When it comes to the world of peptide research, the selection of appropriate fluorescent dyes is crucial for various applications, including cellular imaging, molecular diagnostics, and therapeutic interventions. This article delves into the nuances of choosing the right fluorescent dyes for peptides, offering insights into the latest research and practical considerations.

Key Takeaways:

• The choice of fluorescent dye depends on the specific application and properties of the peptide.
• Commonly used dyes include FITC, FAM, TAMRA, and Cyanine dyes.
• The impact of the dye on the function and location of the peptide should be carefully evaluated.

Understanding Fluorescent Dyes in Peptide Research

The Role of Fluorescent Dyes

Fluorescent dyes are pivotal in peptide research for visualizing and tracking biological processes. They enable the observation of peptides in various environments, from in vitro studies to in vivo applications.

Selection Criteria

When selecting a fluorescent dye, consider factors like wavelength, brightness, photostability, and the potential impact on peptide structure and function.

Popular Fluorescent Dyes for Peptides

FITC, FAM, TAMRA, and Cyanine Dyes

These dyes are widely used due to their effective labeling properties and compatibility with various imaging techniques. For more information, visit LifeTein’s page on fluorescent dyes.

Alexa Dyes are another common option, though these are typically conjugated to peptides via cysteine residues or Lys(N3), like Cyanine dyes.

Novel Dyes and Custom Solutions

Advancements in dye technology have led to the development of novel dyes offering enhanced properties. Custom solutions may also be available for specific research needs.

Impact of Dyes on Peptide Function

Alteration of Peptide Properties

Research indicates that fluorescent labels can significantly alter the physicochemical properties of peptides, affecting their function and localization. For instance, a study by H. Szeto et al. (read more) highlights how different fluorescent labels can lead to varied intracellular targeting and function in cell-penetrating tetrapeptides.

Mitochondrial Targeting and Protection

Certain dyes have been shown to target specific cellular components, such as mitochondria, influencing peptide behavior and therapeutic potential.

Practical Considerations in Dye Selection

Compatibility with Experimental Conditions

The chosen dye must be compatible with the experimental conditions, including pH, temperature, and the presence of other biomolecules.

Cost and Availability

Consider the cost and availability of dyes, especially for large-scale studies or specialized applications. For a range of options, explore LifeTein’s custom synthesis page.

Typically, FITC, FAM, and TAMRA are less costly than dyes like Cyanine or AlexaFluor.

Frequently Asked Questions

• How do I choose the right fluorescent dye for my peptide?

• Consider the application, desired wavelength, required properties of the dye, and the potential impact on the peptide’s function.

• Can the dye alter the function of my peptide?

• Yes, fluorescent labels can change the peptide’s properties and intracellular behavior.

• Are there custom dye options available for specific needs?

• Yes, custom dye solutions can be developed for unique research requirements.

For further reading on the impact of fluorescent dyes on peptides, consider the research by M. Berezin et al. on the selection of small peptide molecular probes (read the study).

Szeto, H.H., Schiller, P.W., Zhao, K. and Luo, G. (2005), Fluorescent dyes alter intracellular targeting and function of cell-penetrating tetrapeptides. The FASEB Journal, 19: 118-120. https://doi.org/10.1096/fj.04-1982fje

Mikhail Y. Berezin, Kevin Guo, Walter Akers, Joseph Livingston, Metasebya Solomon, Hyeran Lee, Kexian Liang, Anthony Agee, and Samuel Achilefu, Rational Approach To Select Small Peptide Molecular Probes Labeled with Fluorescent Cyanine Dyes for in Vivo Optical Imaging. Biochemistry 2011 50 (13), 2691-2700
https://doi.org/10.1021/bi2000966

The chronic autoinflammatory bowel disease ulcerative colitis affects millions around the world. The condition involves autoreactive T cells and macrophages in the colonic mucosa attacking healthy colon cells, leading to inflammation, ulcers, and other debilitating symptoms and complications. While there is no outright cure, the only treatment involved is long-term immunosuppression, which can lead to even more health complications and risk of cancer down the line. One novel solution possible is antigen-specific immunotherapy, where the specific antigens are presented to the T cells in the presence of immunomodulators. Peptide-assisted antigen-specific Immunotherapy of ulcerative colitis by being adsorbed to nanofibers utilized in the colon-specific niche developed for this condition.

