The document discusses aptamers, which are oligonucleotides or peptides that bind to targets with high affinity and specificity due to their unique three-dimensional structures. It describes how aptamers are produced through an in vitro selection process called SELEX and classified based on their structure and selection technique. The document also compares aptamers to antibodies and outlines several applications of aptamers in therapeutics, drug delivery, bioimaging, diagnostics, and more.
In this presentation I have mentioned whatever the possible relevant content required for the aptamer based drug delivery.
Citation Is done at the end of slide.
Content is up to date & true to my belief.
Thanks & Best Regards.
Anurag Pandey
B.Pharm (FACULTY OF PHARMACY, INVERTIS UNIVERSITY)
M.Pharm (INSTITUTE OF PHARMACY, NIRMA UNIVERSITY)
Email :- anurag.dmk05@gmail.com
Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention. Recent advances in the chemical modifications of nucleic acids suggest that one of the major barriers to use, stability, can be overcome. The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and therapeutic application. We should expect more aptamers to be isolated in the near future against an ever increasing repertoire of targets, using these different SELEX approaches with increased speed and efficiency. Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades.
Nucleic acid based therapeutic drug delivery systemtadisriteja9
Nucleic acid based Drug delivery system is one of the trending research area, which i have taken and made as Powerpoint for easy and quick learning purpose
In this presentation I have mentioned whatever the possible relevant content required for the aptamer based drug delivery.
Citation Is done at the end of slide.
Content is up to date & true to my belief.
Thanks & Best Regards.
Anurag Pandey
B.Pharm (FACULTY OF PHARMACY, INVERTIS UNIVERSITY)
M.Pharm (INSTITUTE OF PHARMACY, NIRMA UNIVERSITY)
Email :- anurag.dmk05@gmail.com
Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention. Recent advances in the chemical modifications of nucleic acids suggest that one of the major barriers to use, stability, can be overcome. The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and therapeutic application. We should expect more aptamers to be isolated in the near future against an ever increasing repertoire of targets, using these different SELEX approaches with increased speed and efficiency. Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades.
Nucleic acid based therapeutic drug delivery systemtadisriteja9
Nucleic acid based Drug delivery system is one of the trending research area, which i have taken and made as Powerpoint for easy and quick learning purpose
Aptamers - New Class of Oligonucleotide for Therapeutic and Diagnostic Useajithnandanam
http://technologyinscience.blogspot.com/2014/01/aptamers-new-class-of-oligonucleotide.html
Aptamers are single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets; most aptamers to proteins bind with Kds (equilibrium constant) in the range of 1 pM to 1 nM similar to monoclonal antibodies. These nucleic acid ligands bind to nucleic acid, proteins, small organic compounds, and even entire organisms. Aptamers have many potential uses in intracellular processes studies, medicine and technology. Aptamers are often identified using a technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment). By this techniques aptamers(oligos) having high affinity and specificity to the target is isolated from the sequence pool after several rounds of selection.
Nucleic Acid Based Therapeutic Delivery System.pptxRAHUL PAL
Therapeutic nucleic acids (TNAs) are nucleic acids themselves or closely related compounds used to treat disease. Although various types of TNAs exist, they share a common mechanism of action that is mediated by sequence‐specific recognition of endogenous nucleic acids through Watson–Crick base pairing 7.
What are the advantages of nucleic acid based therapeutics?
The major advantage of nucleic acid-based therapeutics lies in the fact that they can be used to accurately target a tumor or tissue, then have a specific therapeutic protein, biologic, or immune engager expressed only at the site of interest.
This will provide you the introduction about the tumor, its Anatomy & Physiology,How they are monitored?, Classification and grades of tumor, Tumor Targeting Techniques, strategies and Principles. Also provide you some examples of Marketed products.
Myself Omkar Tipugade , PG Student of Department of Pharmaceutics. today I will discus on the topic Gene Therapy . In that we discus about the method for gene therapy & its application for disease treatment.
Liposomes, Structure of liposome, phospholipids, classification of liposomes, method of preparation of liposomes, mechanism of liposome formation, application of liposomes.
