Aptamer-siRNA Conjugates

Of all these novel drug delivery systems, RNAi therapy plays a prominent role in the silencing of certain genes that are responsible for various disease states. Despite the promise, delivery of siRNA to target cells presents a challenge in their utilization in the clinical setting. To solve these problems, BOC Sciences is applying aptamer-siRNA conjugates for the first time. Utilizing the promise of aptamer-siRNA conjugates, BOC Sciences is committed to help customers worldwide to achieve significant progress in development of targeted gene silencing therapies.

What is an Aptamer?

The word "aptamer" was based in part on the Latin aptus and was conceived by the Ellington and Szostak labs in 1990. SELEX involves the selection of an aptamer (oligonucleotide sequence of RNA or DNA) from a random oligonucleotide sequence library made in vitro. Aptamers are 20 to 80 nucleotides, mostly showing 3D structure, fold into a 3D structure like 3D structure of proteins. The aptamers can bind target molecules tightly and specifically, including heavy metal ions, cells, proteins, peptides, bacteria, viruses etc. are similar to antibodies, but they have many outstanding characters such as smaller size, easy of synthesis and low immunogenicity. Given these properties, aptamers are attractive components of nanoconjugates for targeted drug delivery, with the prospect of delivering siRNA selectively to discrete cell types.

What are Aptamer-siRNA Conjugates?

When aptamers and siRNAs are defined as conjugates, they are collectively termed aptamer-siRNA conjugates or aptamer siRNA chimeras (AsiCs). Upon association, AsiCs are internalized by endocytosis and colocalize, at least transiently, in endosome or lysosome compartments. RNA interference (RNAi) pathways identify released endosomal contents as foreign molecules. The ribonuclease (Dicer) then binds to the siRNA of AsiC, and excise the siRNA region from the aptamer and load them to the RNA-induced silencing complex (RISC). RISC is composed of proteins that facilitate the targeted degradation of messenger RNA (mRNA) RISC contains the Argonaute protein (AGO2) which unwinds the siRNA double-strand thereby only the guide strand being left. Consequently, mRNA now has the ability to base par along with the guide strand loaded by RISC, and the cleavage of mRNA will in turn reduce the gene expression of that mRNA. Aptamer-siRNA conjugates have emerged as a new approach for targeted gene therapy and can be used as a powerful tool for design of gene therapy for cancer, cardiovascular diseases and viral infections. Due to this direct conjugation, a simple delivery mechanism can bring precise delivery of siRNA into target cells. These conjugates thus guide siRNA to targets using the high specificity and binding affinity of aptamers, significantly increasing the potential of RNA interference (RNAi) therapy.

BOC Sciences' Aptamer-siRNA Conjugates Services

As a significant contract services provider specializing in the field of science, BOC Sciences has been engaged in a patented service regarding aptamer-sirna conjugates that will fulfill the specific needs of both academic labs and pharmaceutical companies in this wave of plenty of novel siRNA drugs coming in to progress from early development stages towards advanced stages. BOC Sciences combines its years of experience and cutting-edge technology to develop precisely and synthesize aptamer-siRNA conjugates in the areas of gene silencing for targeted applications. We combine a focus on providing the highest quality products and services with a commitment to solving the most difficult R&D challenges in RNA therapeutics. Below is our service flow:

Custom siRNA Synthesis

With a commitment to excellence and innovation, BOC Sciences empowers clients to accelerate their scientific endeavors by providing high-quality, custom-designed siRNA molecules for a wide range of applications.

• Free siRNA design or you can submit selected siRNA sequences.
• Comprehensive modification and labeling: from conventional modification to fluorescent/non-fluorescent labeling, even dual and multiple labeling.
• Synthetic scale: 0.015 µmol, 50 nmol, 100 nmol, 200 nmol, 1 µmol, 5 µmol, 10 µmol, and > 10 µmol.
• Quality control by analytical HPLC, mass spectrometry, annealing in RNase-free water, and double-stranded analysis by analytical gels.

Custom Aptamer Synthesis

BOC Sciences primarily offers the following options in custom aptamer synthesis services:

• Aptamer Custom Synthesis Service: We employ SELEX methods to screen and obtain aptamers with high affinity and specificity for your target ligand from nucleic acid sequence libraries. Your aptamers will be purified via PAGE or HPLC and validated by mass spectrometry.
• Aptamer Modification Service: Various modifications are available, such as biotin modification for EMSA or Southern blotting. Fluorescent modifications are suitable for many imaging and diagnostic studies due to their high sensitivity. Modifications like 3' inverted deoxythymidine, 2'-O-methyl (OCH3) modification, 2'-fluoro modification, and 2'-NH2 modification enhance stability against nucleases.
• Aptamer Optimization Service: This includes basic sequence optimization, structural stability optimization, hydrophobic/hydrophilic part optimization, SELEX optimization, and post-SELEX optimization.
• Aptamer In Vitro Analysis Service: This encompasses stability analysis and cytotoxicity testing.

