Confirmation and clarity came a year later in 1953, when  James D. Watson  and  Francis Crick  correctly hypothesized, in their journal article " Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid ", the  double helix  structure of DNA, and suggested the  copying mechanism  by which DNA functions as hereditary material. Furthermore, Watson and Crick suggested that DNA, the genetic material, is responsible for the synthesis of the thousands of proteins found in cells. They had made this proposal based on the structural similarity that exists between the two macromolecules: both protein and DNA are linear sequences of monomers (amino acids and nucleotides, respectively). [ 8 ]


Once the Hershey–Chase experiment was published, the scientific community generally acknowledged that DNA was the genetic code material. This discovery led to a more detailed investigation of DNA to determine its composition as well as its 3D structure. Using  X-ray crystallography , the structure of DNA was discovered by James Watson and Francis Crick with the help of previously documented experimental evidence by  Maurice Wilkins  and  Rosalind Franklin . [ 9 ]
Knowledge of the structure of DNA led scientists to examine the nature of genetic coding and, in turn, understand the process of protein synthesis.  George Gamow  proposed that the  genetic code  was composed of sequences of three DNA base pairs known as triplets or  codons  which represent one of the twenty amino acids. [ 10 ]  Genetic coding helped researchers to understand the mechanism of  gene expression , the process by which information from a gene is used in  protein synthesis .  Since then, much research has been conducted to modulate steps in the gene expression process. These steps include  transcription ,  RNA splicing ,  translation , and  post-translational modification  which are used to control the chemical and structural nature of proteins. [ 11 ]  Moreover, genetic engineering gives engineers the ability to directly manipulate the genetic materials of organisms using  recombinant DNA  techniques. The first recombinant DNA molecule was created by  Paul Berg  in 1972 when he combined DNA from the monkey virus  SV40  with that of the  lambda phage . [ 12 ]


Experiments on hereditary material during the time of the Hershey–Chase experiment often used bacteriophages as a  model organism .  Bacteriophages lend themselves to experiments on hereditary material because they incorporate their  genetic material  into their  host cell 's genetic material (making them useful tools), they multiply quickly, and they are easily collected by researchers. [ 7 ]


The Hershey–Chase experiment, its predecessors, such as the  Avery–MacLeod–McCarty experiment , and successors served to unequivocally establish that hereditary information was carried by DNA. This finding has numerous applications in  forensic science ,  crime investigation  and  genealogy .  It provided the background knowledge for further applications in DNA forensics, where  DNA fingerprinting  uses data originating from DNA, not protein sources, to deduce  genetic variation . [ 13 ]


Isogenic human disease models  are a family of cells that are selected or engineered to accurately model the  genetics  of a specific patient population,  in vitro . They are provided with a genetically matched 'normal cell' to provide an isogenic system to research disease biology and novel therapeutic agents. [ 1 ]  They can be used to model any disease with a genetic foundation.  Cancer  is one such disease for which isogenic human disease models have been widely used.


Human isogenic disease models have been likened to 'patients in a test-tube', since they incorporate the latest research into human genetic diseases and do so without the difficulties and limitations involved in using non-human models. [ 2 ]


Historically, cells obtained from animals, typically mice, have been used to model cancer-related pathways. However, there are obvious limitations inherent in using animals for modelling genetically determined diseases in humans. Despite a large proportion of genetic conservation between humans and mice, there are significant differences between the biology of mice and humans that are important to cancer research. For example, major differences in  telomere  regulation enable murine cells to bypass the requirement for  telomerase  upregulation, which is a rate-limiting step in human cancer formation. As another example, certain ligand-receptor interactions are incompatible between mice and humans. Additionally, experiments have demonstrated important and significant differences in the ability to transform cells, compared with cells of murine origin. For these reasons, it remains essential to develop models of cancer that employ human cells. [ 3 ]


Isogenic cell lines are created via a process called homologous gene-targeting. Targeting vectors that utilize homologous recombination are the tools or techniques that are used to knock-in or knock-out the desired disease-causing mutation or SNP ( single nucleotide polymorphism ) to be studied. Although disease mutations can be harvested directly from cancer patients, these cells usually contain many background mutations in addition to the specific mutation of interest, and a matched normal cell line is typically not obtained. Subsequently, targeting vectors are used to ' knock-in ' or ' knock out ' gene mutations enabling a switch in both directions; from a normal to cancer genotype; or vice versa; in characterized human cancer cell lines such as  HCT116  or Nalm6. [ 4 ]


There are several gene targeting technologies used to engineer the desired mutation, the most prevalent of which are briefly described, including key advantages and limitations, in the summary table below.


rAAV can introduce subtle point mutations, SNPs as well as small insertions with high efficiency. Moreover, many peer reviewed studies have shown that rAAV does not introduce any confounding off target genomic events. [ citation needed ]


Appears to be the preferred method being adopted in academia, Biotech and Pharma on a precision versus time versus cost basis. [ citation needed ] |


This process can therefore generate 3 genotypes (+/+; -/+ and -/-); enabling therefore the analysis of haplo-insufficient gene function.


Current limitation is the need to sequentially target single alleles making generation of knock-out cell lines a two-step process.|


Homologous recombination (HR) is a kind of genetic recombination in which genetic sequences are exchanged between two similar segments of DNA. HR plays a major role in eukaryotic cell division, promoting genetic diversity through the exchange between corresponding segments of DNA to create new, and potentially beneficial combinations of genes. [ citation needed ]


HR performs a second vital role in DNA repair, enabling the repair of double-strand breaks in DNA which is a common occurrence during a cell's lifecycle. It is this process which is artificially triggered by the above technologies and bootstrapped in order to engender 'knock-ins' or 'knockouts' in specific genes 5, 7.


