Peptide libraries are a cornerstone in the field of biochemical research, offering a versatile tool for various applications ranging from drug discovery to therapeutic development. Understanding the utility and benefits of peptide libraries can significantly enhance research outcomes in these areas.

Key Takeaways:

• Peptide libraries provide a diverse range of peptide sequences for high-throughput screening.
• They are essential in drug discovery, epitope mapping, and studying protein-protein interactions.
• Peptide libraries facilitate the identification of bioactive peptides and optimization of peptide-based therapeutics.

What is a Peptide Library?

A peptide library is a collection of peptides with a systematic variation of amino acid sequences. It is used to study a wide range of biochemical and pharmaceutical properties.

Types of Peptide Libraries

Peptide libraries can be random, overlapping, positional, or alanine-scanned, each serving different research purposes.

Applications of Peptide Libraries

Drug Discovery

Peptide libraries are instrumental in identifying and optimizing new drug candidates, particularly in targeting specific proteins or receptors.

Vaccine Development

They are used to identify peptide sequences that can elicit an immune response, aiding in the design of effective vaccines.

Advantages of Using Peptide Libraries

High-Throughput Screening

Peptide libraries allow for the simultaneous screening of thousands of peptides, significantly speeding up the research process.

Versatility

They can be customized to include a wide range of modifications, such as phosphorylation, methylation, and cyclization.

Designing a Peptide Library

Selection of Amino Acids

The choice of amino acids in a peptide library is crucial and depends on the specific research objective.

Library Size and Diversity

The size and diversity of a peptide library determine its effectiveness in screening and identifying active peptides.

Explore LifeTein’s Custom Peptide Synthesis Services for more on designing peptide libraries.

Peptide Library in Research and Development

Epitope Mapping

Peptide libraries are used to identify the specific parts of antigens that are recognized by antibodies, crucial for vaccine development and diagnostic assays.

Protein-Protein Interactions

They enable the study of protein-protein interactions, which is essential in understanding cellular processes and identifying therapeutic targets.

Technological Advances in Peptide Libraries

High-Throughput Synthesis

Modern peptide libraries are synthesized using advanced technologies, allowing for rapid production of large numbers of peptides.

Customization

Peptide libraries can be customized to include specific sequences, modifications, and lengths tailored to the unique requirements of a research project.

Advanced Synthesis Techniques

Modern synthesis techniques, such as continuous-flow peptide synthesis, enable the efficient and high-quality production of peptide libraries.

Discover more about advanced synthesis techniques at LifeTein’s Peptide Library Service.

Challenges and Solutions in Peptide Library Utilization

Managing Complexity

The complexity of peptide libraries can be challenging, but advanced computational tools and algorithms are available to manage and analyze the vast data generated.

Quality Control

Ensuring the quality and purity of peptides in a library is crucial. Techniques like HPLC and mass spectrometry are employed for stringent quality control.

Frequently Asked Questions

• What is the main advantage of using a peptide library?

• The main advantage is the ability to screen a vast number of peptides simultaneously for various biochemical properties.

• Can peptide libraries be used in personalized medicine?

• Yes, peptide libraries can be used to identify peptide-based therapeutics tailored to individual genetic profiles.

• How are peptide libraries synthesized?

• They are synthesized using methods like solid-phase peptide synthesis, allowing for the incorporation of diverse amino acids and modifications.

In summary, peptide libraries are invaluable tools in modern scientific research, offering unparalleled opportunities for discovery and innovation in various fields. Their ability to screen a vast array of peptide sequences rapidly makes them indispensable in drug discovery, vaccine development, and the study of protein interactions. With the advancement of synthesis technologies and analytical tools, the potential of peptide libraries continues to expand, paving the way for new breakthroughs in science and medicine.

Enhancing the water solubility of peptide sequences is a critical aspect of peptide-based therapeutic development and biochemical research. This article explores various strategies and scientific insights into making peptides more water soluble.

Key Takeaways:

• Water solubility of peptides is influenced by their amino acid composition and sequence.
• Incorporating hydrophilic amino acids can significantly enhance solubility.
• Peptide modifications and the use of solubility-enhancing agents are effective strategies.

Understanding Peptide Solubility

The Importance of Solubility

Water solubility is crucial for the biological function and therapeutic application of peptides. Soluble peptides are more bioavailable and easier to handle in laboratory settings.