Mimetic peptides utilized in antigen-specific immunotherapy

LifeTein provided the group with the Cationic TGF-β1 mimetic peptide, whose role in the niche is to bind to a key receptor and suppress the activation of CD8+ T cells. This also polarizes the macrophages involved as well. The final results proved that not only could the auto reactive T cells be inhibited, but healthy colon cells could help repair previous damages of ulcerative colitis afterwards. All of this was achieved while actively avoiding the cancer risks of standard immunosuppressive approaches. Hopefully, this modular method can be pivotal in future developments for safer and more effective uses of immunotherapies.

Kin Man Au, Justin E. Wilson, Jenny P.-Y. Ting, Andrew Z. Wang. An Injectable Subcutaneous Colon-Specific Immune Niche For The Treatment Of Ulcerative Colitis doi: https://doi.org/10.1101/2023.10.03.560652

The incurable neurodegenerative Alzheimer’s disease has long been associated with β-amyloid build-up in the brain. While this has been known, direct evidence supporting the close relationship of Alzheimer’s disease and the role of β-amyloid has been hard to come by, until now. Recent Aβ-immunotherapy trials have shown that removing aggregated β-amyloid from symptomatic patients can slow down the disease.

β-amyloid removal slows the progression of Alzheimer’s disease

This breakthrough holds many implications for future treatment and handling of Alzheimer’s disease. While the new evidence is far from being a cure itself, it presents the opportunity for long-term prevention and potential immunoprevention towards the disease. This is in part due to how early the signs and abnormal β-amyloid build-up can begin in Alzheimer’s patients, as well as how complex the disease itself can become. However many clinical trials and experiments it may take, groups like LifeTein will always be ready to help supply researchers with the materials they need to make this future possible.

Jucker, M., & Walker, L. C. (2023). Alzheimer’s disease: From immunotherapy to immunoprevention. In Cell (Vol. 186, Issue 20, pp. 4260–4270). Elsevier BV. https://doi.org/10.1016/j.cell.2023.08.021

Many sensory declines accompany aging, one of which is sight. That being said, there is a need for much more research on the visual changes associated with such aging. Specifically, the changes in rod bipolar cells and their ribbon synapses due to aging are an area of interest, along with the complex calcium systems at work. Using a zebrafish model, peptides help determine the effects of aging on vision and the retina.

Zebrafish were used for this experiment thanks to their unique roles as model organisms; they share 70% genomic similarity with humans, and their short lifespan offers the chance to study life cycles in a few short years while still comparable to human aging over decades. Researchers compared data between middle-aged (MA, 18-months-old) and older-aged (OA, 36-months-old) zebrafish, equating to human ages of approximately 38 and 75 years of age, respectively. Using TAMRA ribbon-binding peptides from LifeTein, the team was able to observe changes between the two ages of zebrafish.

What was discovered was a decreased number of synaptic ribbons and increased ribbon length in the OA models. Further, there were many alterations to the local calcium dynamics of the system, implying a more complex change to vision deterioration than initially expected. The model shows how subtle changes could have vast implications for disease models where these alterations may be amplified and surely sheds more light on how human vision may decline with age.

Abhishek P Shrestha, Nirujan Rameshkumar, Johane Martins Boff, Rhea Rajmanna, Thadshayini Chandrasegaran, Frederick E Courtney, David Zenisek, Thirumalini Vaithianathan
bioRxiv 2023.09.01.555825; doi: https://doi.org/10.1101/2023.09.01.555825

LifeTein’s Innovative Peptide Antagonist: A New Ally in the Fight Against Breast Cancer Metastasis

Breast cancer stands as the most commonly diagnosed cancer in women worldwide and is a leading cause of cancer-related deaths. Among its subtypes, triple-negative breast cancer (TNBC) is particularly notorious for being aggressive and prone to metastasizing to vital organs like the brain, lungs, bones, and liver. Despite being more responsive to chemotherapy, TNBC’s propensity for metastasis poses a significant challenge in cancer treatment.