A recent key advance was discovered using AAV-homologous recombination vectors, which increases the low natural rates of HR in differentiated human cells when combined with gene-targeting vectors-sequences. [ citation needed ]


Factors leading to the recent commercialization of isogenic human cancer cell disease models for the pharmaceutical industry and research laboratories are twofold. [ citation needed ]


Firstly, successful patenting of enhanced targeting vector technology has provided a basis for commercialization of the cell-models which eventuate from the application of these technologies. [ citation needed ]


Secondly, the trend of relatively low success rates in pharmaceutical RnD and the enormous costs have created a real need for new research tools that illicit how patient sub-groups will respond positively or be resistant to targeted cancer therapeutics based upon their individual genetic profile. [ citation needed ]


The  Luria–Delbrück experiment  (1943) (also called the  Fluctuation Test ) demonstrated that in  bacteria , genetic  mutations  arise in the absence of  selective pressure  rather than being a response to it. Thus, it concluded  Darwin 's theory of  natural selection  acting on random mutations applies to bacteria as well as to more complex organisms.  Max Delbrück  and  Salvador Luria  won the 1969  Nobel Prize in Physiology or Medicine  in part for this work.


Suppose a single bacterium is introduced to a growth medium with rich nutrients, and allowed to grow for

 N
 ×

 {\displaystyle N\times }

  of its doubling time, we would obtain

 2

 N

 {\displaystyle 2^{N}}

  offsprings. Then, we introduce a  challenge  by bacteriophages. This would kill off most bacteria, but leave some alive. We can then smear the growth medium over a new growth medium, and count the number of colonies as the number of survivors.


In the Lamarckian scenario, each bacterium faces the challenge alone. Most would perish, but a few would survive the ordeal and found a new colony. In the Darwinian scenario, resistance to the phage would randomly occur during the replication. Those that inherited the resistance would survive, while those that did not would die.


In the Lamarckian scenario, assuming each bacterium has an equally small probability of survival, then the number of new colonies is Poisson distributed, which decays exponentially at large number of survivors.


In the Darwinian scenario, assuming that the probability of mutation is small enough that we expect only a single mutation during the entire replication phase, and that, for simplicity, we really do get just a single mutation, then with probability

 1

 /

 2

 {\displaystyle 1/2}

  there is a single survivor, with probability

 1

 /

 4

 {\displaystyle 1/4}

  there are 2 survivors, etc. That is, the probability scales as

 1
 number of survivors

 {\displaystyle {\frac {1}{\text{number of survivors}}}}

 .


In particular, if the distribution of survivor number turns out to decay more like a power law than like an exponential, then we can conclude with high statistical likelihood that Darwinian scenario is true. This is a rough overview of the Luria–Delbrück experiment. (Section 4.4  [ 1 ] )


By the 1940s the ideas of inheritance and mutation were generally accepted, though the role of DNA as the hereditary material had not yet been established. It was thought that bacteria were somehow different and could develop heritable genetic mutations depending on the circumstances they found themselves: in short, was the mutation in bacteria pre-adaptive (pre-existent) or post-adaptive (directed adaption)? [ 2 ]


In their experiment, Luria and Delbrück inoculated a small number of bacteria ( Escherichia coli ) into separate  culture  tubes. After a period of growth, they plated equal volumes of these separate cultures onto  agar  containing the T1  phage  (virus). If resistance to the virus in bacteria were caused by an induced activation in bacteria i.e. if resistance were not due to heritable genetic components, then each plate should contain roughly the same number of resistant colonies.
Assuming a constant rate of mutation, Luria hypothesized that if mutations occurred after and in response to exposure to the selective agent, the number of survivors would be distributed according to a  Poisson distribution  with the  mean  equal to the  variance . This was not what Delbrück and Luria found: Instead the number of resistant colonies on each plate varied drastically: the variance was considerably greater than the mean.


Luria and Delbrück proposed that these results could be explained by the occurrence of a constant rate of random mutations in each generation of bacteria growing in the initial culture tubes. Based on these assumptions Delbrück derived a  probability distribution  (now called the  Luria–Delbrück distribution [ 3 ] [ 4 ] ) that gives a relationship between  moments  consistent with the experimentally obtained values. Therefore, the conclusion was that mutations in bacteria, as in other organisms, are  random  rather than directed. [ 5 ]


The results of Luria and Delbrück were confirmed in more graphical, but less quantitative, way by Newcombe. Newcombe  incubated  bacteria in a  Petri dish  for a few hours, then  replica plated  it onto two new Petri dishes treated with phage. The first plate was left unspread, and the second plate was then respread, that is, bacterial cells were moved around allowing single cells in some colony to form their own new colonies. If colonies contained resistant bacterial cells before entering into contact with the phage virus, one would expect that some of these cells would form new resistant colonies on the respread dish and so to find a higher number of surviving bacteria there. When both plates were incubated for growth, there were actually as much as 50 times greater number of bacterial colonies on the respread dish. This showed that bacterial mutations to virus resistance had randomly occurred during the first incubation. Once again, the mutations occurred before selection was applied. [ 6 ]


More recently, the results of Luria and Delbrück were questioned by Cairns and others, who studied mutations in sugar  metabolism  as a form of environmental stress. [ 7 ]  Some scientists suggest that this result may have been caused by selection for gene amplification and/or a higher  mutation rate  in cells unable to divide. [ 8 ]  Others have defended the research and propose mechanisms which account for the observed phenomena consistent with   adaptive mutagenesis . [ 9 ]


This distribution appears to have been first determined by  Haldane . [ 10 ]  An unpublished manuscript was discovered in 1991 at  University College London  describing this distribution. The derivation is different but the results are difficult to compute without the use of a computer.


