Complementary Oligonucleotides Research Articles

Development of an Integrated CMUTs-based Resonant Biosensor for Label-Free Detection of DNA with Improved Selectivity by Ethylene-Glycol Alkanethiols

Gravimetric resonant-inspired biosensors have attracted increasing attention in industrial and point-of-care applications, enabling label-free detection of biomarkers such as DNA and antibodies. Capacitive micromachined ultrasonic transducers (CMUTs) are promising tools for developing miniaturized high-performance biosensing complementary metal oxide silicon (CMOS) platforms. However, their operability is limited by inefficient functionalization, aggregation, crosstalk in the buffer, and the requirement for an external high-voltage (HV) power supply. In this study, we aimed to propose a CMUTs-based resonant biosensor integrated with a CMOS front-end interface coupled with ethylene-glycol alkanethiols to detect single-stranded DNA oligonucleotides with large specificity. The topography of the functionalized surface was characterized by energy-dispersive X-ray microanalysis. Improved selectivity for on-chip hybridization was demonstrated by comparing complementary and non-complementary single-stranded DNA oligonucleotides using fluorescence imaging technology. The sensor array was further characterized using a five-element lumped equivalent model. The 4-mm2 application-specific integrated circuit chip was designed and developed through 0.18 μm HV Bipolar-CMOS-Double diffused MOS (DMOS) technology (BCD) to generate on-chip 20V HV boosting and to track feedback frequency under a standard 1.8 V supply, with a total power consumption of 3.8 mW in a continuous mode. The measured results indicated a detection sensitivity of 7.943 × 10−3 μmol∙ L−1∙Hz−1 over a concentration range of 1 to 100 μmol∙L−1. In conclusion, the label-free biosensing of DNA under dry conditions was successfully demonstrated using a microfabricated CMUT array with a 2 MHz frequency on CMOS electronics with an internal HV supplier. Moreover, ethylene-glycol alkanethiols successfully deposited self-assembled monolayers on aluminum electrodes, which has never been attempted thus far on CMUTs, to enhance the selectivity of bio-functionalization. The findings of this study indicate the possibility of full-on-chip DNA biosensing with CMUTs.

Critical Issues on the Surface Functionalization of Plasmonic Au-Ag/TiO2 Thin Films with Thiolated Oligonucleotide-Based Biorecognition Elements.

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. M1A is generally located in the T loop of cytosolic tRNA and between acceptor and D stems of mitochondrial tRNAs, it is involved in tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template followed by qPCR using the ligation product as the template. M1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using sub-nanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires.

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.

Poly(ethylene Glycol) (PEG)–OligoRNA Hybridization to mRNA Enables Fine‐Tuned Polyplex PEGylation for Spleen‐Targeted mRNA Delivery

Organ‐selective targeting of mRNA polyplexes has been rarely explored despite the substantial potential of polymer‐based systems in mRNA delivery. In this study, spleen‐selective delivery of polyplexes is achieved by employing mRNA engineering to coat them with poly(ethylene glycol) (PEG). In this approach, mRNA is hybridized with PEGylated complementary RNA oligonucleotides (PEG–OligoRNAs), followed by the addition of linear poly(ethyleneimine). In this method, it is ensured that nearly all added PEG strands bind to the polyplexes, thereby enabling precise control of PEG amounts on the surface. Following systemic injection into mice, non‐PEGylated polyplexes yield robust protein expression in the lung and spleen. Intriguingly, adding a small number of PEG–OligoRNAs drastically reduces protein expression efficiency in the lung while preserving it in the spleen, realizing spleen targeting of mRNA polyplexes. Furthermore, PEGylated polyplexes demonstrate their potential utility in mRNA vaccination. In mechanistic analyses, non‐PEGylated polyplexes immediately agglomerate in the blood and deposit in the lung. Coating polyplexes with a small amount of short PEG effectively prevents these processes. Notably, even slight changes in PEG amounts and lengths dramatically impact the physicochemical properties and biological functionalities of the polyplexes, emphasizing the benefits of an mRNA engineering‐based approach for fine‐tuning polyplex PEG coating.

Sensing Levofloxacin with an RNA Aptamer as a Bioreceptor.

