INTRODUCTION:

Bioconjugation bridges the gap between the immense potential of DNA and the targeted action of small molecules. By attaching these small compounds to DNA, researchers can create powerful tools for diagnostics, drug delivery, and gene therapy. The method chosen for this attachment hinges on several factors. Click chemistry, a popular technique, offers remarkable versatility. It leverages a reliable reaction between azide and alkyne groups to link a broad range of molecules to DNA. Alternatively, NHS ester chemistry provides a simpler approach for attaching carboxylic acids to amines on the DNA strand. However, this method might not provide the precise control over the attachment site that some applications require. Ultimately, the most suitable bioconjugation strategy depends on the specific properties of the small molecule being attached, the desired location for attachment on the DNA, and the need for biocompatibility within living cells. Considering these factors ensures the creation of a truly effective DNA-small molecule conjugate. Christian et al. provided a comprehensive review of click chemistry applications in DNA and RNA bioconjugation.¹

Farooqi et al. explored the use of click chemistry for drug delivery applications, including DNA-based conjugates.² Staudinger ligation method for bioconjugation was described by Prescher et al.³

The bioconjugation of DNA with small compounds merges the fields of chemistry and biology to create functionalized biomolecular hybrids with diverse applications. The research paper by Saxon et al. discusses the use of hydrazinopyrazole ligation for protein conjugation, which can be adapted for DNA bioconjugation.⁴ This burgeoning area of research involves the covalent attachment of small molecules to DNA, enabling the generation of tailored entities with precise properties. Methodologies for bioconjugation span traditional chemical modification techniques to more recent innovations such as enzymatic approaches and biorthogonal reactions. Li et al. explores the use of DNA for the self-assembly of functional nanostructures, which can involve bioconjugation.⁵ These methods offer researchers versatile tools to manipulate DNA-small compound conjugates under mild conditions, allowing for precise control over conjugate formation.

Applications of DNA-small compound conjugates span multiple disciplines, showcasing their versatility and potential impact. In therapeutics, these conjugates hold promise for targeted drug delivery and controlled release, minimizing off-target effects and enhancing therapeutic efficacy. Moreover, they have been instrumental in gene editing technologies like CRISPR-Cas systems, facilitating precise genome modifications. In diagnostics, DNA-small molecule conjugates serve as sensitive and specific probes for detecting nucleic acids, proteins, and small molecules, offering valuable tools for disease diagnosis and monitoring. Additionally, these conjugates find utility in materials science for the fabrication of nanoscale structures, biosensors, and molecular devices, highlighting their role in advancing bottom-up assembly approaches. The review article by Mutller et al. explores bioconjugation applications in radiopharmaceuticals, which can involve DNA conjugation for targeted delivery.⁶

Despite significant progress, challenges remain in optimizing bioconjugation strategies for clinical translation and addressing issues related to stability and scalability. Future research directions aim to overcome these challenges and explore new applications in emerging fields such as synthetic biology, nanomedicine, and precision medicine. Continued innovation in bioconjugation methodologies and interdisciplinary collaboration holds the key to unlocking the full potential of DNA-small compound conjugates, paving the way for ground breaking advancements in science and technology. Review article published by Bergström et al. focuses on bioconjugation strategies for targeted delivery of therapeutic oligonucleotides, which involve conjugating DNA with targeting moieties.⁷

In the current investigation, our focus revolves around the coupling of single-stranded DNA molecules with small heterocyclic compounds through peptide linkages while operating in a solution-phase environment. This method involves a step-by-step process where the DNA molecules, typically single-stranded due to their ease of manipulation, are chemically bonded to the small heterocyclic compounds. The linkage is facilitated through peptide bonds, which are formed between the DNA molecules and the compounds, resulting in a covalent attachment. This coupling process is conducted in a solution phase, indicating that the reactions take place within a liquid medium rather than on a solid support (Figure 1). This approach offers several advantages, including flexibility in reaction conditions, efficient mixing of reactants, and scalability for larger-scale synthesis. By employing this methodology, we aim to explore the potential applications of these DNA-small compound conjugates, particularly in areas such as drug delivery, diagnostics, and nanotechnology.

RESULT AND DISCUSSION:

COMU (7-(Carboxynorpholino)-1H-benzotriazole-1-yloxymethylene)-dimethylaminonium hexafluorophosphate) and collidine (2,4,6-trimethylpyridine) can be used together for peptide coupling reactions. COMU is a member of the uronium-based coupling reagent family. It functions by activating the carboxylic acid group of one peptide fragment, forming a reactive intermediate. This intermediate then reacts with the free amine group of another peptide fragment, forming the desired peptide bond. COMU is known for its efficiency and ability to minimize racemization (undesirable rearrangement) at the chiral center of the amino acid.