Testing Solubility

When initially testing solubility, trying distilled water first is almost always a great initiative.

It is recommended to test the solubility of a small portion of the sample rather than dissolving the entire sample and to choose an initial solvent that can be easily removed by lyophilization. This allows easy recovery of the peptide from the solvent.

Factors Affecting Solubility

The solubility of peptides depends on various factors, including the amino acid composition, sequence, peptide length, and the presence of hydrophobic or hydrophilic residues.

Strategies for Enhancing Solubility

Incorporating Hydrophilic Amino Acids

Introducing hydrophilic amino acids like lysine, arginine, and glutamic acid can make peptides more water soluble.

Sequence Optimization

Modifying the sequence and length of the peptide can also impact its solubility. Shorter peptides with optimized sequences tend to be more soluble.

For more insights into peptide solubility, consider the study by Asuka Inada et al. on the water solubility of complexes between a peptide mixture and poorly water-soluble drugs (read more).

Peptide Modifications

N-terminal Acetylation and C-terminal Amidation

These modifications can shield the peptide from enzymatic degradation and enhance solubility.

Use of Solubility-Enhancing Tags

Attaching solubility tags like polyethylene glycol (PEG) can significantly improve the solubility of peptides.

Computational Approaches

Molecular Dynamics Simulations

Advanced computational methods like molecular dynamics simulations can predict the solubility of peptides based on their structure and composition.

Machine Learning Algorithms

Machine learning algorithms can analyze large datasets to predict and optimize peptide solubility.

For further reading on peptide solubility, explore the research by Yan Jiao et al. on zein-derived peptides as nanocarriers to increase the water solubility and stability of lutein (read the study).

Practical Considerations

pH and Ionic Strength

Adjusting the pH and ionic strength of the solution can significantly influence peptide solubility. Peptides tend to be more soluble at pH values away from their isoelectric point, or neutral pH levels as well.

Determining the overall charge of a peptide will greatly assist in assessing the solubility, LifeTein has a comprehensive guide on how to find the charge.

Temperature

Temperature can also affect solubility. In some cases, increasing the temperature can enhance the solubility of peptides.

Frequently Asked Questions

• How do amino acid properties affect peptide solubility?

• Amino acids with hydrophilic side chains increase solubility, while hydrophobic ones decrease it. Overall charge will affect solubility as well.

• Can peptide length influence its solubility?

• Yes, shorter peptides generally have higher solubility.

• Are there chemical modifications that can enhance peptide solubility?

• Yes, modifications like N-terminal acetylation, C-terminal amidation, and the addition of solubility tags can improve solubility.

For additional insights into peptide solubility, consider the study by R. Sarma et al. on peptide solubility limits and backbone interactions (read the study).

Inada, A., Wang, M., Oshima, T., & Baba, Y. (2016). Water Solubility of Complexes between a Peptide Mixture and Poorly Water-Soluble Ionic and Nonionic Drugs. In Journal of Chemical Engineering of Japan (Vol. 49, Issue 6, pp. 544–551). Informa UK Limited. https://doi.org/10.1252/jcej.15we313

Jiao, Y., Zheng, X., Chang, Y., Li, D., Sun, X., & Liu, X. (2018). Zein-derived peptides as nanocarriers to increase the water solubility and stability of lutein. In Food & Function (Vol. 9, Issue 1, pp. 117–123). Royal Society of Chemistry (RSC). https://doi.org/10.1039/c7fo01652b

Sarma, R., Wong, K.-Y., Lynch, G. C., & Pettitt, B. M. (2018). Peptide Solubility Limits: Backbone and Side-Chain Interactions. In The Journal of Physical Chemistry B (Vol. 122, Issue 13, pp. 3528–3539). American Chemical Society (ACS). https://doi.org/10.1021/acs.jpcb.7b10734

Penetratin, a cell-penetrating peptide (CPP), RQIKIWFQNRRMKWKKGG, has emerged as a significant tool in molecular biology and drug delivery. This article provides a comprehensive overview of Penetratin, its properties, applications, and the latest research insights.
Key Takeaways:
• Penetratin is a powerful CPP derived from the Antennapedia protein of Drosophila.
• It is known for its ability to traverse cellular membranes efficiently.
• Penetratin is used in drug delivery, particularly in targeting cancer cells and crossing the blood-brain barrier.

