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We report a thorough investigation of the role of single-stranded thymidine (ssT) linkers in the stability and flexibility of minimal, multistranded DNA nanostructures. We systematically explore the impact of varying the number of ssTs in three-way junction motifs (3WJs) on their formation and properties. Through various UV melting experiments and molecular dynamics simulations, we demonstrate that while the number of ssTs minimally affects thermodynamic stability, the increasing ssT regions significantly enhance the structural flexibility of 3WJs. Utilizing this knowledge, we design triangular DNA nanoparticles with varying ssTs, all showing exceptional assembly efficiency except for the 0T triangle. All triangles demonstrate enhanced stability in blood serum and are nonimmunostimulatory and nontoxic in mammalian cell lines. The 4T 3WJ is chosen as the building block for constructing other polygons due to its enhanced flexibility and favorable physicochemical characteristics, making it a versatile choice for creating cost-effective, stable, and functional DNA nanostructures that can be stored in the dehydrated forms while retaining their structures. Our study provides valuable insights into the design and application of nucleic acid nanostructures, emphasizing the importance of understanding stability and flexibility in the realm of nucleic acid nanotechnology. Our findings suggest the intricate connection between these ssTs and the structural adaptability of DNA 3WJs, paving the way for more precise design and engineering of nucleic acid nanosystems suitable for broad biomedical applications. 

Nucleic Acid (NA) nanotechnology is a rapidly emerging field demonstrating application of polynucleotides as a versatile biopolymer to fabricate nanostructures of various dimensions and shapes in a programmable and highly predictable way. The folding of DNA or RNA strands into a stable double helix configuration mainly relies on the Watson-Crick (Canonical) base pair composition (G=C and A-T or A-U in the case of RNA), base stacking, and metal ion concentrations. The thermodynamic parameters of DNA B-form helix formation and A-form helix of RNA can be computed using empirically defined sets of nearest neighboring parameters encompassed within the 2D structure predicting programs for example mfold, NUPAC. However, these programs are lacking parameters for a hybrid DNA/RNA base pairing and non-canonical base interactions. In this report, we focused our study to evaluate thermodynamic parameters of several in silico designed three-way junction (3WJ) DNA and hybrid DNA-RNA structural elements. The designed 3WJ motifs contain three helical stems linked with 4,3,2,1, and 0 single stranded Thymidine (T) or Uridine (U) nucleotides. We will report assembly efficiency of the 3WJs investigated by gel shift assay and thermodynamic parameters measured by UV-melting technique. Our experiments reveal that the amount of Ts and Us linkages in the three-way junction dictate the stability of the overall 3WJ conformations. This study is important as we expect it will contribute to the existing set of parameters used for NA structure prediction algorithms as well as provide a guidance for rational design of NA nanostructures. 

Intrinsic properties of RNA to form Watson-Crick base pairing allows it to self-assemble into specific and programmable nano-sized complexes. However, the naturally occurring RNA strands are not stable, and they hydrolyze quickly in blood serum. This limits RNA application, for example, as a nanovehicle for targeted drug delivery. The replacement of the hydroxyl group at the 2’ position of the ribose to 2’-Fluoro (2’-F) or 2’-Methoxy (2’-Met) can drastically elevate the resistance of the RNA to nucleases and improve overall stability. However, to synthesize such modified RNAs, a mutated version of traditionally used T7 RNA polymerase is often required. The recombinant RNApol will not discriminate between regular riboNucleotideTriphosphates (rNTPs) vs modified-rNTPs and, hence, can be implemented to transcribe modified RNA strands. Herein, we describe overexpression and isolation of RGVG and Y639F RNA polymerases from E.Coli cells using metal-ion immobilized affinity chromatography. We demonstrated that in optimized conditions, these RNA polymerases can be used to obtain milligram quantities of 2’-F and 2’-Met RNA polymers possessing high levels of resistance to nuclease degradation in blood serum. 

Nucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. “Click” chemistry has become popular conjugation approach due to its simplicity and high conjugation efficiency. However, the major drawback of the conjugation of oligonucleotides is the purification of the products, as traditionally used chromatography techniques are usually time-consuming and laborious, requiring copious quantities of materials. Herein, we introduce a simple and rapid purification methodology to separate the excess of unconjugated small molecules and toxic catalysts using a molecular weight cut-off (MWCO) centrifugation approach. As proof of concept, we deployed “click” chemistry to conjugate a Cy3-alkyne moiety to an azide-functionalized oligodeo-xynucleotide (ODN), as well as a coumarin azide to an alkyne-functionalized ODN. The calculated yields of the conjugated products were found to be 90.3 ± 0.4% and 86.0 ± 1.3% for the ODN-Cy3 and ODN-coumarin, respectively. Analysis of purified products by fluorescence spectroscopy and gel shift assays demonstrated a drastic amplitude of fluorescent intensity by multiple folds of the reporter molecules within DNA nanoparticles. This work is intended to demonstrate a small-scale, cost-effective, and robust approach to purifying ODN conjugates for nucleic acid nanotechnology applications.