To combat the growing threat of antibiotic resistance, environmental testing for antibiotic contamination is gaining an increasing role. This study aims to develop an easy-to-use assay for the detection of the fluoroquinolone antibiotic levofloxacin. Levofloxacin is used in human and veterinary medicine and has been detected in wastewater and river water. An RNA aptamer against levofloxacin was selected using RNA Capture-SELEX. The 73 nt long aptamer folds into three stems with a central three-way junction. It binds levofloxacin with a Kd of 6 µM and discriminates the closely related compound ciprofloxacin. Furthermore, the selection process was analyzed using a next-generation sequencing approach to better understand the sequence evolution throughout the selection. The aptamer was used as a bioreceptor for the development of a lateral flow assay. The biosensor exploited the innate characteristic of RNA Capture-SELEX to select aptamers that displace a complementary DNA oligonucleotide upon ligand binding. The lateral flow assay achieved a limit of visual detection of 100 µM. While the sensitivity of this assay constrains its immediate use in environmental testing, the present study can serve as a template for the selection of RNA aptamer-based biosensors.

Sensing of gene expression in live cells using electrical impedance spectroscopy and DNA-functionalized gold nanoparticles

A novel electrical impedance spectroscopy-based method for non-destructive sensing of gene expression in living cells is presented. The approach used takes advantage of the robustness and responsiveness of electrical impedance spectroscopy and the highly specific and selective nature of DNA hybridization. The technique uses electrical impedance spectroscopy and gold nanoparticles functionalized with single-stranded DNA complementary to an mRNA of interest to provide reliable, real-time, and quantifiable data on gene expression in live cells. The system was validated by demonstrating specific detection of the uidA mRNA, which codes for the β-glucuronidase (GUS) enzyme, in Solanum lycopersicum MsK8 cells. Gold nanoparticles were functionalized with single-stranded DNA oligonucleotides consisting of either a sequence complementary to uidA mRNA or an arbitrary sequence. The DNA-functionalized gold nanoparticles were mixed with cell suspensions, allowing the gold nanoparticles to penetrate into the cells. The impedance spectra of suspensions of cells with gold nanoparticles inserted within them were then studied. In suspensions of uidA-expressing cells and gold nanoparticles functionalized with the complementary single-stranded DNA oligonucleotide, the impedance magnitude in the frequency range of interest was significantly higher (146 %) in comparison to all other controls. Due to its highly selective nature, the methodology has the potential to be used as a precision agricultural sensing system for accurate and real-time detection of markers of stress, viral infection, disease, and normal physiological activities.

Development of a one-pot and sequence-specific LAMP assay based on the self-quenching probe integrated by the complementary oligonucleotide for an enhanced fluorescence quenching

Single-modified fluorogenic primer (Sfp) enables accurate identification of LAMP amplicons without being affected by non-specific products. However, the fluorescence self-quenching by nucleobases for Sfp is generally of low efficiency, and the high background signal makes it a great challenge to achieve visual inspection with naked eyes. In the present study, the oligonucleotide (Ao) complementary to Sfp was designed, which would hybridize to Sfp and dramatically heighten the quenching effect, leading to a low background signal in negative reaction. Instead, for positive reaction, Sfp is incorporated into the double-stranded amplicons, resulting in dequenching and consequently, enhanced fluorescence. The detection scheme can be further improved by a dual-color fluorescence strategy, allowing visual detection of 1 pg rainbow trout DNA in a closed-tube format within 30 min. Therefore, our LAMP-Ao-Sfp assay represents a useful tool for rapid and sensitive detection, and can serve as a reliable method for on-site detection in low-resource settings.

Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity.

Small nucleolytic ribozymes are RNAs that cleave their own phosphodiester backbone. While proteinaceous enzymes are regulated by a variety of known mechanisms, methods of regulation for ribozymes remain unclear. Twister is one ribozyme class for which many structural and catalytic properties have been elucidated. However, few studies have analyzed the activity of twister ribozymes in the context of a native flanking sequence, even though ribozymes as transcribed in nature do not exist in isolation. Interactions between the ribozyme and its neighboring sequences can induce conformational changes that inhibit self-cleavage, providing a regulatory mechanism that could naturally determine ribozyme activity in vivo and in synthetic applications. To date, eight twister ribozymes have been identified within the staple crop rice (Oryza sativa). Herein, we select several twister ribozymes from rice and show that they are differentially regulated by their flanking sequence using published RNA-seq data sets, structure probing, and cotranscriptional cleavage assays. We found that the Osa 1-2 ribozyme does not interact with its flanking sequences. However, sequences flanking the Osa 1-3 and Osa 1-8 ribozymes form inactive conformations, referred to here as "ribozymogens", that attenuate ribozyme self-cleavage activity. For the Osa 1-3 ribozyme, we show that activity can be rescued upon addition of a complementary antisense oligonucleotide, suggesting ribozymogens can be controlled via external signals. In all, our data provide a plausible mechanism wherein flanking sequence differentially regulates ribozyme activity in vivo. More broadly, the ability to regulate ribozyme behavior locally has potential applications in control of gene expression and synthetic biology.