Introduction
What is Penetratin?
Penetratin is a short peptide derived from the third helix of the homeodomain of the Antennapedia protein in Drosophila. It is one of the most studied CPPs due to its ability to penetrate cellular membranes.
The Structure and Sequence
Penetratin is rich in positively charged residues, which play a crucial role in its membrane penetration capabilities. The amino acid sequence is as follows: RQIKIWFQNRRMKWKKGG


Mechanism of Action
Cellular Uptake
Penetratin is known to interact with negatively charged membrane components, facilitating its entry into cells. This interaction is crucial for its function as a CPP.
Translocation Mechanism
The exact mechanism of Penetratin’s translocation across cell membranes is still a subject of research. It is believed to involve direct penetration rather than endocytosis.

Applications of RQIKIWFQNRRMKWKKGG:
Drug Delivery
Penetratin has been extensively studied for its potential in drug delivery, especially for targeting tumor cells and delivering therapeutic agents across the blood-brain barrier.
Gene Therapy
Its ability to carry large molecules like nucleic acids makes it a promising tool for gene therapy applications.
For more information on Penetratin and its applications, visit LifeTein’s page on Penetratin.
Research Insights
Penetratin in Cancer Therapy
Studies have shown that Penetratin can selectively target cancer cells, making it a potential tool for targeted cancer therapy. For instance, a study by Bashiyar Almarwani et al. (read more) investigates Penetratin’s insertion into cancer cell membranes.
Crossing the Blood-Brain Barrier
Penetratin’s ability to cross the blood-brain barrier opens avenues for treating neurodegenerative diseases. Research by S. Bera et al. (read the study) provides insights into its structural elucidation in model membranes.
Challenges and Considerations
Selectivity and Efficiency
While Penetratin is efficient in penetrating cells, its selectivity, especially in distinguishing between healthy and cancer cells, is a critical area of research.
Safety and Toxicity
Understanding the safety profile and potential toxicity of Penetratin is essential, particularly for its use in clinical applications.
For a deeper understanding of Penetratin’s properties, explore LifeTein’s overview of Cell Permeable Peptides (CPPs).
Frequently Asked Questions
1. What makes Penetratin a unique CPP?
• Its high efficiency in penetrating cellular membranes and the ability to carry large molecules.
2. Can Penetratin be used in treating brain diseases?
• Yes, its ability to cross the blood-brain barrier makes it a candidate for treating neurological disorders.
3. Is Penetratin selective in targeting cells?
• Current research is focused on enhancing its selectivity, particularly in distinguishing between healthy and cancer cells.
For further reading on Penetratin’s interaction with cell membranes, consider the research by I. Alves et al. on its membrane binding and internalization efficacy (read the study).

Almarwani, B.; Hamada, Y.Z.; Phambu, N.; Sunda-Meya, A. Investigating the Insertion Mechanism of Cell-Penetrating Peptide Penetratin into Cell Membranes: Implications for Targeted Drug Delivery. Biophysica 2023, 3, 620-635. https://doi.org/10.3390/biophysica3040042

Swapna Bera, Rajiv K. Kar, Susanta Mondal, Kalipada Pahan, and Anirban Bhunia. Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood–Brain Barrier. Biochemistry 2016 55 (35), 4982-4996
https://doi.org/10.1021/acs.biochem.6b00518

Alves ID, Bechara C, Walrant A, Zaltsman Y, Jiao C-Y, Sagan S (2011) Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study. PLoS ONE 6(9): e24096. https://doi.org/10.1371/journal.pone.0024096

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 landscape of in situ hybridization (ISH), specifically fluorescence in situ hybridization (FISH), has undergone a transformative shift with the introduction of Nucleic Acid Mimics (NAMs). These modified probes, encompassing Peptide Nucleic Acid (PNA), Locked Nucleic Acid (LNA), 2′-O-Methyl-RNA, UNA (unlocked nucleic acid), and Phosphorodiamidate Morpholino Oligomers (PMOs), have emerged as groundbreaking tools, overcoming the limitations associated with traditional DNA and RNA probes.