Aptamer siRNA Conjuction

Offered by BOC Sciences, Aptamer-siRNA conjugates are useful tools for targeted gene regulation and therapy. Here, we develop a method for the site-specific conjugation of siRNA molecules, the high-adhesive siRNAs, with target aptamers based on their coincident and complementary chemical properties that allow for efficient delivery and for selective binding. For this we utilized numerous engineering strategies to help improve the loading efficiency of the siRNA onto the aptamers. Strategies are carefully crafted to impart essential structural and chemical features to these conjugates, maintaining their stability and function. In this study, we not only develop coupling methodologies that provides the robustness necessary for potential medical applications but also focus on the functional optimization of aptamer-siRNAs conjugates. This requires optimization of binding affinity, cellular uptake, and intracellular release kinetics, among other factors to enhance their therapeutic utility.

Characterization Service

• Tertiary structural analysis
• Thermodynamic analysis
• Combined with affinity measurements
• Competition assays

Advantages of Aptamer-siRNA Conjugates

• No immunogenicity and toxicity
• Lower production cost
• Relatively easily screened, synthesized, programmable designed, and chemically modified

According to professional technical support, BOC Sciences provides existing siRNA based on target genes and high quality aptamer design and synthesis. We will also fine-tune the chemical junction that enables an unmodified RNAi molecule to be attached directly to the 3' end of the aptamer strand or to the antisense strand that complements the RNAi strand. Through years of experience in aptamer design and coupling, BOC Sciences is well-prepared to perform a one-stop service for aptamer-siRNA conjugates services. Please feel free to contact us or send us an inquiry directly and we will be happy to assist you.

Case Study

Case study 1: Enhancing Targeted Therapeutics for Cancer Using Aptamer-Chimerized siRNA

Fabrication of the LNA-modified APT siRNA chimera. (Nithya, S.; et al, 2015)

This case study explores a novel targeted therapy approach for cancer. Researchers fused aptamers (DNA or RNA molecules with specificity) with drugs, antisense oligos, or nanoparticles to generate targeted therapeutics for cancer. They found that fusing aptamers with siRNA increased the availability of siRNA to target cells, thus enhancing therapeutic efficacy. EpCAM RNA aptamer (EpApt or Ep) was chosen as the target for siRNA fusion due to its suitable secondary structure. Studies indicated that stathmin and survivin proteins are oncogenic in retinoblastoma (RB), breast cancer, and other cancers. Consequently, researchers fused EpCAM Apt with siRNA targeting survivin and stathmin, enhancing stability through Locked Nucleic Acid (LNA) modification. Experimental validation demonstrated that LNA-modified fusion aptamers remained stable for up to 96 hours and were internalized into RB, WERI-Rb1, and breast cancer MDAMB453 cell lines. Further investigation using recombinant dicer enzyme showed the generation of siRNA by these constructs. In vitro quantitative polymerase chain reaction and immunofluorescence microscopy analysis revealed effective silencing of stathmin and survivin in RB and breast cancer models. These fusion constructs significantly inhibited proliferation of breast cancer cells. Thus, LNA-modified aptamer-based siRNA delivery facilitates cell targeting and warrants further investigation in animal models.

FAQ

1. Can aptamers bind to RNA?

Yes, aptamers are able to bind to RNA. Aptamers are nucleic acid or nucleic acid analog sequences (RNA, DNA, PNA, or locked nucleic acid) that show high affinity with its target molecule. Interactions between secondary and tertiary structures render them bind. When present in the right conditions, aptamers can specifically bind to defined areas of RNA, a property that can be exploited in applications to detect, study structural features, and intervene in RNA.

2. Are RNA aptamers single stranded?

Yes, RNA aptamers are in fact inherently single-stranded molecules. Intramolecular interactions within the single RNA strand create complex secondary and tertiary structures. This single-stranded structural feature allows them to be folded into unique 3D structures, which contribute to their high specificity and high affinity for target molecules.

3. What does an aptamer do?

Aptamers are small, single-stranded DNA or RNA molecules able to interact with their targets with high affinity and specificity. They work by identifying and attaching to their target molecules through a phenomenon called ligand recognition. Once they are bound, aptamers can either act redundantly or inhibit their target molecule differently, depending on the context and on their application.

4. What are the benefits of Aptamer-siRNA Chimera?

• Targeted Delivery: The aptamer component specifically binds to cell surface receptors on the target cells, ensuring that the siRNA is delivered where it is needed most.
• Reduced Off-Target Effects: By targeting specific cells, aptamer-siRNA chimeras minimize the impact on non-target cells, reducing potential side effects.
• Enhanced Efficacy: Direct delivery to the target cells improves the gene-silencing efficiency of the siRNA, leading to better therapeutic outcomes.

Reference

1. Nithya, S.; et al. Targeting Cancer Cells Using LNA-Modified Aptamer-siRNA Chimeras. Nucleic Acid Therapeutics. 2015, 25 (6).

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