Complexes and supramolecular associates of dodecyl-containing oligonucleotides with serum albumin

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with a high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins via electrophoretic mobility shift assay. The results testify to the formation of albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that interaction of human serum albumin with a duplex of complementary dodecyl-containing oligonucleotides resulted in associates of round shape with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and with polydeoxyadenylic acid and dodecyl-containing oligothymidylate also resulted in formation of supramolecular associates with a size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm. The obtained data allow considering dodecyl-containing oligonucleotides and albumin as potential components for the development of methods to design self-assembled systems for solving problems of molecular biology, biomedicine, and the development of unique theranostics with targeted action.

Obinutuzumab-Based Drug-Free Macromolecular Therapeutics Synergizes with Topoisomerase Inhibitors.

Drug-free macromolecular therapeutics (DFMT) utilizes modified monoclonal antibodies (or antibody fragments) to generate antigen-crosslinking-induced apoptosis in target cells. DFMT is a two-component system containing a morpholino oligonucleotide (MORF1) modified antibody (Ab-MORF1) and human serum albumin conjugated with multiple copies of complementary morpholino oligonucleotide (MORF2), (HSA-(MORF2)x ). The two components recognize each other via the Watson-Crick base pairing complementation of their respective MORFs. One HSA-(MORF2)x molecule can hybridize with multiple Ab-MORF1 molecules on the cell surface, thus serving as the therapeutic crosslink-inducing mechanism of action. Herein, various anti-neoplastic agents in combination with the anti-CD20 Obinutuzumab (OBN)-based DFMT system are examined. Three different classes of chemotherapies are examined: DNA alkylating agents; proliferation pathway inhibitors; and DNA replication inhibitors. Chou-Talalay combination index mathematics is utilized to determine which drugs engaged synergistically with OBN-based DFMT. It is determined that OBN-based DFMT synergizes with topoisomerase inhibitors and DNA nucleotide analogs but is antagonistic with proliferation pathway inhibitors. Cell mechanism experiments are performed to analyze points of synergism or antagonism by investigating Ca2+ influx, mitochondrial health, lysosomal stability, and cell cycle arrest. Finally, the synergistic drug combinatorial effects of OBN-based DFMT with etoposide in vivo are demonstrated using a human xenograft non-Hodgkin's lymphoma mouse model.

Enhancement of in vivo targeting properties of ErbB2 aptamer by chemical modification.

Aptamers have great potential for diagnostics and therapeutics due to high specificity to target molecules. However, studies have shown that aptamers are rapidly distributed and excreted from blood circulation due to nuclease degradation. To overcome this issue and to improve in vivo pharmacokinetic properties, inverted deoxythymidine (idT) incorporation at the end of aptamer has been developed. The goal of this study was to evaluate the biological characterization of 3'-idT modified ErbB2 aptamer and compare with that of unmodified aptamer via nuclear imaging. ErbB2-idT aptamer was labeled with radioisotope F-18 by base-pair hybridization using complementary oligonucleotide platform. The hyErbB2-idT aptamer demonstrated specific binding to targets in a ErbB2 expressing SK-BR-3 and KPL4 cells in vitro. Ex vivo biodistribution and in vivo imaging was studied in KPL4 xenograft bearing Balb/c nu/nu mice. 18F-hyErbB2-idT aptamer had significantly higher retention in the tumor (1.36 ± 0.17%ID/g) than unmodified 18F-hyErbB2 (0.98 ± 0.19%ID/g) or scrambled aptamer (0.79 ± 0.26% ID/g) at 1 h post-injection. 18F-hyErbB2-idT aptamer exhibited relatively slow blood clearance and delayed excretion by the renal and hepatobiliary system than 18F-hyErbB2 aptamer. In vivo PET imaging study showed that 18F-hyErbB2-idT aptamer had more stronger PET signals on KPL4 tumor than 18F-hyErbB2 aptamer. The results of this study demonstrate that attachment of idT at 3'-end of aptamer have a substantial influence on biological stability and extended blood circulation led to enhanced tumor uptake of aptamer.