Peptide Nucleic Acids (PNAs):

Peptide Nucleic Acids (PNAs) stand at the forefront of this molecular revolution, offering a fusion of DNA specificity and peptide versatility. The distinctive PNA backbone, composed of peptide linkages, ensures unparalleled stability and resistance to enzymatic degradation. With superior hybridization properties, PNAs bind to complementary DNA or RNA sequences with exceptional affinity, making them indispensable for applications ranging from targeted gene therapy to diagnostic assays and antisense technologies.

Unlocking the Potential: PNA’s Key Features:

Stability and Resistance:
PNAs, characterized by a neutral polyamide backbone, showcase remarkable stability against nucleases and enzymatic degradation. This attribute enhances the half-life of PNA molecules, ensuring their efficacy in diverse experimental conditions.

High-Affinity Binding:
The hybridization capabilities of PNAs are unparalleled, facilitating strong, sequence-specific binding to target nucleic acids. PNA’s shorter length allows for enhanced cell penetration and consistent hybridization performance, even under low salt concentrations. Its unique melting temperature response to single nucleotide changes enables precise probe design.

Versatility in Applications:
PNAs find applications across a spectrum of research areas, including molecular diagnostics, gene editing, and nanotechnology. Their adaptability for specific sequences and functions makes PNAs an invaluable asset in the molecular biologist’s toolkit.

Locked Nucleic Acid (LNA):

Described in 1997, Locked Nucleic Acid (LNA) boasts a ribose ring locked in a specific conformation, ensuring water solubility and low toxicity. LNA’s design flexibility, including modifications like phosphorothioate, enhances resistance to nucleases without compromising affinity. Combining LNA with 2′-O-Methyl-RNA provides flexibility in melting temperature adjustments for optimized hybridization efficiency.

UNA and Other NAMs:

UNA, an acyclic RNA analog, offers flexibility, though it may impact nucleic acid duplex stability. Modifications like 2′-pyrene and 3′-O-amino-UNA address stability concerns, presenting potential applications in FISH experiments. Additionally, Phosphorodiamidate Morpholino Oligomers (PMOs), characterized by non-ionic properties and resistance to nucleases, have shown success in bacterial and fungal infection detection via FISH.

Challenges and Progress:

Despite the exceptional qualities of NAMs, particularly PNA and LNA, their widespread adoption in FISH for microorganism detection has been slower than anticipated. Challenges include hydrophobicity and water solubility issues for PNA probes, along with a general lack of awareness among laboratories. Nevertheless, studies showcasing PNA’s superior performance over traditional DNA probes underscore the promising potential of NAMs in microbial detection.

Conclusion:

As research in the field progresses, ongoing efforts are addressing the limitations associated with NAMs. Their unique features position them as compelling alternatives for FISH-based microorganism detection, holding the promise of unlocking new frontiers in advanced genetic research.

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

Live-cell imaging using peptides serves as an invaluable asset in the world of life science research, offering a window into the dynamic lives of cells in conditions closely resembling their natural habitat. By capturing real-time images of living cells, researchers can explore intricate aspects of cell behavior, growth, and movement.

In contrast to the static imaging of fixed cells and tissues, where photobleaching poses a significant challenge, live-cell imaging calls for a different approach. While fixed cell imaging demands high-intensity illumination and prolonged exposure, such conditions prove detrimental to the vitality of live cells. Consequently, live-cell microscopy finds itself in a constant balancing act, seeking to attain image quality while safeguarding cell well-being. This balance often imposes limitations on spatial and temporal resolutions in live-cell experiments.

Live-cell imaging encompasses a wide range of contrast-enhanced techniques in optical microscopy. The majority of investigations rely on various forms of fluorescence microscopy, often in conjunction with transmitted light methodologies. The ongoing evolution of imaging techniques and the development of fluorescent probes ensure that live-cell imaging retains its significance as a critical tool in the field of biology.

1. The Fundamentals of Fluorescence Microscopy

One of the fundamental considerations is determining the precise amount of excitation light required to obtain a meaningful image. It’s worth noting that high-intensity light, especially in the near-UV range, can harm cells and potentially induce DNA damage. However, in live-cell fluorescence microscopy, the primary source of phototoxicity stems from the photobleaching of fluorophores. A critical protective measure involves deactivating the illuminating light when not in use. Employing shutters to control light exposure proves to be a pivotal element in live-cell imaging.