A small number of cells are used to inoculate parallel cultures in a non-selective medium. [ 11 ]  The cultures are grown to saturation to obtain equal cell densities. The cells are plated onto selective media to obtain the number of mutants ( r ). Dilutions are plated onto rich medium to calculate the total number of viable cells (  N t  ). The number of mutants that appear in the saturated culture is a measure of both the mutation rate and when the mutants arise during the growth of the culture: mutants appearing early in the growth of the culture will propagate many more mutants than those that arise later during growth. These factors cause the frequency ( r / N t  ) to vary greatly, even if the number of mutational events ( m ) is the same. Frequency is not a sufficiently accurate measure of mutation and the mutation rate ( m / N t ) should always be calculated.


The estimation of the mutation rate ( μ ) is complex. Luria and Delbruck estimated this parameter from the  mean  of the distribution but this estimator was subsequently shown to be biased.


The Lea–Coulson method of the  median  was introduced in 1949. [ 12 ]  This method is based on the equation


This method has since been improved on but these more accurate methods are complex. The Ma–Sandri–Sarkar  maximum likelihood  estimator is currently the best known  estimator . [ 14 ]  A number of additional methods and estimates from experimental data have been described. [ 15 ]


Two web-applications for the calculation of the mutation rate are freely available: Falcor  [ 11 ]  and  bz-rates . Bz-rates implements a generalized version of the Ma–Sandri–Sarkar  maximum likelihood  estimator that can take into account the relative differential growth rate between mutant and wild-type cells as well as a generating function estimator that can estimate both the mutation rate and the differential growth rate. A worked example is shown in this paper by Jones  et al . [ 16 ]


In all these models the mutation rate ( μ ) and growth rate ( β ) were assumed to be constant. The model can be easily generalized to relax these and other constraints. [ 17 ]  These rates are likely to differ in non experimental settings. The models also require that  N t   μ  >> 1 where  N t  is the total number of organisms. This assumption is likely to hold in most realistic or experimental settings.


Luria and Delbrück [ 5 ]  estimated the mutation rate (mutations per bacterium per unit time) from the equation


where  β  is the cellular growth rate,  n 0  is the initial number of bacteria in each culture,  t  is the time, and


where  N s  is the number of cultures without resistant bacteria and  N  is the total number of cultures.


Lea and Coulson's model [ 12 ]  differed from the original in that they considered a collection of independent  Yule processes  (a filtered  Poisson process ). Numerical comparisons of these two models with realistic values of the parameters has shown that they differ only slightly. [ 18 ]  The  generating function  for this model was found by Bartlett in 1978 [ 19 ]  and is


where  μ  is the mutation rate (assumed to be constant),  φ  = 1 −  e − βt  with  β  as the cellular growth rate (also assumed to be constant) and  t  as the time.


The determination of  μ  from this equation has proved difficult but a solution was discovered in 2005 [ citation needed ] . Differentiation of the generating function with respect to  μ  allows the application of the  Newton–Raphson  method which together with the use of a  score function  allows one to obtain  confidence intervals  for  μ .


The mechanism of resistance to the phage T1 appears to have been due to mutations in the  fhu A gene - a membrane protein that acts as the T1 receptor. [ 20 ]  The  ton B gene product is also required for infection by T1. The FhuA protein is actively involved in the transport of  ferrichrome ,  albomycin  and  rifamycin . [ 21 ]  It also confers sensitivity to  microcin J25  and  colicin M  and acts as a receptor for the phages T5 and phi80 as well as T1.


The FhuA protein has a beta-barrel domain (residues 161 to 714) that is closed by a globular cork domain (residues 1 to 160). [ 22 ]  Within the cork domain is the TonB binding region (residues 7 to 11). The large membrane spanning monomeric β-barrel domains have 22 β-strands of variable length, several of which extend significantly beyond the membrane hydrophobic core into the extracellular space. There are 11 extracellular loops numbered L1 to L11. The L4 loop is where the T1 phage binds.


The  T4  r II system  is an  experimental system  developed in the 1950s by  Seymour Benzer  for studying the substructure of the  gene . The experimental system is based on  genetic crosses  of different  mutant   strains  of  bacteriophage T4 , a virus that infects the bacteria  Escherichia coli .


