Purification of Self-Assembled DNA Tetrahedra Using Gel Electrophoresis.

DNA nanostructures have found applications in a variety of fields such as biosensing, drug delivery, cellular imaging, and computation. Several of these applications require purification of the DNA nanostructures once they are assembled. Gel electrophoresis-based purification of DNA nanostructures is one of the methods used for this purpose. Here, we describe a step-by-step protocol for a gel-based method to purify self-assembled DNA tetrahedra. With further optimization, this method could also be adapted for other DNA nanostructures. © 2022 Wiley Periodicals LLC. Basic Protocol: Purification of self-assembled DNA tetrahedra.

Surface-enhanced Raman spectroscopy for drug discovery: peptide-RNA binding.

The ever-growing demand for new drugs highlights the need to develop novel cost- and time-effective techniques for drug discovery. Surface-enhanced Raman spectroscopy (SERS) is an emerging ultrasensitive and label-free technique that allows for the efficient detection and characterization of molecular interactions. We have recently developed a SERS platform for detecting a single protein molecule linked to a gold substrate (Almehmadi et al. Scientific Reports 2019). In this study, we extended the approach to probe the binding of potential drugs to RNA targets. To demonstrate the proof of concept, two 16-amino acid residue peptides with close primary structures and different binding affinities to the RNA CUG repeat related to myotonic dystrophy were tested. Three-microliter solutions of the RNA repeat with these peptides at nanomolar concentrations were probed using the developed approach, and the binding of only one peptide was demonstrated. The SER spectra exhibited significant fluctuations along with a sudden strong enhancement as spectra were collected consecutively from individual spots. Principal component analysis (PCA) of the SER spectral datasets indicated that free RNA repeats could be differentiated from those complexed with a peptide with 100% accuracy. The developed SERS platform provides a novel opportunity for label-free screening of RNA-binding peptides for drug discovery. Schematic representation of the SERS platform for drug discovery developed in this study.

Fluorescence-Based Binding Characterization of Small Molecule Ligands Targeting CUG RNA Repeats.

Pathogenic CUG and CCUG RNA repeats have been associated with myotonic dystrophy type 1 and 2 (DM1 and DM2), respectively. Identifying small molecules that can bind these RNA repeats is of great significance to develop potential therapeutics to treat these neurodegenerative diseases. Some studies have shown that aminoglycosides and their derivatives could work as potential lead compounds targeting these RNA repeats. In this work, sisomicin, previously known to bind HIV-1 TAR, is investigated as a possible ligand for CUG RNA repeats. We designed a novel fluorescence-labeled RNA sequence of r(CUG)10 to mimic cellular RNA repeats and improve the detecting sensitivity. The interaction of sisomicin with CUG RNA repeats is characterized by the change of fluorescent signal, which is initially minimized by covalently incorporating the fluorescein into the RNA bases and later increased upon ligand binding. The results show that sisomicin can bind and stabilize the folded RNA structure. We demonstrate that this new fluorescence-based binding characterization assay is consistent with the classic UV Tm technique, indicating its feasibility for high-throughput screening of ligand-RNA binding interactions and wide applications to measure the thermodynamic parameters in addition to binding constants and kinetics when probing such interactions.

A mini DNA-RNA hybrid origami nanobrick.

DNA origami is typically used to fold a long single-stranded DNA scaffold into nanostructures with complex geometries using many short DNA staple strands. Integration of RNA into nucleic acid nanostructures is also possible, but has been less studied. In this research, we designed and characterized a hybrid RNA-scaffolded origami nanostructure with dimensions of ∼12 nm. We used 12 DNA staple strands to fold a 401 nt RNA scaffold into a ten-helix bundle with a honeycomb cross section. We verified the construction of the nanostructure using gel electrophoresis and atomic force microscopy. The DNA-RNA hybrid origami showed higher resistance to ribonuclease compared to a DNA-RNA duplex control. Our work shows potential use in folding long RNA, such as messenger RNA, into origami nanostructures that can be delivered into targeted cells as medicine or a vaccine.

Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA).

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5' end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA-thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.

Correction: Click and photo-release dual-functional nucleic acid nanostructures.

Correction for 'Click and photo-release dual-functional nucleic acid nanostructures' by Vibhav A. Valsangkar et al., Chem. Commun., 2019, DOI: 10.1039/c9cc03806j.

Click and photo-release dual-functional nucleic acid nanostructures.

We functionalize nucleic acid nanostructures with click chemistry (for attachment of cargos) and a photocleavable linker (for release). We demonstrate cargo attachment using a fluorescein dye and release using UV trigger from an RNA three-way junction, a DNA star motif and a DNA tetrahedron. Such multifunctional nucleic acid nanostructures have potential in targeted drug delivery.

Integration of a photocleavable element into DNA nanoswitches.

Reconfigurable DNA nanostructures can be designed to respond to external stimuli such as nucleic acids, pH, small molecules and enzymes. In this study, we incorporated photocleavable linkers in DNA strands that trigger a conformational change in binary DNA nanoswitches. We demonstrate control of the output using UV light, with potential applications in biosensing and molecular computation.

Cyano Modification on Uridine Decreases Base-Pairing Stability and Specificity through Neighboring Disruption in RNA Duplex.

5-Cyanomethyluridine (cnm5 U) and 5-cyanouridine (cn5 U), the two uridine analogues, were synthesized and incorporated into RNA oligonucleotides. Base-pairing stability and specificity studies in RNA duplexes indicated that cnm5 U slightly decreased the stability of the duplex but retained the base-pairing preference. In contrast, cn5 U dramatically decreased both base-pairing stability and specificity between U:A and other noncanonical U:G, U:U, and U:C pairs. In addition, the cn5 U:G pair was found to be stronger than the cn5 U:A pair and the other mismatched pairs in the context of a RNA duplex; this implied that cn5 U might slightly prefer to recognize G over A. Our mechanistic studies by molecular simulations showed that the cn5 U modification did not directly affect the base pairing of the parent nucleotide; instead, it weakened the neighboring base pair in the 5' side of the modification in the RNA duplexes. Consistent with the simulation data, replacing the Watson-Crick A:U pair to a mismatched C:U pair in the 5'-neighboring site did not affect the overall stability of the duplex. Our work reveals the significance of the electron-withdrawing cyano group in natural tRNA systems and provides two novel building blocks for constructing RNA-based therapeutics.

Click-based functionalization of a 2'-O-propargyl-modified branched DNA nanostructure.

DNA has emerged as a versatile building block for programmable self-assembly. DNA-based nanostructures have been widely applied in biosensing, bioimaging, drug delivery, molecular computation and macromolecular scaffolding. A variety of strategies have been developed to functionalize these nanostructures. In this study, we report a facile click-based strategy to incorporate a metal chelating ligand and a fluorescent tag into a three-point-star DNA tile containing 2'-O-propargyl groups. Such a strategy opens up the possibility of functionalizing pre-assembled DNA strands to construct platforms for metal or drug delivery.

Water-bridged hydrogen bond formation between 5-hydroxylmethylcytosine (5-hmC) and its 3'-neighbouring bases in A- and B-form DNA duplexes.

5-Hydroxylmethylcytosine (5hmC) has been recognized as the sixth base with important biological functions in many tissues and cell types. We present here the high-resolution crystal structures and molecular simulation studies of both A-form and B-form DNA duplexes containing 5hmC. We observed that 5hmC interacts with its 3'-neighboring bases through water-bridged hydrogen bonds and these interactions may affect the further oxidation of 5hmC.