Comparative Study of Surface Activation Steps for Thermally Grown Oxide Interface and Optimal Silanization

Silanization is one of the widely explored surface modification strategies for biofunctionalization of oxide interfaces. For biosensor applications, silanes with active terminal groups such as amine, thiol, carboxylic, and aldehyde groups are utilized in routine. In near‐field sensing schemes like biologically sensitive field‐effect transistors, it is crucial to generate a homogeneous layer of silane to confine the biointeractions in close vicinity of the sensor interface. The homogeneity of such biofunctional layer is determined by the surface activation and silanization protocol being applied. Herein, the impact of the surface activation process and silanization on electrical characteristics of field‐effect devices is studied comprehensively using an electrolyte‐oxide‐semiconductor (EOS) capacitor with a high‐quality gate oxide. The thermally grown silicon oxide (SiO2) interface is activated using acidic mixtures and plasma treatment, while the subsequent silanization steps are investigated comparatively using two different silanes (3‐aminopropyl triethoxysilane (APTES) and 3‐glycidyloxypropyl trimethoxysilane (GPTMS) in wet‐chemical and vapor‐phase processes. Furthermore, the optimized silanization process is utilized to immobilize an oligo strand at the EOS capacitor surface, followed by the hybridization of complementary oligo strands. The optimized protocol holds the potential for large‐scale production of functional oxide interfaces for various applications.

Complexes and Supramolecular Associates of Dodecyl-Containing Oligonucleotides with Serum Albumin.

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.

Simulations suggest robust microtubule attachment of kinesin and dynein in antagonistic pairs

Intracellular transport is propelled by kinesin and cytoplasmic dynein motors that carry membrane-bound vesicles and organelles bidirectionally along microtubule tracks. Much is known about these motors at the molecular scale, but many questions remain regarding how kinesin and dynein cooperate and compete during bidirectional cargo transport at the cellular level. The goal of the present study was to use a stochastic stepping model constructed by using published load-dependent properties of kinesin-1 and dynein-dynactin-BicD2 (DDB) to identify specific motor properties that determine the speed, directionality, and transport dynamics of a cargo carried by one kinesin and one dynein motor. Model performance was evaluated by comparing simulations to recently published experiments of kinesin-DDB pairs connected by complementary oligonucleotide linkers. Plotting the instantaneous velocity distributions from kinesin-DDB experiments revealed a single peak centered around zero velocity. In contrast, velocity distributions from simulations displayed a central peak around 100 nm/s, along with two side peaks corresponding to the unloaded kinesin and DDB velocities. We hypothesized that frequent motor detachment events and relatively slow motor reattachment rates resulted in periods in which only one motor is attached. To investigate this hypothesis, we varied specific model parameters and compared the resulting instantaneous velocity distributions, and we confirmed this systematic investigation using a machine-learning approach that minimized the residual sum of squares between the experimental and simulation velocity distributions. The experimental data were best recapitulated by a model in which the kinesin and dynein stall forces are matched, the motor detachment rates are independent of load, and the kinesin-1 reattachment rate is 50 s−1. These results provide new insights into motor dynamics during bidirectional transport and put forth hypotheses that can be tested by future experiments.

Proximity Ligation Assay (PLA) for Fillets of Drosophila Larvae.

The ability to detect protein-protein interactions is critical for understanding the mechanisms underlying protein and cell function. Current methods to assay protein-protein interactions, such as co-immunoprecipitation (Co-IP) and fluorescence resonance energy transfer (FRET), have limitations; for example, Co-IP is an in vitro technique and may not reflect the situation in vivo, and FRET typically suffers from low signal-to-noise ratio. The proximity ligation assay (PLA) is an in situ method for inferring protein-protein interaction with a high signal-to-noise ratio. The PLA technique can indicate that two different proteins are closely associated by the ability of two secondary antibody oligonucleotide probes to hybridize when they are close to each other. This interaction generates a signal with rolling-circle amplification using fluorescent nucleotides. Although a positive result does not indicate that two proteins directly interact, it implies a potential in vivo interaction that can then be verified in vitro. PLA uses primary antibodies against the two proteins (or epitopes) of interest, one raised in mouse and the other raised in rabbit. When these antibodies bind to proteins that lie within 40 nm of each other in the tissue, complementary oligonucleotides conjugated individually to mouse and rabbit secondary antibodies can anneal to form a template for rolling-circle amplification. Using fluorescently labeled nucleotides, rolling circle amplification generates a strong fluorescent signal in areas of the tissue where the two proteins are found together that is detected using conventional fluorescence microscopy. This protocol describes how to perform PLA in vivo on the central nervous system and peripheral nervous system of third-instar larvae of the fruit fly Drosophila melanogaster.