One type of mutation in the T4 bacteriophage identified by researchers in phage genetics by the 1950s was known as  r  (for  rapid ), which caused the phage to destroy bacteria more quickly than normal.  These could be spotted easily because they would produce larger  plaques  rather than the smaller plaques characteristic of the  wild type  virus.  Through  genetic mapping , the researchers had identified specific regions in the T4 chromosome, called the  r I,  r II, and  r III  loci , associated with the  r  mutants.  In 1952, while performing experiments with  r II mutants, Seymour Benzer found a strain that did not behave normally.  By 1953, after the publication of  Watson  and  Crick 's  proposed structure  of  DNA , Benzer hit on the idea that the apparently defective  r  mutants might have been the result of crossing two different  r II mutants, each of which had part of the  r II gene intact, so that the hybrid strain did not exhibit the  r  phenotype at all because it combined the intact parts of the  r II gene. [ 1 ]


From there, Benzer saw that it would be possible to generate many independent  r  mutants, and by measuring the  recombination frequency  between different  r  strains, he could map the substructure of a single gene.  Although the chance of successful recombination between any mating pair of rII mutants is small, a single petri dish could be the basis for millions of trials at once.  They could be screened easily by using a specific strain of  E. coli , known as K12 (λ), that was susceptible to wild type T4 but not to  r  mutants. [ 2 ]


Benzer's concept was quite controversial within classical genetic thought, in which each gene is treated as a singular point along a chromosome, not a divisible stretch of nucleic acids (as implied by the work of Watson and Crick).  Initially,  Max Delbrück —a respected phage geneticist and leader of the so-called  phage group  of which Benzer was a part—found Benzer's idea outrageous. [ 3 ]


Beginning in 1954, Benzer put the T4  r II system to use, creating and crossing hundreds of  r  mutants and developing an increasingly detailed map of the structure of the  r II gene.  In his early work, he identified two separate but very close loci within the  r II region, which he suggested were  nucleotide sequences  that encoded different  polypeptides ; he called these " cistrons ". [ 4 ]


Benzer identified a number of different types of  r  mutants.  Some he classified as  deletions , others as  point mutations .  By various crosses of the many different strains exhibited deletions and point mutations, Benzer located each point mutation into a sub-region of one of the cistrons, and ordered the point mutations within that sub-region.   Benzer also proposed  missense  and  nonsense mutations  from his  r II studies.  The T4  r II system enabled Benzer to identify recombination frequencies as low as .02%, much lower than in typical genetics experiments.  This was equivalent to detecting recombination between only one or two base pairs. [ 5 ]


In the early 1950s the prevailing view was that the genes in a chromosome acted like discrete entities, indivisible by recombination and arranged like beads on a string.  The experiments of Benzer using mutants defective in the T4 rII system, during 1955-1959, showed that individual genes have a simple linear structure and are likely to be equivalent to a linear section of DNA [ 6 ] [ 7 ]  (see also  Phage group ).


After Benzer demonstrated the power of the T4  r II system for exploring the fine structure of the gene, others adapted the system to explore related problems.  For example,  Francis Crick  and others used one of the peculiar  r  mutants Benzer had found (a deletion that fused the A and B cistrons of  r II) to  demonstrate the triplet nature  of the  genetic code . [ 8 ]


The principal that three sequential bases of DNA code for each amino acid was demonstrated in 1961 using frameshift mutations in the rIIB gene of bacteriophage T4 [ 9 ] [ 10 ]  (also see  Crick, Brenner et al. experiment ).


Richard Feynman , the renowned Caltech theoretical physicist, worked on the T4 rII system during the summer of 1961, and his experimental results were included in a publication by Edgar et al. [ 11 ]   These authors showed that recombination frequencies between rII mutants are not strictly additive.  The recombination frequency from a cross of two rII mutants (a x d) is usually less than the sum of recombination frequencies for adjacent internal sub-intervals (a x b) + (b x c) + (c x d).  Although not strictly additive, a systematic relationship was observed [ 12 ]  that likely reflects the underlying molecular mechanism of recombination (see  genetic recombination  and  synthesis dependent strand annealing ).


Tetracycline-controlled transcriptional activation  is a method of inducible  gene expression  where  transcription  is reversibly turned on or off in the presence of the  antibiotic   tetracycline  or one of its derivatives (e.g.  doxycycline ). [ 1 ]


Tetracycline-controlled gene expression is based upon the mechanism of resistance to tetracycline antibiotic treatment found in  gram-negative bacteria . In nature, the P tet   promoter  expresses TetR (the  repressor ) and TetA, the protein that pumps tetracycline antibiotic out of the cell. [ 2 ]


The difference between Tet-On and Tet-Off is not whether the  transactivator  turns a gene on or off, as the name might suggest; rather, both proteins  activate  expression. The difference relates to their respective response to tetracycline or  doxycycline  (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the  absence  of Dox, whereas Tet-On activates in the  presence  of Dox.


The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. [ 3 ]
The Tet-Off system for controlling expression of genes of interest in mammalian cells was developed by Professors  Hermann Bujard  [ de ]  and  Manfred Gossen  at the  University of Heidelberg  and first published in 1992. [ 4 ]


The Tet-Off system makes use of the  tetracycline transactivator (tTA)  protein, which is created by fusing one  protein , TetR (tetracycline repressor), found in  Escherichia coli  bacteria, with the activation domain of another protein,  VP16 , found in the  herpes simplex virus . [ 5 ]


The resulting tTA protein is able to bind to DNA at specific  TetO   operator  sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a  minimal promoter  such as the  CMV promoter . The entirety of several TetO sequences with a minimal promoter is called a  tetracycline response element  (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter.


In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes.


A Tet-On system works similarly, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator  only if not  bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the rtTA protein is capable of binding the operator  only if  bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred over Tet-Off for its faster responsiveness.


Tet-Off  expression systems  are also used in generating transgenic mice which conditionally express gene of interest.