Additionally, it’s crucial to eliminate undesired wavelengths of light and opt for emission filters that are fine-tuned to maximize the signal. Mitigating photobleaching can be achieved by reducing oxygen levels, and minimizing background fluorescence can be accomplished by excluding phenol red and serum from the culture medium. It’s also essential to prevent any contamination of the illuminating light with even minute traces of ultraviolet or infrared wavelengths. The contained photobleaching chemistry within the β-barrel structure of fluorescent proteins (FPs) or peptides makes them less phototoxic.

The most effective strategy for reducing photobleaching and the associated photodamage is to minimize excitation light exposure by carefully managing exposure time and light intensity while maintaining a satisfactory signal-to-noise ratio tailored to the specific research question.

2. The Live Cell Imaging Microscope

When selecting an optical microscopy system for live-cell imaging, three key factors come into play: detector sensitivity (signal-to-noise ratio), specimen viability, and the speed required for image acquisition. To optimize the signal-to-noise ratio, it’s crucial to select filters that closely match the spectral profiles of the fluorophores in use. Most epi-illumination microscopes and confocal systems acquire data in four dimensions.

Time-lapse imaging, involving the capture of cellular events over various timeframes, is commonly employed. This technique enables the repetitive imaging of cell cultures at specific time intervals. Wavelengths for excitation and emission filters should be finely tuned to match the fluorophore, thus reducing unnecessary light exposure. To minimize photodamage to the specimen, it’s advisable to use the lowest magnification that suits the specific experiment.

3. Managing the Microscope Environment

Maintaining a physiological environment is imperative for cultured cells and tissues to behave naturally. Thus, the control of factors such as temperature and tissue culture medium composition plays a critical role in obtaining meaningful data in live-cell imaging experiments. Mammalian cell lines are typically maintained at 37°C. Variations in temperature and vibrations can negatively affect focus stability. To address these issues, options like stage-top incubators and fully enclosed microscopes can be considered. Most tissue culture media are buffered to a physiological pH using sodium bicarbonate and 5% CO2.

4. Unmasking the Potential of Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is a remarkable technique that enhances the spatial resolution of fluorescence microscopes to under 10 nanometers. This substantial improvement in resolution makes FRET particularly appealing for studying co-localization events in biological samples, especially within living cells. However, in live-cell studies, the risk of FRET measurements being invalidated by acceptor fluorophore recovery, similar to FRAP experiments, must be carefully considered. Therefore, the use of acceptor photobleaching in live-cell experiments may not always be suitable.

Useful FRET calculator that provides a listing of key FRET pair information: www.fpbase.org/fret/

Furthermore, modern fluorescent dyes, such as the Alexa dye series, offer advantages like enhanced quantum efficiency and brightness, making them indispensable for specific techniques. LifeTein offers an extensive range of fluorescent labeling options, including FITC, FAM, TAMRA, Cyanine Dye Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, EDANS/Dabcyl, MCA, AZDye, BODIPY FL or Alexa Fluor (Alexa488, Alexa532, Alexa546, Alexa594, Alexa633, Alexa647), ATTO Dyes (Atto465, Atto488, Atto495, Atto550, Atto647), and DyLight (DyLight 488, DyLight 550). However, chemical fluorescent dyes often exhibit higher cytotoxicity, possibly due to the cytoplasm’s reduced protection from reactive free radical breakdown products when compared to FPs, where the fluorophore is encapsulated within the FP beta-barrel structure. A range of cell-permeable fluorescent molecules is available for the specific labeling of intracellular organelles.

5. Navigating Photobleaching

Photobleaching is an inherent aspect of live-cell imaging. Even the most advanced fluorescent molecules convert only a fraction of the absorbed energy into fluorescence. Some of this energy inevitably triggers chemical reactions that lead to fluorophore breakdown. Thus, aside from maintaining a physiological environment and confirming the specific labeling and function of the protein or organelle of interest, the most critical factor for successful live-cell imaging and obtaining meaningful data is the minimization of excitation light. This requires a thorough understanding of the microscope and optimization of components that regulate exposure, wavelength selection, and the collection of emitted photons.

A proper experimental setup is equally vital. Waiting for the visual system to adapt to darkness before attempting to locate a faint sample is advisable, given that modern cameras are often more sensitive than the human eye. However, it’s important to avoid using live camera modes to find your sample. Every photon is precious.