Recent studies, including notable research from the Medical University of Lodz in Poland, have identified a key factor in TNBC metastasis: increased F11R/JAM-A activity. This protein plays a crucial role in the early stages of cancer cell migration across blood vessels, a precursor to metastasis. Enter LifeTein, a pioneering force in peptide technology, which has made a groundbreaking contribution to this research area.

LifeTein provided a specialized peptide antagonist, named P4D, designed to specifically target and inhibit F11R/JAM‑A. The effectiveness of P4D was rigorously tested in lab models. Remarkably, this antagonist not only curbed the proliferation of TNBC cells but also significantly reduced their survival by directly targeting F11R/JAM-A. The result was a notable hindrance in the metastasis process in the mouse models used for the study.

This breakthrough has significant implications. The success of P4D in these preliminary studies suggests potential for future clinical trials and paves the way for more targeted, effective treatments for TNBC, possibly extending to the development of tailored antibodies. LifeTein’s contribution to this field exemplifies its commitment to advancing cancer therapy, offering new hope to those battling with TNBC.

For more detailed insights, refer to the original study by Bednarek, R., Wojkowska, D.W., Braun, M. et al., titled “Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein,” published in Cancer Cell International.

Bednarek, R., Wojkowska, D.W., Braun, M. et al. Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein. Cancer Cell Int 23, 160 (2023).

As methods of medicine advance, targeted drug delivery becomes a more appealing and achievable option over its non-selective counterpart. It can focus solely on increasing therapeutic concentration in the target area while greatly eliminating any exposure to healthy tissue, and thus drastically lowering side effects as well. The effective and simple mechanisms of click chemistry are a great way to design payloads for these targeted drug delivery methods. With the use of enzyme-degradable peptides in click chemistry drug delivery, lasting therapeutics can remain in the system for local sustained release over time as well.

Enzyme-degradable peptides for sustained drug delivery

The team at Rutgers focused on a two-phase method to set up the targeted drug delivery. First, ROS-sensitive PEGDA and acrylate-PEG-azide are aimed at the target area, driven by elevated free radical levels. Once the pretargeting is complete, a payload tethered to DBCO is delivered and captured via azide-DBCO reactions. Enzyme-degradable peptides were provided by LifeTein and incorporated into both steps for the ongoing release of the captured payloads.

The results showed success in the models tested, with the initial dosage still effective in capturing the payload several days later. This system demonstrated the versatility of a two-phase method, where long-term effects are even further avoided by incorporating enzyme-degradable peptides. The proof of concept displayed here has great promise for the future of drug delivery and just goes to show how applicable click chemistry is to even more fields.

Emily T. DiMartini, Kelly Kyker-Snowman & David I. Shreiber (2023) A click chemistry-based, free radical-initiated delivery system for the capture and release of payloads, Drug Delivery, 30:1, DOI: 10.1080/10717544.2023.2232952

During cell division, microtubules in the chromosome attach to a region called the centromere. While most species have a single size-restricted centromere, or a monocentromere, some species exist with multiple centromeres distributed across the chromosome, called holocentromeres. What is even more interesting is how holocentric chromosomes are considered to have evolved from the monocentric organisms, and this transition occurred independently across distant lineages, such as green algae, protozoans, invertebrates, as well as flowering plant families. One group aimed to study these holocentromeres more via the lilioid Chionographis japonica. Their goal was to better understand the convergent evolution of holocentromeres studied with peptides.

Peptides help explore holocentromeres

The group determined that the chromosomal localization of the target centromere is usually marked with histone H3 (CENH3). With this knowledge, they utilized peptides and antibodies of CENH3 provided by LifeTein to create models of the transition of C. japonica from interphase to prophase and study the possible mechanisms as well. They found the holocentromere was made up only of a few, evenly spaced CENH3-positive megabase-sized satellite arrays. Overall, the reason for the convergent evolution of holocentromeres from a monocentromere may stem from multiple factors, but more experiments like the ones presented will surely provide further analysis into this complex and fascinating case of convergent evolution.
Kuo, YT., Câmara, A.S., Schubert, V. et al. Holocentromeres can consist of merely a few megabase-sized satellite arrays. Nat Commun 14, 3502 (2023). https://doi.org/10.1038/s41467-023-38922-7