The Tet-On Advanced transactivator (also known as rtTA2 S -M2) is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-Off. In addition, its expression is considered to be more stable in eukaryotic cells due to being  human codon optimized  and utilizing three minimal transcriptional activation domains. It was discovered in 2000 as one of two improved mutants by H. Bujard and his colleagues after random mutagenesis of the Tet repressor part of the transactivator gene. [ 6 ]  Tet-On 3G (also known as rtTA-V10  [ 7 ] ) is similar to Tet-On Advanced but was derived from rtTA2 S -S2 rather than rtTA2 S -M2. It is also human codon optimized and composed of three minimal VP16 activation domains. However, the Tet-On 3G protein has five amino acid differences compared to Tet-On Advanced which appear to increase its sensitivity to Dox even further. Tet-On 3G is sensitive to 100-fold less Dox and is seven-fold more active than the original Tet-On. [ 8 ]


Other systems such as the T-REx system by Life Technologies work in a different fashion. [ 9 ]  The gene of interest is flanked by an upstream CMV promoter and two TetO2 sites. Expression of the gene of interest is repressed by the high affinity binding of TetR homodimers to each TetO2 sequences in the absence of tetracycline. Introduction of tetracycline results in binding of one tetracycline on each TetR homodimer followed by release of TetO2 by the TetR homodimers. Unbinding of TetR homodimers and TetO2 result in derepression of the gene of interest.


A modified version of T-REx is the Linearizer  synthetic biological circuit , optimized for gene expression tuning in eukaryotic (budding yeast, human, etc) cells. By incorporating TetO2 sites into the promoter driving TetR expression, it creates  negative feedback , which ensures homogeneous expression (low noise) and a linear dose-response to tetracycline analogs.  [ 10 ]


In the most commonly used  plasmids , the tetracycline response element consists of seven repeats of the 19bp bacterial TetO sequence ( TCCCTATCAGTGATAGAGA ) separated by spacer sequences (for example: ACGATGTCGAGTTTAC). It is the TetO that is recognized and bound by the TetR portion of Tet-On or Tet-Off. The TRE is usually placed upstream of a minimal promoter that has very low basal expression in the absence of bound Tet-Off (or Tet-On).


The Tet system has advantages over  Cre ,  FRT , and  ER  (estrogen receptor) conditional gene expression systems. In the Cre and FRT systems, activation or knockout of the gene is irreversible once recombination is accomplished, whereas, in Tet and ER systems, it is reversible. The Tet system has very tight control on expression, whereas ER system is somewhat leaky. [ 11 ]  However, the Tet system, which depends on transcription and subsequent translation of a target gene, is not as fast-acting as the ER system, which stabilizes the already-expressed target protein upon hormone administration. Also, since the 19bp tet-o sequence is naturally absent from mammalian cells,  pleiotropy  is thought to be minimized compared to hormonal methods of control. When using the Tet system in cell culture, it is important to confirm that each batch of  fetal bovine serum  is tested to confirm that contaminating  tetracyclines  are absent or are too low to interfere with inducibility.


The mechanism of action for the antibacterial effect of tetracyclines relies on disrupting protein translation in bacteria, thereby damaging the ability of microbes to grow and repair; however protein translation is also disrupted in eukaryotic  mitochondria  leading to effects that may  confound  experimental results. [ 12 ]


TROPI , or "Analysis of a Novel Sensory Mechanism in Root Phototropism", is an experiment on the  International Space Station  (ISS) to investigate the growth and development of plant seedlings under various gravity and lighting combinations. [ 1 ]  It was launched on  Space Shuttle Endeavour  during the  STS-130  mission and was performed on the ISS during  Expedition 22 . Frozen plant samples from the TROPI experiment were returned on the landing of the  STS-131  mission on  Space Shuttle Discovery .


Arabidopsis thaliana  seeds, (thale cress, the  genome  of which has been  DNA  sequenced as a reference organism for the study of plant biology and genetics), were germinated and grown under various lighting and gravity conditions, using  centrifugal  gravity-simulation [ 2 ]  and  LEDs  of various wavelengths (colors) and intensities to model lighting conditions. The specific aim of this project was to investigate  phototropism  in plants grown in  microgravity  conditions without the complications of a 1-g environment. [ 3 ]  Experiments performed were used to explore the mechanisms of both blue-light- and red-light-induced phototropism in plants.


John Z. Kiss of  Miami University  (Oxford OH) is Principal Investigator, Richard E. Edelmann of Miami University and Melanie J. Correll of the University of Florida are co-investigators; Kenny Vassigh of  NASA  is the project manager and Marianne Steele of NASA is the project scientist.  The payload was developed by the NASA  Ames Research Center , Moffett Field, CA.  The experiment was performed in the European Modular Cultivation System (EMCS) [ 4 ]  built by the  European Space Agency  (ESA). The Norwegian User Support Operation Centre (N-USOC), [ 5 ]  located in  Trondheim, Norway , controlled the EMCS during the TROPI experiments on the ISS.


