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The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.This Review provides an overview of four platform technologies that are currently used in the clinic for delivery of nucleic acid therapeutics, describing their properties, discussing technical advancements that led to clinical approval, and highlighting examples of approved genetic drugs that make use of these technologies.

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR–Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples.

The introduction of AlphaFold 2 1 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design 2 , 3 , 4 , 5 – 6 . Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein–ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein–nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody–antigen prediction accuracy compared with AlphaFold-Multimer v.2.3 7 , 8 . Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework. AlphaFold 3 has a substantially updated architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues with greatly improved accuracy over many previous specialized tools.

Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s. The authors describe how to easily prepare a large number of dipsticks from cellulose-based filter paper and use them to rapidly purify nucleic acids from a variety of sources.

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health. This Review summarizes the rapidly expanding toolbox of assays for CRISPR-based diagnostics and highlights their point-of-care applications.

The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type–specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.

Nature has achieved exquisite sequence control in the synthesis of polymers like DNA. In contrast, synthetic polymers rarely have the same fidelity in their chemistry or uniformity in chain-length distribution, especially when more than one monomer is involved. Lutz et al. ( 1238149 ) review the progress that has been made in making sequence-controlled polymers of increasing length and complexity. These developments have come from both advances in synthetic chemistry methods and the exploitation of biological machinery. Sequence-controlled polymers are macromolecules in which monomer units of different chemical nature are arranged in an ordered fashion. The most prominent examples are biological and have been studied and used primarily by molecular biologists and biochemists. However, recent progress in protein- and DNA-based nanotechnologies has shown the relevance of sequence-controlled polymers to nonbiological applications, including data storage, nanoelectronics, and catalysis. In addition, synthetic polymer chemistry has provided interesting routes for preparing nonnatural sequence-controlled polymers. Although these synthetic macromolecules do not yet compare in functional scope with their natural counterparts, they open up opportunities for controlling the structure, self-assembly, and macroscopic properties of polymer materials.

DNA-templated synthesis takes advantage of the programmability of DNA-DNA interactions to accelerate chemical reactions under diluted conditions upon sequence-specific hybridization. While this strategy has proven advantageous for a variety of applications, including sensing and drug discovery, it has been so far limited to the use of nucleic acids as templating elements. Here, we report the rational design of DNA templated synthesis controlled by specific IgG antibodies. Our approach is based on the co-localization of reactants induced by the bivalent binding of a specific IgG antibody to two antigen-conjugated DNA templating strands that triggers a chemical reaction that would be otherwise too slow under diluted conditions. This strategy is versatile, orthogonal and adaptable to different IgG antibodies and can be employed to achieve the targeted synthesis of clinically-relevant molecules in the presence of specific IgG biomarker antibodies. DNA-templated synthesis takes advantage of sequence-specific hybridization to accelerate chemical reactions. Here, the authors present templated synthesis controlled through antibody-antigen interactions.

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5′-untranslated regions (5′-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.