Aquasomes are nanoparticulate carrier system but instead of being simple nanoparticles these are three layered self assembled structures, comprised of a solid phase nanocrystalline core coated with oligomeric film to which biochemically active molecules are adsorbed with or without modification.
Aptamers - New Class of Oligonucleotide for Therapeutic and Diagnostic Useajithnandanam
http://technologyinscience.blogspot.com/2014/01/aptamers-new-class-of-oligonucleotide.html
Aptamers are single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets; most aptamers to proteins bind with Kds (equilibrium constant) in the range of 1 pM to 1 nM similar to monoclonal antibodies. These nucleic acid ligands bind to nucleic acid, proteins, small organic compounds, and even entire organisms. Aptamers have many potential uses in intracellular processes studies, medicine and technology. Aptamers are often identified using a technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment). By this techniques aptamers(oligos) having high affinity and specificity to the target is isolated from the sequence pool after several rounds of selection.
Nucleic Acid Based Therapeutic Delivery System.pptxRAHUL PAL
Therapeutic nucleic acids (TNAs) are nucleic acids themselves or closely related compounds used to treat disease. Although various types of TNAs exist, they share a common mechanism of action that is mediated by sequence‐specific recognition of endogenous nucleic acids through Watson–Crick base pairing 7.
What are the advantages of nucleic acid based therapeutics?
The major advantage of nucleic acid-based therapeutics lies in the fact that they can be used to accurately target a tumor or tissue, then have a specific therapeutic protein, biologic, or immune engager expressed only at the site of interest.
This will provide you the introduction about the tumor, its Anatomy & Physiology,How they are monitored?, Classification and grades of tumor, Tumor Targeting Techniques, strategies and Principles. Also provide you some examples of Marketed products.
Myself Omkar Tipugade , PG Student of Department of Pharmaceutics. today I will discus on the topic Gene Therapy . In that we discus about the method for gene therapy & its application for disease treatment.
Liposomes, Structure of liposome, phospholipids, classification of liposomes, method of preparation of liposomes, mechanism of liposome formation, application of liposomes.
Aquasomes are nanoparticulate carrier system but instead of being simple nanoparticles these are three layered self assembled structures, comprised of a solid phase nanocrystalline core coated with oligomeric film to which biochemically active molecules are adsorbed with or without modification.
Apoptosis is characterized by several biochemical features including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering and the eventual engulfment of the cell by phagosomes.
Microplate capture and detection assay is used to detect the specifi.pdfanonaeon
Microplate capture and detection assay is used to detect the specific protein-DNA interactions
and identifies the proteins. The method includes immobilization of DNA probes on the surface of
microplates (having wells) coated with streptavidin. The cellular extract, prepared in binding
buffer, is added for enough time to facilitate binding protein to bind to the oligonucleotide
followed by washing of wells several times to remove nonspecifically bound proteins. A protein
specific antibody is used for detection of protein.
Comparison of advantages and disadvantages of microplate capture and detection assay and
EMSA:
A: Advantages
1: Microplate capture and detection assay is highly sensitive, fast and throughput technique that
is based on ELISA while EMSA is based on comparative velocity of migration of DNA-protein
complexes through non denaturing gel electrophoresis.
2: Both techniques can skip the use of radioacitve probes by using biotinylated or fluorescently
labeled DNA probes.
3: EMSA uses DNA probe mutational analysis for testing the binding affinity of proteins.
4: Though EMSA can detect the presence of low quantities of DNA binding proteins from
lysates, Microplate capture and detection assay can detect even lesser abundance of protein in the
well by using with enzyme-labeled antibodies and a chemiluminescent substrate.
Disadvantages:
1: Both techniques require supershift antibodies i.e. antibodies which can selectively bind to
DNA bound native proteins.
2: The technique gives little information about corresponding changes in transcription factor-
DNA.
3: The techniques have been used for a few target proteins so far while EMSA is applicable
under in vitro conditions only.