In the long term, the results from TROPI will help in the development of future space,  Moon , and  Mars  life-support systems, in which plants are used to help remove  carbon dioxide  and generate  oxygen  via photosynthesis for maintenance of atmospheric and other conditions, reducing the need for very expensive re-supply from Earth. [ 6 ]


Scientific research on the ISS


Nucleotides  are  organic molecules  composed of a nitrogenous base, a  pentose  sugar and a  phosphate . They serve as  monomeric  units of the  nucleic acid   polymers  –  deoxyribonucleic acid  (DNA) and  ribonucleic acid  (RNA),  both of which are essential  biomolecules  within all  life-forms  on  Earth . Nucleotides are obtained in the diet and are also synthesized from common  nutrients  by the  liver . [ 1 ]


Nucleotides are composed of three subunit molecules: a  nucleobase , a  five-carbon sugar  ( ribose  or  deoxyribose ), and a phosphate group consisting of one to three  phosphates .  The four nucleobases in DNA are  guanine ,  adenine ,  cytosine , and  thymine ; in RNA,  uracil  is used in place of thymine.


Nucleotides also play a central role in  metabolism  at a fundamental, cellular level. They provide chemical energy—in the form of the  nucleoside triphosphates ,  adenosine triphosphate  (ATP),  guanosine triphosphate  (GTP),  cytidine triphosphate  (CTP), and  uridine triphosphate  (UTP)—throughout the cell for the many cellular functions that demand energy, including:  amino acid ,  protein  and  cell membrane  synthesis, moving the cell and cell parts (both internally and intercellularly), cell division, etc.. [ 2 ]  In addition, nucleotides participate in  cell signaling  ( cyclic guanosine monophosphate  or cGMP and  cyclic adenosine monophosphate  or cAMP) and are incorporated into important  cofactors  of enzymatic reactions (e.g.,  coenzyme A ,  FAD ,  FMN ,  NAD , and  NADP + ).


In experimental  biochemistry , nucleotides can be  radiolabeled  using  radionuclides  to yield radionucleotides.


5-nucleotides  are also used in  flavour enhancers  as  food additive  to enhance the  umami  taste, often in the form of a yeast extract. [ 3 ]


A nucleo tide  is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a  nucleobase  (the two of which together are called a  nucleo side ), and one  phosphate group . With all three joined, a nucleotide is also termed a "nucleo side   mono phosphate", "nucleoside  di phosphate" or "nucleoside  tri phosphate", depending on how many phosphates make up the phosphate group. [ 4 ]


In  nucleic acids , nucleotides contain either a  purine  or a  pyrimidine  base—i.e., the nucleobase molecule, also known as a nitrogenous base—and are termed  ribo nucleotides if the sugar is ribose, or  deoxyribo nucleotides if the sugar is deoxyribose. Individual phosphate molecules repetitively connect the  sugar-ring  molecules in two adjacent nucleotide monomers, thereby connecting the nucleotide monomers of a nucleic acid end-to-end into a long chain. These chain-joins of sugar and phosphate molecules create a 'backbone' strand for a single- or  double helix . In any one strand, the chemical orientation ( directionality ) of the chain-joins runs from the  5'-end  to the  3'-end  ( read : 5 prime-end to 3 prime-end)—referring to the five carbon sites on sugar molecules in adjacent nucleotides. In a double helix, the two strands are oriented in opposite directions, which permits  base pairing  and  complementarity  between the base-pairs, all which is essential for  replicating  or  transcribing  the encoded information found in DNA. [ citation needed ]


Nucleic acids then are  polymeric   macromolecules  assembled from nucleotides, the  monomer-units of nucleic acids . The purine bases  adenine  and  guanine  and pyrimidine base  cytosine  occur in both DNA and RNA, while the pyrimidine bases  thymine  (in DNA) and  uracil  (in RNA) occur in just one. Adenine forms a  base pair  with thymine with two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds.


In addition to being building blocks for the construction of nucleic acid polymers, singular nucleotides play roles in cellular energy storage and provision, cellular signaling, as a source of phosphate groups used to modulate the activity of proteins and other signaling molecules, and as enzymatic  cofactors , often carrying out  redox  reactions.  Signaling  cyclic nucleotides  are formed by binding the phosphate group twice to the same sugar  molecule , bridging the 5'- and 3'-  hydroxyl groups  of the sugar. [ 2 ]   Some signaling nucleotides differ from the standard single-phosphate group configuration, in having multiple phosphate groups attached to different positions on the sugar. [ 5 ]   Nucleotide cofactors include a wider range of chemical groups attached to the sugar via the  glycosidic bond , including  nicotinamide  and  flavin , and in the latter case, the ribose sugar is linear rather than forming the ring seen in other nucleotides.


Nucleotides can be  synthesized  by a variety of means, both  in vitro  and  in vivo . [ citation needed ]


In vitro,  protecting groups  may be used during laboratory production of nucleotides. A purified  nucleoside  is protected to create a  phosphoramidite , which can then be used to obtain analogues not found in nature and/or to  synthesize an oligonucleotide . [ citation needed ]


In vivo, nucleotides can be synthesized  de novo  or recycled through  salvage pathways . [ 1 ]  The components used in de novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and  amino acid  metabolism, and from ammonia and carbon dioxide. Recently it has been also demonstrated that cellular bicarbonate metabolism can be regulated by mTORC1 signaling. [ 6 ]  The liver is the major organ of de novo synthesis of all four nucleotides. De novo synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid, onto which a phosphorylated ribosyl unit is covalently linked. Purines, however, are first synthesized from the sugar template onto which the ring synthesis occurs. For reference, the syntheses of the  purine  and  pyrimidine  nucleotides are carried out by several enzymes in the  cytoplasm  of the cell, not within a specific  organelle . Nucleotides undergo breakdown such that useful parts can be reused in synthesis reactions to create new nucleotides. [ citation needed ]


