprise PROTAC warheads covalently linked to separate, complementary DNA strands, brought together in space via DNA hybridisation. OligoPROTACs are able to degrade the POI in a distance-dependant manner. Furthermore, we demonstrate the first-instance of a dynamic off-switch mechanism for PROTAC activity, enabled by toehold-mediated strand displacement using a third DNA strand. This work highlights the potential of DNA nanotechnology to enhance the safety and functionality of PROTAC systems, paving the way for more refined and translatable therapeutic strategies.

The flexible and modular design of synthetic cells, comprising lipid vesicles capable of imitating the structure and function of living cells, facilitates their application as drug delivery devices. The ability to control the synthesis of biomolecules within synthetic cells using a tissue-penetrating stimulus opens up additional levels of functionality that has the potential to improve biological potency and circumvent drug leakage from preloaded vesicles. To this end, we have designed spherical nucleic acids comprising DNA promoter sequences decorating magnetic nanoparticle cores. These spherical nucleic acids allowed us to harness the heat dissipated from magnetic hyperthermia (a clinically approved anticancer therapy) to regulate cell-free protein synthesis and release cargo on demand. Furthermore, this magnetic regulation of biosynthesis was achieved using clinically tolerable magnetic field strengths and frequencies. We then deployed an opaque blocking material that is impenetrable by current activation methods to highlight the potential of this technology for targeting and controlling the in situ synthesis of biomolecules using tissue-penetrating magnetic fields deep within the body.

Polyethylene glycol (PEG) is widely used in liposome formulation due to its blocking properties and ability to prolong circulation in vivo to create biomimetic liposomes and drug delivery devices. Similarly, membrane-embedded DNA nanotechnology is increasingly used to modulate cellular behaviour and communication. However, there is a gap in knowledge in how PEG-lipid formulations can be optimized for both liposome properties and control of selective DNA hybridization. To address this, we systematically investigated the effect of liposome PEG content on DNA mediated tethering of liposomes to glass surfaces. We formulated liposomes of two different lipid compositions (DOPE/DOPC or DPhPC), with varying amounts of PEGylated lipid (0%-50%). We measured the effect of increased PEG content on liposome size and polydispersity through dynamic light scattering (DLS). Small amounts of PEG (0%-20%) introduced repulsive forces that reduced size, while large amounts of PEG (30%-50%) increased polydispersity. PEG-liposomes were then decorated with cholesterol-DNA strands and labeled with either intercalating lipid dyes or fluorescently labeled lipids. Binding to surfaces via complementary DNA strands was quantified using total internal reflection fluorescence (TIRF) microscopy. We found that PEGylation of DNA-liposomes could either block or enhance surface binding, depending on the amount of PEG. DNA-liposomes with reduced surface binding included DPhPC/DiD with 10% or 20% PEG-lipid. In contrast, DNA-liposome surface binding increased for DOPE/DOPC/DiD with increasing PEG%. This study highlights that while PEG can act to stabilize liposome formulations, its ability to block specific DNA-binding interactions on membranes is variable and dependent on membrane composition.

Nucleic acids have emerged as a powerful class of therapeutics. Through simple base pair complementarity, nucleic acids allow the targeting of a variety of pathologically relevant proteins and RNA molecules. However, despite the preliminary successes of nucleic acids as drugs in the clinic, limited biodistribution, inadequate delivery mechanisms, and target engagement remain key challenges in the field. A key area of research has been the chemical optimization of nucleic acid backbones to significantly enhance their "drug-like" properties. Alternatively, this review focuses on the next generation of nucleic acid chemical modifications: covalent biochemical conjugates. These conjugates are being applied to improve the delivery, functionality, and targeting. Exploiting research on heterobifunctionals, such as PROTACs, RIBOTACs, molecular glues, etc., has the potential to dramatically expand nucleic acid drug functionality and target engagement capabilities. Such next-generation chemistry-based enhancements have the potential to unlock nucleic acids as effective and versatile therapeutic agents.

The advancement of synthetic cells as drug delivery devices hinges on the development of targeting strategies, in particular the controlled synthesis of biomolecules in-situ using a deeply penetrative stimulus. To address this, we have designed spherical nucleic acids comprising DNA promoter sequences decorating magnetic nanoparticle cores. By harnessing the heat dissipated from magnetic hyperthermia (a clinically-approved anticancer therapy) we tightly controlled cell-free protein synthesis. We then deployed a tissue phantom that is impenetrable by current activation methods to demonstrate the potential of this technology for the remote control of synthetic cells using deeply tissue-penetrating magnetic fields. This paves the way for targeting and controlling the in-situ synthesis of biomolecules deep within the body. ### Competing Interest Statement The authors have declared no competing interest.