Solution
Microplate capture and detection assay is used to detect the specific protein-DNA interactions
and identifies the proteins. The method includes immobilization of DNA probes on the surface of
microplates (having wells) coated with streptavidin. The cellular extract, prepared in binding
buffer, is added for enough time to facilitate binding protein to bind to the oligonucleotide
followed by washing of wells several times to remove nonspecifically bound proteins. A protein
specific antibody is used for detection of protein.
Comparison of advantages and disadvantages of microplate capture and detection assay and
EMSA:
A: Advantages
1: Microplate capture and detection assay is highly sensitive, fast and throughput technique that
is based on ELISA while EMSA is based on comparative velocity of migration of DNA-protein
complexes through non denaturing gel electrophoresis.
2: Both techniques can skip the use of radioacitve probes by using biotinylated or fluorescently
labeled DNA probes.
3: EMSA uses DNA probe mutational analysis for testing the binding affinity of proteins.
4: Though EMSA can detect the presence of low quantities of DNA binding proteins from
lysates, Microplate capture and detection assay can detect even lesser abundance of protein in the
well by us.
Gel electrophoresis is a method for separation and analysis
of macromolecules (DNA, RNA and proteins) and
their fragments, based on their size and charge.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
Follow us on: Pinterest
Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
Contact us if you are interested:
Email / Skype : kefaya1771@gmail.com
Threema: PXHY5PDH
New BATCH Ku !!! MUCH IN DEMAND FAST SALE EVERY BATCH HAPPY GOOD EFFECT BIG BATCH !
Contact me on Threema or skype to start big business!!
Hot-sale products:
NEW HOT EUTYLONE WHITE CRYSTAL!!
5cl-adba precursor (semi finished )
5cl-adba raw materials
ADBB precursor (semi finished )
ADBB raw materials
APVP powder
5fadb/4f-adb
Jwh018 / Jwh210
Eutylone crystal
Protonitazene (hydrochloride) CAS: 119276-01-6
Flubrotizolam CAS: 57801-95-3
Metonitazene CAS: 14680-51-4
Payment terms: Western Union,MoneyGram,Bitcoin or USDT.
Deliver Time: Usually 7-15days
Shipping method: FedEx, TNT, DHL,UPS etc.Our deliveries are 100% safe, fast, reliable and discreet.
Samples will be sent for your evaluation!If you are interested in, please contact me, let's talk details.
We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
3. Aptamers are oligonucleotides, such as ribonucleic acid (RNA) and
single-strand deoxyribonucleic acid (ssDNA) or peptide molecules that
can bind to their targets with high affinity and specificity due to their
specific three-dimensional structures.
Especially, RNA and ssDNA aptamers can differ from each other in
sequence and folding pattern, although they bind to the same target.
It is derived from
Latin word, aptus/apto – to fit and
In Greek, meros – part (Smallest Unit Of Repeating Structure)
Aptamers can be combined with ribozymes to self-cleave in the
presence of their target molecule. They range in size from 20 to 80 bases
(6-26kda).
4. • Aptamer are produced by an in vitro selection or Systematic
Evolution Of Ligand By Exponential Enrichment(SELEX).
• It will produce ssRNA with specific binding target.
• This is an iterative process of binding, partitioning, amplifying novel
nucleic acids and regeneration.
• The pool of DNA is transcribed into an RNA pool and it subsequently
exposed to the target ligand of interest.