The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts with the formation of carbamoyl phosphate from  glutamine  and CO 2 . Next,  aspartate carbamoyltransferase  catalyzes a condensation reaction between  aspartate  and  carbamoyl phosphate  to form  carbamoyl aspartic acid , which is cyclized into  4,5-dihydroorotic acid  by  dihydroorotase . The latter is converted to  orotate  by  dihydroorotate oxidase . The net reaction is:


Orotate is covalently linked with a phosphorylated ribosyl unit. The covalent linkage between the ribose and pyrimidine occurs at position C 1 [ 7 ]  of the  ribose  unit, which contains a  pyrophosphate , and N 1  of the pyrimidine ring.  Orotate phosphoribosyltransferase  (PRPP transferase) catalyzes the net reaction yielding orotidine monophosphate (OMP):


Orotidine 5'-monophosphate  is decarboxylated by orotidine-5'-phosphate decarboxylase to form uridine monophosphate (UMP). PRPP transferase catalyzes both the ribosylation and decarboxylation reactions, forming UMP from orotic acid in the presence of PRPP. It is from UMP that other pyrimidine nucleotides are derived.  UMP is phosphorylated by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP.  First, the diphosphate from UDP is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis:


CTP is subsequently formed by the amination of UTP by the catalytic activity of  CTP synthetase . Glutamine is the NH 3  donor and the reaction is fueled by ATP hydrolysis, too:


Cytidine monophosphate (CMP) is derived from cytidine triphosphate (CTP) with subsequent loss of two phosphates. [ 8 ] [ 9 ]


The atoms that are used to build the  purine nucleotides  come from a variety of sources:


The  de novo synthesis  of  purine nucleotides  by which these precursors are incorporated into the purine ring proceeds by a 10-step pathway to the branch-point intermediate  IMP , the nucleotide of the base  hypoxanthine .  AMP  and  GMP  are subsequently synthesized from this intermediate via separate, two-step pathways. Thus, purine  moieties  are initially formed as part of the  ribonucleotides  rather than as  free bases .


Six enzymes take part in IMP synthesis. Three of them are multifunctional:


The pathway starts with the formation of  PRPP .  PRPS1  is the  enzyme  that activates  R5P , which is formed primarily by the  pentose phosphate pathway , to PRPP by reacting it with  ATP . The reaction is unusual in that a pyrophosphoryl group is directly transferred from ATP to C 1  of R5P and that the product has the  α  configuration about C1. This reaction is also shared with the pathways for the synthesis of  Trp ,  His , and the  pyrimidine nucleotides . Being on a major metabolic crossroad and requiring much energy, this reaction is highly regulated.


In the first reaction unique to purine nucleotide biosynthesis,  PPAT  catalyzes the displacement of PRPP's  pyrophosphate  group (PP i ) by an amide nitrogen donated from either  glutamine  (N),  glycine  (N&C),  aspartate  (N),  folic acid  (C 1 ), or CO 2 . This is the committed step in purine synthesis. The reaction occurs with the inversion of configuration about ribose C 1 , thereby forming  β - 5-phosphorybosylamine  (5-PRA) and establishing the anomeric form of the future nucleotide.


Next, a glycine is incorporated fueled by ATP hydrolysis, and the carboxyl group forms an amine bond to the NH 2  previously introduced. A one-carbon unit from folic acid coenzyme N 10 -formyl-THF is then added to the amino group of the substituted glycine followed by the closure of the imidazole ring. Next, a second NH 2  group is transferred from glutamine to the first carbon of the glycine unit. A carboxylation of the second carbon of the glycin unit is concomitantly added. This new carbon is modified by the addition of a third NH 2  unit, this time transferred from an aspartate residue. Finally, a second one-carbon unit from formyl-THF is added to the nitrogen group and the ring is covalently closed to form the common purine precursor inosine monophosphate (IMP).


Inosine monophosphate is converted to adenosine monophosphate in two steps. First, GTP hydrolysis fuels the addition of aspartate to IMP by adenylosuccinate synthase, substituting the carbonyl oxygen for a nitrogen and forming the intermediate adenylosuccinate. Fumarate is then cleaved off forming adenosine monophosphate. This step is catalyzed by adenylosuccinate lyase.


Inosine monophosphate is converted to guanosine monophosphate by the oxidation of IMP forming xanthylate, followed by the insertion of an amino group at C 2 .  NAD +  is the electron acceptor in the oxidation reaction. The amide group transfer from glutamine is fueled by ATP hydrolysis.


In humans, pyrimidine rings (C, T, U) can be degraded completely to CO 2  and NH 3  (urea excretion). That having been said, purine rings (G, A) cannot. Instead, they are degraded to the metabolically inert  uric acid  which is then excreted from the body. Uric acid is formed when GMP is split into the base guanine and ribose. Guanine is deaminated to xanthine which in turn is oxidized to uric acid. This last reaction is irreversible. Similarly, uric acid can be formed when AMP is deaminated to IMP from which the ribose unit is removed to form hypoxanthine. Hypoxanthine is oxidized to xanthine and finally to uric acid. Instead of uric acid secretion, guanine and IMP can be used for recycling purposes and nucleic acid synthesis in the presence of PRPP and aspartate (NH 3  donor). [ citation needed ]