Tick infestations are a recurring roadblock of human development around the world, with estimated damages in the global economic landscape being as high as 30 billion USD. Specifically, India has long been susceptible to tick-borne diseases, due to multiple species invading the livestock. These regional parasites are major vectors for Crimean–Congo hemorrhagic fever virus (CCHFV), a disease with a devastating case fatality rate of 10–40%. While the main way of combatting the infestation of ticks and their carried disease has always been pesticides, often to an invasive degree of their own, scientists are working diligently for ways to produce a vaccine for this deadly and prevalent outbreak. One such method that has been explored is multi-epitopic peptide vaccines that combat Crimean–Congo hemorrhagic fever virus, specifically through the potential immune stimulatory responses they cause.

Multi-epitopic peptides help boost immune system

LifeTein provided the group with the two designed multi-epitopic peptides, VT1 and VT2. Using the two peptides, the group put them into two working vaccines in an effort to explore how effective they were at fighting back the ticks. With rabbits, they found strong immunity conferred by the vaccine, displayed by quick larval detachment, delayed tick feeding, low engorgement weights, and overall efficacy against both tick larvae and adults. The results show just how effective treatment with the vaccines are against ticks carrying CCHFV, and the compatibility with rabbits is a great starting point.

In an ideal experiment, the group would have tested on cattle, since that is a much more affected group by these ticks. Regardless, the suitability and stability displayed warrants more attention be put into these multi-epitopic peptide vaccines. Their efficacy displayed against infestations as such is sure to save the global economy billions, as well as countless lives. Immunization in this route is surely more appealing than that of constant and overwhelming pesticides being put in place at every conceivable turn. LifeTein is excited to see where else peptide-based vaccines can be implemented and what other unique properties they can bring to the table.

Nandi A, Manisha, Solanki V, Tiwari V, Sajjanar B, Sankar M, Saini M, Shrivastava S, Bhure SK, Ghosh S. Protective Efficacy of Multiple Epitope-Based Vaccine against Hyalomma anatolicum, Vector of Theileria annulata and Crimean–Congo Hemorrhagic Fever Virus. Vaccines. 2023; 11(4):881. https://doi.org/10.3390/vaccines11040881

Cell-penetrating peptides (CPPs) are a longstanding part of the biochemical world, with their potential for delivering bioactive agents into cells, their importance will likely never diminish. However, not many advancements are being made to CPPs, rather scientists and teams alike are always looking to develop more efficient CPPs when possible. This is where a hot topic of recent comes into play, researchers utilized a deep-learning-based CPP prediction method, one they have named AiCPP, to effectively develop novel CPPs while reducing false-positive predictions. Using LifeTein cell-penetrating peptides and machine learning, AiCPP was able to combine data with other lists of CPPs to generate successful new sequences that the team at hand was able to test effectively.

LifeTein CPPs help train AI to generate new sequences

The group utilized a sliding window approach on their wealth of data and included a list of peptides with low similarity to CPPs as a negative to reduce false positives obtained. These techniques helped AiCPP stand out against other machine learning methods before it, and they found AiCPP can further optimize CPP sequences with higher efficacy as well. Valuable information can be gathered by studying the patterns by which this machine learning determines are effective for CPPs.

Though novel, the limitations of utilizing this and other machine-learning methods should be understood as well. Foremost, this and other CPP prediction studies do not answer any important questions about the mechanisms of cell permeation, or how each CPP specifically achieves this. It is also worth noting that the effectiveness of a given CPP is limited to the type of cell is it trying to penetrate, for example, MCF-7 used in the referenced study. Though, these shortcomings are possible to overcome, and in the future, AiCPP may become more advanced and be able to offer even more research, with LifeTein keeping up as well and ready to assist any team that may need their new CPPs developed.

Park H, Park J-H, Kim MS, Cho K, Shin J-M. In Silico Screening and Optimization of Cell-Penetrating Peptides Using Deep Learning Methods. Biomolecules. 2023; 13(3):522. https://doi.org/10.3390/biom13030522