• And by Affinity column chromatography unbound sequences are
removed. the strongest binding sequences are identified and bound
sequences are eluted and amplified by RT-PCR
6. CLASSIFICATION OF APTAMERS BASED ON DIFFERENT TECHNIQUES
Aptamers
Aptamer
SELEX
Aptamer-Small
molecule
conjugated system
Cytotoxic
drug
Linkers
Chemical
Labile
linkers
Acid
cleavable
Disulfide
linkers
Enzyme
labile linkers
Peptide
linkers
Beta
Glucuronide
linkers
Non-Labile
Linkers
Aptamer-NPs
conjugated
system
Inorganic
nanomaterial
Gold NPs
Nanoscale
Iron oxide
Mesoporou
s silica NPs
Quantum
Dots
Organic
Nanomaterials
Liposomes
PLGA(Poly Lactic
Co-Glycolic Acid)
Polymer micelles
Dendrimers
Serum Albumin NPs
DNA NPs
7. CLASSIFICATION OF APTAMERS BASED ON STRUCTURE
APTAMERS
NUCLEIC
ACID
RNA DNA XNA
AMINO
ACID
PEPTIDES AFFIMERS
8. Aptamers selection methods
Capillary
Electrophoresis based
SELEX
Conventional In
Vitro SELEX
Affinity chromatography and
Magnetic Bead based SELEX
Cell SELEX
Nitrocellulose
Membrane Filtration
based SELEX
Microfluidic
based
SELEX
9. A. CONVENTIONAL in vitro SELEX
SELEX or in vitro selection is a technique used to isolate aptamers with high affinity for a
given target from approximately 1012–1015 combinatorial oligonucleotide libraries.
In general, the SELEX process is comprised of three steps that are repeated in order to
search for nucleotides that are better able to bind to the target.
1. In the first step (library generation), a library, except the initial compound in the library,
is converted into single-strand nucleotides that consist of random sequence regions,
usually 30–40 mers, flanked by the primer binding site.
2. In the second step (binding and separation), the target-bound library components are
separated from the unbound components.
3. Finally, in the third step (amplification), the target-bound library component is amplified
by the PCR to create a new library to be used in the next round. Aptamers are
continuously developed through this on-going process, and their characteristics are
identified using various biological assays.
11. b. Nitrocellulose Membrane Filtration-Based SELEX
• A nitrocellulose membrane is often used to immobilize proteins in Western blots and
atomic force microscopy (AFM) because it provides simple and rapid protein
immobilization by its non-specific affinity for amino acids.
• In 1968, a method using nitrocellulose membranes was employed by Kramlova’s group
to easily and quickly separate a protein from RNA molecules.
• Because the targets were primarily proteins in the early stage of SELEX, the use of a
nitrocellulose membrane was applied during the separation step. However, these
membranes have some limitations, such as being incapable of binding small molecules
and peptides, and they generally require at least 12 selection rounds.
12.
13. C.AFFINITY CHROMATOGRAPHY BASED SELEX
Affinity chromatography is a method for separating the components from a
biochemical mixture. It is primarily used for the purification of recombinant proteins,
based on a highly specific biological interaction, such as that between a receptor and a
ligand, or an antigen and an antibody.
The immobile phase is generally composed of agarose-based beads, and the beads are
packed onto a column for the washing and elution processes.
In the binding and separation steps of SELEX, affinity chromatography assists in the
selection of only the library components with an affinity for the target, by immobilizing
target molecules on the beads.
In the case of immobilization of proteins, various tags such as glutathione S-transferase
(GST) and the His-tag are utilized and, in the case of small organic molecules, the
targets are covalently fixed on the beads via a chemical reaction, such as coupling with
EDC (1-Ethyl-3-(3 Dimethylaminopropyl) carbo-di-imide) . Thus, SELEX for small
molecules, as well as proteins, can be carried out using this method. Several aptamers
were selected using this method by making an affinity column containing target-
immobilized beads.
15. D. MAGNETIC BEADS BASED SELEX
Magnetic beads are also used to immobilize the target via an interaction or a
chemical reaction between an affinity tag and the substrate on the beads.
The use of magnetic beads is an especially powerful tool for easy and rapid
isolation of target-immobilized beads with a magnet.
aptamer selection has been involved in linking this magnetic bead-based strategy
to the SELEX technique. Particularly because the library bound to the target is
easily and simply separated from the unbound one by a magnet, many attempts
at selecting target specific aptamer have followed
17. E. Capillary Electrophoresis-Based SELEX
Capillary electrophoresis (CE) possesses several appealing advantages over the
other analytical separation methods in the aspects of speed, resolution, capacity,
and minimal sample dilution.
