Linggen Kong

Peptide nucleic acid (PNA) and DNAzymes have recently been used to develop an artificial DNA nuclease system named PNA-assisted double-stranded DNA nicking by DNAzymes (PANDA) for genetic engineering. Interestingly, the PANDA system demonstrated a higher sequence fidelity than CRISPR/Cas9, with the ability to discriminate single-nucleotide mismatches. To evaluate the source of PANDA's sequence fidelity, we conducted kinetic experiments that separately examined the kinetics of PNA invasion and DNAzyme cleavage, each under rate-limiting conditions. Our results show that PNA serves as an initial mismatch "inspector," while DNAzyme adds complementary specificity during the cleavage process. Notably, PNA and DNAzyme recognize mismatches at opposite ends of their binding regions, enabling cooperative discrimination of mismatches across the entire target site, including regions that are typically difficult to distinguish by other methods. This dual recognition mechanism enhances PANDA's sequence fidelity, particularly in single-nucleotide mismatch discrimination. These findings establish PANDA as a promising molecular tool for precise, targeted DNA manipulation, offering a robust platform for applications that require stringent sequence specificity.

Site‐directed mutagenesis (SDM) is a key driver for many biochemical investigations and biotechnological applications. Despite decades of development, it is still difficult to perform SDM on some sequences, including highly repetitive ones, because polymerase chain reaction (PCR) is often used as the most effective method for SDM, but results in complicated products with these sequences. To overcome this limitation, we report herein a PCR‐free SDM method that uses DNA‐cleaving DNAzymes. Since these DNAzymes lack the capability of cleaving double‐stranded DNA, we employed single‐stranded DNAs as assisting DNAs to open superhelical plasmids, allowing the DNA‐cleaving DNAzymes to cleave the plasmid. Such a system, named single‐stranded DNA‐assisted double‐stranded DNA nicking by DNAzymes (DANDA), expanded the substrate scope of DNAzymes to double‐stranded, superhelical plasmids. We showed that DANDA is highly customizable and target‐specific, which allows successful generation of mutations on a plasmid containing PCR‐incompatible repetitive sequences. By using solely unmodified DNA oligos that are more cost‐effective and easier to prepare than other systems that employ either PNA or restriction enzymes, this DANDA system further expands the applications of DNA‐cleaving DNAzymes in synthetic biology for different biochemical and biotechnological applications.

Site‐directed mutagenesis (SDM) is a key driver for many biochemical investigations and biotechnological applications. Despite decades of development, it is still difficult to perform SDM on some sequences, including highly repetitive ones, because polymerase chain reaction (PCR) is often used as the most effective method for SDM, but results in complicated products with these sequences. To overcome this limitation, we report herein a PCR‐free SDM method that uses DNA‐cleaving DNAzymes. Since these DNAzymes lack the capability of cleaving double‐stranded DNA, we employed single‐stranded DNAs as assisting DNAs to open superhelical plasmids, allowing the DNA‐cleaving DNAzymes to cleave the plasmid. Such a system, named single‐stranded DNA‐assisted double‐stranded DNA nicking by DNAzymes (DANDA), expanded the substrate scope of DNAzymes to double‐stranded, superhelical plasmids. We showed that DANDA is highly customizable and target‐specific, which allows successful generation of mutations on a plasmid containing PCR‐incompatible repetitive sequences. By using solely unmodified DNA oligos that are more cost‐effective and easier to prepare than other systems that employ either PNA or restriction enzymes, this DANDA system further expands the applications of DNA‐cleaving DNAzymes in synthetic biology for different biochemical and biotechnological applications.

DNA aptamers struggle to detect CoA due to the dominance of adenosine in SELEX. Using capture SELEX with rigorous counter selections, a CoA-specific aptamer is obtained and converted to a fluorescent sensor, allowing visualizing CoA in living cells.

Glycosylated RNAs (glycoRNAs) have recently emerged as a new class of molecules of substantial interest owing to their potential roles in cellular processes and diseases. However, studying glycoRNAs is challenging owing to the lack of effective research tools including, but not limited to, imaging techniques to study the spatial distribution of glycoRNAs. Recently, we reported the development of a glycoRNA imaging technique, called sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA), to visualize sialic acid-containing glycoRNAs with high sensitivity and specificity. Here we describe the experimental design principles and detailed step-by-step procedures for ARPLA-assisted glycoRNA imaging across multiple cell types. The procedure includes details for target selection, oligo design and preparation, optimized steps for RNA in situ hybridization, glycan recognition, proximity ligation, rolling circle amplification and a guideline for image acquisition and analysis. With properly designed probe sets and cells prepared, ARPLA-based glycoRNA imaging can typically be completed within 1 d by users with expertise in biochemistry and fluorescence microscopy. The ARPLA approach enables researchers to explore the spatial distribution, trafficking and functional contributions of glycoRNAs in various cellular processes.

Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization methods. We report sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA) to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggest that surface glycoRNA is inversely associated with tumor malignancy and metastasis. Investigation of the relationship between glycoRNAs and monocyte–endothelial cell interactions suggests that glycoRNAs may mediate cell–cell interactions during the immune response.

The discovery of synthetic molecules that can bind biomolecules such as proteins with high specificity and affinity is very important in our understanding and modulation of the functions of these biomolecules. DNA-encoded library technologies have been used to screen such molecules from different libraries; however, the current methods have not been able to truly tap into Darwinian selection to regenerate molecular diversity after each screening. In this article, the authors designed a mating strategy, used by natural evolution, by shuffling two hybridizable DNA-encoded libraries (DEL) in each selection round to achieve lineage crossing based diversification, where the actual peptide ligands under selection that are modified to assemble with DEL can thus take advantage of such effects to form diversified combinations, which boosts the selection space when the original libraries are relatively small. Though only low-molecule-weight libraries were used in this study, further development of the mating strategy has the potential to better interrogate higher-molecule-weight diversity space to cover larger interfaces of interactions and more possibilities for protein-based therapeutics.

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

The Trx and Grx systems are deeply involved in bacterial responses to H ₂ O ₂ -induced oxidative stress. However, little is known about their roles in response to other ROS, such as organic peroxides (OPs). In this study, we used S. oneidensis as a research model to investigate the interplay between Trx/Grx and OxyR/OhrR. We show that Trxs mediate the redox status of transcriptional OP-responding regulator OhrR. Although none of the trx or grx genes are directly controlled by OxyR or OhrR, expression of trxA and trxC is induced by tert -butyl hydroperoxide ( t -BHP). We further show that the trxA and trxC genes respond to effects of glutathione (GSH) depletion rather than oxidation. These findings underscore the particular importance of Trx in the bacterial OP stress response.