Unfortunately, photobleaching remains an unavoidable challenge, often determining the number of images that can be acquired. There is no simple solution to eliminate photobleaching completely. The specific excitation and emission bands, as well as the intracellular environment, influence the apparent brightness and, consequently, the apparent photobleaching of specific fluorescent proteins.”

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

Please view this video on how to design cell penetrating peptides. The transcript is listed below.

Transcript

Slide 1:

Thank you for joining me. My topic today is the cell-penetrating peptides. My main focus will be the peptide design, peptide synthesis, and its applications.

Slide 2:

First, let me briefly introduce LifeTein. LifeTein was founded in 2008. We have been in the peptide industry for more than ten years. We specialize in peptide synthesis, chemical synthesis, antibody production, and protein services.

Slide 3:

Our main focus is peptide synthesis service. However, over the years, we have expanded to other protein-related areas like protein, antibody services, and products.

Slide 4:

So let us quickly get into the topic: cell-penetrating peptides. What is a cell-penetrating peptide or cpp? From the definition, CPP is a short peptide. They can be about 4-40 aa. The short peptide can enter the cell membrane. They can deliver bioactive cargoes.

Slide 5:

CPPs can also be used to deliver bioactive cargos like siRNAs, DNA, polypeptides, liposomes, nanoparticles, and others, in cells for therapeutic or experimental purposes.

Slide 6:

There are a few popular models for CPP’s entry. 1. The inverted micelle model. The CPPs are positively charged. They interact with the negatively charged phospholipids in the membrane. 2. Direct entry or direct translocation. For example, sequences with multiple Arginines can cause a short-time membrane cytolysis and enter the cells directly. 3. By the traditional method of endocytosis. I will not talk about the details.

Slide 7:

Here are a few examples of the CPP. The most famous examples are HIV tat sequence. The TAT peptide is arginine-rich and can directly penetrate the plasma membrane and stabilize DNA.

Another example is the arginine-rich peptide R8 or R9. We can add stearic acid to the N-terminus.  Stearic acid is a saturated fatty acid with an 18-carbon chain. If you would like to do live cell imaging, we can add fluorescent dye such as Fitc, Alexa fluor, or Cy dye at the N-terminus or C-terminus. I will get to the details.

Slide 8:

CPPs can enter the regular cell membranes. Some other peptides are tissue-targeting peptides. For example, this brain-homing peptide can cross the blood-brain barrier. Other peptides can cross the skin as transdermal peptides, target heart tissues as cardiac targeting peptides, and nuclear localization signal peptides.

Slide 9:

On this slide, I will talk more about the peptide design. There are many ways to make the peptide permeable. In the case of DNA or RNA, you can simply mix the CPPs with oligos. Many transfection reagents are using this mechanism. Simply put, DNA is negatively charged and peptide is positively charged. If you mix them together, they will form small micelles for cell penetration.

However, most of our work is to put the CPPs and your target together by covalent links. For this example, we put eight arginines at the N-terminus of your peptide. A linker called Ahx is added as a spacer. Some users prefer no spacers. It seems that both worked for the purpose. The eight arginines can be put at the C-terminus as well. According to the feedback from our users, most of the N-terminal CPP worked well. A few worked well for the C-terminal conjugation. I guess it depends on the projects.

This example is the Npys linker modification. The cysteine is added to your peptide. We conjugate two sequences together to form a disulfide bond. This is especially useful for the cancer study. Cancer cells have a lower pH of 6.7-7.1. Normal cells have a higher pH of 7.4. Under the acidic environment, the disulfide bond can be cleaved. If your target peptide is a cancer drug candidate, the CPP can introduce the drug cargo to the cancer cell and release the target within the cell. These disulfide-based prodrugs are important for cancer therapy.

If the cysteine is not available for your case, we can add a compound called lysine azide. This method needs click chemistry.

Slide 10:

There are two kinds of click chemistry. The one with copper as the catalyst and the one without. The preferred method is copper-free click chemistry. It is called DBCO and azide reactions. The final conjugate will have a large linker. Many scientists have concerns about the bulky size of the linker. However, some drugs contain bulky linkers without issues or side effects.

Back to Slide 9:

Let us go back to the sequence. The design does not have to be this way. The lysine azide can be any place in the sequence. If you have a head-to-tail cyclic peptide, you can add the lysine azide in the middle. The final product will be like a lollipop, with the CPP as the tail. If the N-terminus is very important to you, you can add the azide at the C-terminus.