This method can separate ionic species by their charge, frictional forces, and
hydrodynamic radius under the influence of an electric field.
With this method, an aptamer can be selected by a mobility shift among the
mixture of a target, the library, and the target-library complex.
In particular, the greatest virtue of applying this method to SELEX is that the
successful selection of the aptamer can be achieved within very few rounds,
generally 2–4 rounds, compared to other methods.
19. f. Microfluidic-Based SELEX
In order to select an aptamer more effectively, SELEX using a
microfluidic or chip system was developed.
Since this method is mainly processed on a chip, it is able to enhance
selection efficiency on a small scale.
For example, the DNA aptamer-specific bound to neurotoxin type B
was obtained after a single round of selection using the Continuous-
flow Magnetic Activated Chip-based Separation (CMACS) device
designed by Soh’s group.
Microfluidic technology-based SELEX is catching on as an advanced
method to select aptamers rapidly and automatically.
21. G. Cell-SELEX
Cell-SELEX is aimed at searching for an aptamer against a whole cell, whereas the
primary targets of other SELEX methods are single highly-purified proteins.
In other words, the targets of Cell-SELEX are extracellular proteins on the cell
surface or unique structures of the cell. In the majority of cases, Cell-SELEX
processes have washing (for adhesive cells) or centrifugation (for suspension cells)
steps during the separation of aptamers, because target immobilization is not
practicable in the solid phase.
In addition, counter selections are necessary in each round to avoid selection of
aptamers that non-specifically recognize the cell surface and that are commonly
located at the surfaces of many cells.
Therefore, this method is complicated, because of the impossibility of immobilizing
targets, and due to counter selection. However, the resulting aptamers, once
selected, are powerful for cell-specific diagnosis, cell-targeted drug delivery, and
cell-specific therapy.
23. h. Other Method-Based SELEX
Several methods, such as Atomic Force Microscopy (AFM),
electrophoretic mobility shift assays (EMSA), and surface plasmon
resonance (SPR), have been performed in connection with SELEX.
Although these strategies have the advantage of reducing the
number of selection rounds, the effectiveness of these methods in
selecting the aptamer has not been clearly demonstrated.
24. Advantages of Aptamers
Easier and more economical to produce.
Compared to antibodies, toxicity and low immunogenicity of particular
antigens do not interfere with the aptamer selection
Aptamers are capable of greater specificity and affinity than antibodies.
Aptamers can easily be modified chemically to yield improved, custom
tailored properties.
Aptamers can specifically bound to either small molecules and complex
multimeric structures.
Improved transport properties allowing cell specific targeting and improved
tissue penetration.
Aptamers are much more stable at ambient temperature than antibodies.
Ability to inactivate proteins, without altering genetic material.
25. Disadvantages of Aptamers
Lower levels of affinities than antibodies.
Aptamers will not bind to some target molecules.
Aptamers identification is expensive and labour intensive.
26. COMPARISON OF APTAMERS AND ANTIBODY
APTAMER ANTIBODIES
Aptamer are oligonucleotide and protein Antibodies are protein in nature
Uniform activity regardless of batch varies from batch to batch.
Investigator determines target site of protein Immune system determines target site of protein.
Wide variety of chemical modifications to molecule
for diverse functions
Limited modifications of molecule
No evidence of immunogenicity. Significant immunogenicity
They are more stable at high temperature and they
can be regenerated easily after denaturation.
Temperature sensitive
Entire selection is a chemical process carried out in
vitro and can therefore target any protein .
Selection requires a biological system, therefore
difficult to raise antibodies to toxins (not tolerated
by animal) or non-immunogenic target.
Aptamers are single stranded DNA or RNA
oligonucleotide or peptides.
Antibodies are monoclonal or polyclonal.
27. Applications of aptamers
1. Aptamers: as drug therapeutics:
Due to specific and tight affinity to target molecules, and low or no
immunogenicity and toxicity, aptamers are expected to be effective
therapeutics reagents.