Variation of Tetrahydrofurans in Thyclotides Enhances Oligonucleotide Binding and Cellular Uptake of Peptide Nucleic Acids.

Selective incorporation of conformational constraints into thyclotides can be used to modulate their binding to complementary oligonucleotides, increase polarity, and optimize uptake into HCT116 cells without assistance from moieties known to promote cell uptake. The X-ray structure and biophysical studies of a thyclotide-DNA duplex reveal that incorporation of tetrahydrofurans into an aegPNA backbone promotes a helical conformation that enhances binding to complementary DNA and RNA. Selective incorporation of tetrahydrofurans into the aegPNA backbone allows polarity to be increased incrementally so that uptake into HCT116 cells can be optimized. The enhanced binding, polarity, and cellular uptake properties of thyclotides were used to demonstrate effective inhibition of microRNA-21 in HCT116 cells.

OligoHydrogelArray (OHA) for Parallelized Solid‐Phase Extraction of Oligonucleotides

AbstractImmobilization of oligonucleotides on solid surfaces is an important step in many experimental workflows in biology, such as gene expression analysis, genotyping, and drug discoveries. Capturing oligonucleotides in a highly efficient miniaturized format is still challenging. In this work, preparation of functionalized miniaturized acrylamide hydrogels on a droplet microarray (DMA) chip is reported. By further modification of these hydrogels through the Cu‐catalyzed alkyne‐azide cycloaddition, oligonucleotides are attached covalently, creating the OligoHydrogelArray (OHA). OHA enables the hybridization of oligonucleotides with defined sequences in a parallelized, miniaturized, and high‐throughput manner with an overall loading capacity and extraction efficiency suitable for most biological and chemical applications. This system enables the parallelized solid‐phase extraction of target oligonucleotides out of a mixture for purification and enrichment of DNA/RNA/PNA for application in fields such as transcriptomics. In the current study, are establish the chemical procedure for reproducible preparation of OHA and determine key characteristics, such as height, diameter, and shape of miniaturized gel pads as well as the loading capacity of oligonucleotides within the material. Finally, the ability to extract complementary oligonucleotide strands out of solution through hybridization is demonstrated to set the course for future applications.

Fluorescent labeling of RNA and DNA on the Hoogsteen edge using sulfinate chemistry.

We have devised a single pot, low-cost method to add azide groups to unmodified nucleic acids without the need for enzymes or chemically modified nucleoside triphosphates. This involves reacting an azide-containing sulfinate salt with the nucleic acid, leading to replacement of C-H bonds on the nucleobase aromatic rings with C-R, where R is the azide-containing linker derived from the original sulfinate salt. With the addition of azide functional groups, the modified nucleic acid can easily be reacted with any alkyne-labeled compound of interest, including fluorescent dyes as shown in this work. This methodology enables the fluorescent labeling of a wide variety of nucleic acids, including natively folded RNAs, under mild conditions with minimal effects upon biochemical function and ribozyme catalysis. To demonstrate this, we show that a pair of labeled complementary ssDNA oligonucleotides (oligos) can hybridize to form dsDNA, even when labeled with multiple fluorophores per oligo. In addition, we also demonstrate that two different group II introns can splice when prelabeled internally with fluorophores, using our method. Broadly, this demonstrates that sulfinate modification of RNA is compatible with ribozyme function and Watson-Crick pairing, while preserving the labile backbone.

Light-Controlled Cell–Cell Assembly Using Photocaged Oligonucleotides

The interactions between heterogeneous cell populations play important roles in dictating various cell behaviors. Cell–cell contact mediates communication through the exchange of signaling molecules, electrical coupling, and direct membrane-linked ligand–receptor interactions. In vitro culturing of multiple cell types with control over their specific arrangement is difficult, especially in three-dimensional (3D) systems. While techniques that allow one to control the arrangement of cells and direct contact between different cell types have been developed that expand upon simple co-culture methods, specific control over heterojunctions that form between cells is not easily accomplished with current methods, such as 3D cell-printing. In this article, DNA-mediated cell interactions are combined with cell-compatible photolithographic approaches to control cell assembly. Specifically, cells are coated with oligonucleotides containing DNA nucleobases that are protected with photocleavable moieties; this coating facilitated light-controlled cell assembly when these cells were mixed with cells coated with complementary oligonucleotides. By combining this technology with digital micromirror devices mounted on a microscope, selective activation of specific cell populations for interactions with other cells was achieved. Importantly, this technique is rapid and uses non-UV light sources. Taken together, this technique opens new pathways for on-demand programming of complex cell structures.