Figure 1: Scheme for Bioconjugation of BNIMZ and BNTZA with 5'AmC3-poly-T₁₅ DNA

Collidine is a hindered tertiary amine that acts as a base in the reaction. It helps deprotonate the carboxylic acid group, making it more susceptible to activation by COMU. Collidine is a weaker base compared to some commonly used options like N,N-Diisopropylethylamine (DIEA). The milder deprotonation by collidine can potentially lead to less racemization at the chiral center compared to stronger bases. We have used solution phase peptide coupling over solid phase. Solution phase peptide coupling offers several advantages over solid phase coupling due to its flexibility and ease of handling. In solution phase coupling, all reactants are dissolved in a liquid medium, allowing for efficient mixing and reaction monitoring. This method offers greater versatility in reaction conditions, facilitating the use of a wide range of substrates and reagents. Additionally, solution phase coupling eliminates the need for solid supports and resin-bound intermediates, simplifying the synthesis process and reducing purification challenges. Moreover, solution phase coupling can be easily scaled up for larger-scale synthesis, making it more suitable for industrial applications. Overall, the simplicity, flexibility, and scalability of solution phase peptide coupling make it an attractive choice for synthetic chemists seeking efficient and cost-effective peptide synthesis methods.

Further for characterizations, we have used the MALDI-TOF MS. It provides significant advantages for analyzing biomolecules like DNA compared to other methods such as ESI. Its high sensitivity enables the detection of DNA at low concentrations, while its wide mass range allows for the analysis of DNA fragments of various sizes. Minimal fragmentation during ionization preserves the integrity of the analyte, crucial for accurate mass determination in DNA sequencing and characterization. Additionally, MALDI-TOF MS offers high throughput, ease of sample preparation, and compatibility with non-volatile matrices, making it suitable for routine analysis of DNA samples in research and clinical settings. Overall, these advantages position MALDI-TOF MS as a valuable tool for biomolecular analysis, particularly in DNA research applications. In both the cases the conjugated DNA was found (Figure 2) with BNIMZ and BNTZA with accuracy.

Figure 2: MALDI Spectra of ssDNA coupled with (A) BNIMZ and (B) BNTZA

Significantly the parent fragment of un-conjugated DNA was found with less abundance. In mass spectrometry data, we found the other molecular fragments also like possible coupling of the heterocyles with free NH₂ group of thymine’s were also found but the abundance of 5’-conjugation with AmC3 group was found significantly. The efficiency of amide bond formation significantly differs between aliphatic and aromatic amines, primarily due to the differences in their basicity, steric hindrance, and the resonance stabilization characteristics. Aliphatic amines are generally more basic than their aromatic counterparts. This is because the lone pair of electrons on the nitrogen in aliphatic amines is more available for protonation or for nucleophilic attack. In contrast, the lone pair on an aromatic amine, here in Thymine in poly-T DNA, is partially delocalized into the aromatic ring, making it less available for reaction. As a result, aliphatic amines (here 5’AmC3) are more reactive towards pepetide coupling towards BNIMZ and BNTZA.

Experimental Procedure:

We have purchased single stranded 15-base pair poly-T DNA with 5’ modification as C3-linker (5’AmC3-poly T DNA) with free NH₂ group in HPLC grade purity from Sigma Aldrich. The ssDNA is directly used for solution phase experiment by dissolving it with de-ionized water. COMU (0.25mmol) was added to a mixture of BNIMZ and BNTZA (0.25mmol), the single stranded poly-T DNA (0.25mmol) and Collidine (0.50mmol) in acetonitrile (2ml) at 0^◦C and the reaction mixture was stirred at 0^◦C for 1h and at r.t. for 2-3h. The mixture was diluted with EtOAc (5ml) and extracted with 1N HC1 (2 × 1ml), 1 N NaHCO₃ (2 × 1ml) and saturated NaCl (2 × 1ml). The EtOAc was then dried with MgSO₄, the solvent was removed, and the crude oligos were directly analyzed by Mass spectrometry. All the solvents and reagents used in the study are HPLC grade. BNIMZ and BNTZA were earlier prepared by our research group by previously reported method.^8-12 Both purified compounds were taken for coupling without any further purification. The scheme for bio-conjugation of DNA with both heterocyclic compounds is depicted in Figure 2. After conjugation, the resultant conjugates were analyzed in MALDI spectrometry by ABI Sciex 5800 TOF instrument. CHCA (α-Cyano-4-hydroxycinnamic acid) matrix was used as matrix for MALDI experiments.

CONCLUSION:

In conclusion, the present study offers a powerful and versatile strategy for successfully coupling single-stranded DNA with small heterocyclic compounds. This approach provides a reliable and efficient method for constructing DNA-conjugates, owing to the inherent reactivity of amino and carboxylic functional groups. COMU and Collidine pair here successfully able to couple BNIMZ and BNTZA with poly-T DNA. By carefully selecting appropriate reagents and optimizing reaction conditions, researchers can achieve site-specific conjugation while minimizing undesired side reactions like racemization. This paves the way for the development of novel DNA-based tools with tailored properties for applications in diagnostics, drug delivery, and gene therapy.

ACKNOWLEDGEMENT:

SS is thankful for the facility provided by Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India.

CONFLICTS OF INTEREST:

The Author Declares no conflict of interest.

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Received on 29.03.2024 Modified on 22.04.2024

Asian J. Research Chem. 2024; 17(2):81-84.

DOI: 10.52711/0974-4150.2024.00016