In 2004, the approval by the Food and Drug Administration (FDA) of
Macugen, a vascular endothelial growth factor (VEGF)-specific aptamer,
for the treatment of neovascular (wet) age-related macular degeneration
(AMD), is a prominent landmark in the application of aptamers.
This drug is a pegylated aptamer, a single strand of nucleic acid with
specificity to VEGF165, which plays a critical role in angiogenesis and
permeability
28. 2. APTAMER: as DRUG DELIVERY SYSTEM:
Aptamers that bind to internalized cell surface receptors have been exploited to deliver drugs
and a variety of other cargo into cells.
For example:
(a)The prostate-specific membrane antigen (PSMA) is an important prostate cancer marker. The
dual aptamer probe—an A10 aptamer for PSMA(+) prostate cancer cells, and a DUP-1 aptamer
for PSMA(−) prostate cancer cells—was invented, and a drug loaded dual aptamer complex was
constructed by loading doxorubicin, an anticancer drug, onto the A10 aptamer strand. As a
result, the doxorubicin can be effectively introduced into the prostate cancer cells.
(b) Design of an siRNA-aptamer conjugate via a modular streptavidin bridge using an antiPSMA
aptamer for prostate cancer cells (LNCaP).
Applications of aptamers
29. 3. Aptamer: uses in Bio-Imaging:
Another application is bio-imaging, using an aptamer that is conjugated to
a fluorophore, or other materials such as gadolinium, which is useful for
magnetic resonance imaging (MRI).
Using aptamers as imaging agents has the advantage of their being non-
toxic, because oligonucleotide moieties are present in the human body.
As aptamers have high specificity for their target, accurate targeting, and
rapid diffusion through the blood circulation, use of these molecules can
increase the certainty of the results obtained during diagnosis or clinical
analysis.
Based on these advantages, aptamers have been studied as imaging agents
for cell imaging as well as single-protein imaging.
Applications of aptamers
30. 4. Aptamers: Ease in Western Blot Analysis:
A Western blot analysis is an analytical technique routinely used to quantify
specific proteins. The procedure includes complicated and elaborate steps and
requires many reagents, such as two types of antibodies.
a new aptamer-based Western blot strategy that has reduced the procedure to
one step, and easily detects the target protein using only one aptamer.
Instead of two types of antibodies, the QD-conjugated RNA aptamer specific for
the His-tag (Polyhistidine tag) was employed. This method has the advantages of
requiring less time, not requiring antibodies or 32P, and introducing the
possibility of multiplexing detection.
Applications of aptamers
31. 5. APTAMERS AS DIAGNOSTIC TOOLS:
As Aptamers are high affinity and specificity, small size, little immunogenicity,
stable structures and ease of synthesis it can be used as diagnostic tools.
Aptamer based detection assays are expected to detect low concentration
pathogens than conventional antibody based detection assay such as ELISA.
TYPE OF APTAMER VIRUS SPECIFIC TARGET MODE OF ACTION
2’-Fluropyrimidine
containing RNA
Aptamer
HIV-1 Strain R5 Glycoprotein 120 Inhibit cell to cell
movement and virus to
cell infection
Peptide Aptamer C1-1 Hepatitis B virus Viral coat Protein Inhibit viral replication
by blocking capsid
formation.
Applications of aptamers
32.
33. 1. Kyung-Mi Song, Seonghwan Lee and Changill Ban; Aptamers and
Their Biological Applications; Sensors 2012, 12, 612-631;
doi:10.3390/s120100612
2. Muir, P.; Li, S.; Lou, S.; Wang, D.; Spakowicz, D.J.; Salichos, L.; Zhang,
J.; Weinstock, G.M.; Isaacs, F.; Rozowsky, J.; et al. The real cost of
sequencing: Scaling computation to keep pace with data
generation. Genome Biol. 2016, 17, 53.
3. Yu, Y.; Liang, C.; Lv, Q.; Li, D.; Xu, X.; Liu, B.; Lu, A.; Zhang, G.
Molecular selection, modification and development of therapeutic
oligonucleotide aptamers. Int. J. Mol. Sci. 2016, 17, 358