Slide 11:

Let us move on to other scenarios. If you would like to track the peptides in live cells, fluorescent dyes can be added. We can do Fitc, Fam, Cy3, cy5, Cy7 and Alexa Fluor. This design will give direct evidence that your target is inside the cells.  

There is a different kind of peptide called peptide nucleic acid or PNA. It is DNA or RNA analog. We can synthesize half as peptide and another half as the PNA.  

This structure is the one I just mentioned earlier. The cyclic tumor targeting RGD peptide can be linked with an R8 cell-penetrating peptide to form a lollipop-shaped structure.

Slide 12:

So far, we have mentioned different ways to conjugate the cargo with cell-penetrating peptides. If your targets are nanoparticles or gold particles. Our requirement is to have active groups like a thiol group or a free amine on it. We have to have the active groups react to the cell-penetrating peptides. It is the same requirement for small compounds.

Slide 13:

The last concept I would like to introduce is the antibody-drug conjugate. This concept is widely accepted in the antibody drug industry. There are three important components: an antibody, a cleavable linker, and the drug.  Once the antibody binds to the target, the drug is released after the hydrolysis by protease.

Slide 14:

The same concept can be used for the peptides. For this concept, we need to screen the best drug candidate for cell entry. The CPP can be tumor-homing peptides, brain-homing peptides, or cardiac targeting peptides I mentioned earlier.

Slide 15:

First, we need to modify the compound. It is better to have a free amine in the compound. Then we can modify the amine group to an azide group. Afterward, we can use the click chemistry for the following conjugation.

Slide 16

LifeTein produced a series of CPPs. They are ready to conjugate your compounds for screening. So far, we have designed and produced more than fifty CPPs.

Slide 17

Step 3 is conjugate peptides with drug candidates.

Slide 18

Once the CPP is conjugated with the drug compound using click chemistry, we can send the final product back to you for further screening. The purpose is to find the best drug delivery system.

Slide 19

To summarize today’s topic, I talked about cell-penetrating peptides with different cargos. As long as you have an active chemical group on the nanoparticles, compounds, or liposomes, we can conjugate the target to any peptide.

Slide 20

That is all for today. Please let me know if you have any questions. Please feel free to contact us by email or phone calls.   

In the world of peptide synthesis, a game-changing innovation has emerged – a remarkable cocktail designed to enhance the cleavage and deprotection of methionine-containing peptides. This groundbreaking concoction, known as Reagent H, is set to transform the landscape of solid-phase peptide synthesis, particularly for those using the 9-fluorenylmethoxycarbonyl (Fmoc) methodology.

Unveiling Reagent H: Your Key to Methionine Side-Chain Protection in Methionine-Containing Peptides

Reagent H, comprised of trifluoroacetic acid (81%), phenol (5%), thioanisole (5%), 1,2-ethanedithiol (2.5%), water (3%), dimethylsulphide (2%), and ammonium iodide (1.5% w/w), has been meticulously crafted to minimize the pesky oxidation of methionine side chains during synthesis. Its exceptional performance is exemplified in the synthesis of a model pentadecapeptide from the active site of DsbC, a pivotal player in protein disulfide bond formation.

The Triumph of Reagent H: Methionine Sulphoxide Conquered

When put to the test, Reagent H outshone its competitors, cocktails K, R, and B. The crude peptides obtained from these widely used mixtures contained a staggering 15% to 55% of methionine sulphoxide. However, Reagent H demonstrated its prowess by yielding pristine peptides devoid of methionine sulphoxide. Remarkably, even when 1.5% w/w NH4I was added to cocktails K, R, and B, they couldn’t match the perfection achieved by Reagent H, although their yield of the desired peptide fell short.

Unraveling the Mysteries: A Closer Look at Methionine-Containing Peptides

But how does Reagent H achieve this remarkable feat? We delve into the proposed mechanism behind its in situ oxidation of cysteine, shedding light on its impressive ability to safeguard methionine side chains while delivering high-quality peptides.

In the world of peptide synthesis, Reagent H stands as a beacon of hope for researchers seeking purity, precision, and protection in their work. Its ability to minimize methionine side-chain oxidation is nothing short of revolutionary, promising a brighter and more efficient future for peptide synthesis enthusiasts. Say goodbye to impurities and hello to perfection with Reagent H.