Plasmids are ubiquitous in biology, where they are used to study gene-function relationships and intricate molecular networks, and hold potential as therapeutic devices. Developing methods to control their function will advance their application in research and may also expedite their translation to clinical settings. Light is an attractive stimulus to conditionally regulate plasmid expression as it is non-invasive, and its properties such as wavelength, intensity, and duration can be adjusted to minimise cellular toxicity and increase penetration. Herein, we have developed a method to site-specifically introduce photocages into plasmids, by resynthesising one strand in a manner similar to Kunkel mutagenesis. Unlike alternative approaches to chemically modify plasmids, this method is sequence-independent at the site of modification and uses commercially available phosphoramidites. To generate our light-activated (LA) plasmids, photocleavable biotinylated nucleobases were introduced at specific sites across the T7 and CMV promoters on plasmids and bound to streptavidin to sterically block access. These LA-plasmids were then successfully used to control expression in both cell-free systems (T7 promoter) and mammalian cells (CMV promoter). These light-activated plasmids might be used to remotely control cellular activity and reduce off-target toxicity for future medical use. Our simple approach to plasmid modification might also be used to introduce novel chemical moieties for advanced function.

Synthetic cells, like their biological counterparts, require internal compartments with distinct chemical and physical properties where different functionalities can be localised. Inspired by membrane-less compartmentalisation in biological cells, here we demonstrate how micro-phase separation can be used to engineer heterogeneous cell-like architectures with programmable morphology and compartment-targeted activity. The synthetic cells selfassemble from amphiphilic DNA nanostructures, producing core-shell condensates due to size-induced de-mixing. Lipid deposition and phase-selective etching are then used to generate a porous pseudo-membrane, a cytoplasm analogue, and membrane-less organelles. The synthetic cells can sustain RNA synthesis via in vitro transcription, leading to cytoplasm and pseudo-membrane expansion caused by an accumulation of the transcript. Our approach exemplifies how architectural and functional complexity can emerge from a limited number of distinct building blocks, if molecular-scale programmability, emergent biophysical phenomena, and biochemical activity are coupled to mimic those observed in live cells.

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ gene expression and, thereby, synthetic cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with ultraviolet light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homoserine lactone synthase, BjaI, quorum-sensing-based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from nonliving matter to living matter, with applications in biology and medicine.

Developing simple methods to silence antisense oligonucleotides (ASOs) using photocages opens up the possibility of precise regulation of biological systems. Here, we have developed a photocaging strategy based on 'handcuffing' two ASOs to a protein. Silencing was achieved by divalent binding of two terminally photocleavable biotin-modified ASOs to a single streptavidin. These 'handcuffed' oligonucleotides showed a drastic reduction in gene knockdown activity in cell-free protein synthesis and were unlocked through illumination, regaining full activity.

Synthetic cells are modular gene-expressing compartments with promising applications in biology and medicine. However, a more diverse toolkit is needed to enhance their capabilities, particularly in terms of controlling their gene expression and employing novel synthetic cell-to-living cell signaling pathways. In this work, photocaged promoters and cell-free synthesis of the acyl homoserine lactone synthase BjaI were used to achieve light-activated communication between synthetic cells and living cells.

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, non-damaging stimuli such as visible light. Here, we have synthesised blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated UV light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.

Cell-free expression of a gene to protein has become a vital tool in nanotechnology and synthetic biology. Remote-control of cell-free systems with multiple, orthogonal wavelengths of light would enable precise, noninvasive modulation, opening many new applications in biology and medicine. While there has been success in developing ON switches, the development of OFF switches has been lacking. Here, we have developed orthogonally light-controlled cell-free expression OFF switches by attaching nitrobenzyl and coumarin photocages to antisense oligonucleotides. These light-controlled OFF switches can be made from commercially available oligonucleotides and show a tight control of cell-free expression. Using this technology, we have demonstrated orthogonal degradation of two different mRNAs, depending on the wavelength used. By combining with our previously generated blue-light-activated DNA template ON switch, we were able to start transcription with one wavelength of light and then halt the translation of the corresponding mRNA to protein with a different wavelength, at multiple timepoints. This precise, orthogonal ON and OFF remote-control of cell-free expression will be an important tool for the future of cell-free biology, especially for use with biological logic gates and synthetic cells.

Within many chemical and biological systems, both synthetic and natural, communication via chemical messengers is widely viewed as a key feature. Often known as molecular communication, such communication has been a concern in the fields of synthetic biologists, nanotechnologists, communications engineers, and philosophers of science. However, interactions between these fields are currently limited. Nevertheless, the fact that the same basic phenomenon is studied by all of these fields raises the question of whether there are unexploited interdisciplinary synergies. In this paper, we summarize the perspectives of each field on molecular communications, highlight potential synergies, discuss ongoing challenges to exploit these synergies, and present future perspectives for interdisciplinary efforts in this area.

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, nondamaging stimuli such as visible light. Here, we have synthesized blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated ultraviolet (UV) light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.