Theories about the  origin of life  require knowledge of chemical pathways that permit formation of life's key building blocks under plausible prebiotic conditions.  The  RNA world  hypothesis holds that in the  primordial soup  there existed free-floating  ribonucleotides , the fundamental molecules that combine in series to form  RNA .  Complex molecules like RNA must have arisen from small molecules whose reactivity was governed by physico-chemical processes.  RNA is composed of  purine  and  pyrimidine  nucleotides, both of which are necessary for reliable information transfer, and thus Darwinian  evolution .  Becker et al. showed how pyrimidine  nucleosides  can be synthesized from small molecules and  ribose , driven solely by wet-dry cycles. [ 10 ]   Purine nucleosides can be synthesized by a similar pathway.  5'-mono- and di-phosphates also form selectively from phosphate-containing minerals, allowing concurrent formation of  polyribonucleotides  with both the purine and pyrimidine bases.  Thus a reaction network towards the purine and pyrimidine RNA building blocks can be established starting from simple atmospheric or volcanic molecules. [ 10 ]


An unnatural base pair (UBP) is a designed subunit (or  nucleobase ) of  DNA  which is created in a laboratory and does not occur in nature. [ 11 ]  Examples include  d5SICS  and  dNaM . These artificial nucleotides bearing hydrophobic  nucleobases , feature two fused  aromatic rings  that form a (d5SICS–dNaM) complex or base pair in DNA. [ 12 ] [ 13 ]   E. coli  have been induced to replicate a plasmid containing UBPs through multiple generations. [ 14 ]  This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. [ 12 ] [ 15 ]


The applications of synthetic nucleotides vary widely and include disease diagnosis, treatment, or precision medicine.


Nucleotide (abbreviated "nt") is a common unit of length for single-stranded nucleic acids, similar to how  base pair  is a unit of length for double-stranded nucleic acids. [ 19 ]


The  IUPAC  has designated the symbols for nucleotides. [ 20 ]  Apart from the five (A, G, C, T/U) bases, often degenerate bases are used especially for designing  PCR primers . These nucleotide codes are listed here.  Some primer sequences may also include the character "I", which codes for the non-standard nucleotide  inosine .  Inosine occurs in  tRNAs  and will pair with adenine, cytosine, or thymine.  This character does not appear in the following table, however, because it does not represent a degeneracy.  While inosine can serve a similar function as the degeneracy "H", it is an actual nucleotide, rather than a representation of a mix of nucleotides that covers each possible pairing needed.


Adenosine 3',5'-bisphosphate  is a form of an  adenosine   nucleotide  with two phosphate groups attached to different carbons in the  ribose  ring. This is distinct from  adenosine diphosphate , where the two phosphate groups are attached in a chain to the 5' carbon atom in the ring.


Adenosine 3',5'-bisphosphate is produced as a product of  sulfotransferase  enzymes from the donation of a sulfate group from the  coenzyme   3'-phosphoadenosine-5'-phosphosulfate . [ 1 ] [ 2 ]  This product is then hydrolysed by  3'(2'),5'-bisphosphate nucleotidase  to give  adenosine monophosphate , which can then be recycled into  adenosine triphosphate . [ 3 ] [ 4 ]


Adenosine 5′-tetraphosphate, Ap4  or  ATPP  is a  nucleotide . It is produced from  ATP  and  triphosphate  (P 3 ) through the action of  acetyl—CoA synthetase . [ 1 ]  Acetyl—CoA synthetase also produces adenosine 5'-pentaphosphate through the reaction of  ADP  and tetraphosphate (P 4 ).


ATPP has been found to play physiological roles in some  mammals .


ATPP is a constituent of  aqueous humor  in  rabbits , where it was found to reduce the  intraocular pressure . [ 2 ]


ATPP has been suggested to play a regulatory role in  rat   aorta .


Adenosine diphosphate ribose  ( ADPR ) is an  ester  molecule formed into chains by the enzyme  poly ADP ribose polymerase . [ 1 ]  ADPR is created from  cyclic ADP-ribose  (cADPR) by the  CD38  enzyme using  nicotinamide adenine dinucleotide  (NAD + ) as a  cofactor . [ 1 ]


ADPR binds to and activates the  TRPM2  ion channel. [ 2 ]  ADPR is the most potent  agonist  of the TRPM2 channel. [ 3 ]  cADPR also binds to TPRM2, and the action of both molecules is  synergistic , with both molecules enhancing the action of the other molecule in activating  the TRPM2 channel. [ 4 ]  Researchers are not sure how the Adenosine diphosphate reacts with the TRPM2 channel, but the ribose sugar may play a role in activating the  TRPM2  ion channel. [ 5 ]


Researchers believe that co-targeting DNA-dependent protein kinase and poly(adenosine diphosphate-ribose) polymerase-1 does not promote apoptosis or mitotic catastrophe of cancer cells after radiation. [ 6 ]


Adenosine monophosphate  ( AMP ), also known as  5'-adenylic acid , is a  nucleotide . AMP consists of a  phosphate  group, the sugar  ribose , and the  nucleobase   adenine . It is an  ester  of  phosphoric acid  and the  nucleoside   adenosine . [ 1 ]  As a  substituent  it takes the form of the prefix  adenylyl- . [ 2 ]


AMP plays an important role in many cellular metabolic processes, being interconverted to  adenosine triphosphate  (ATP) and  adenosine diphosphate  (ADP), as well as  allosterically  activating enzymes such as myophosphorylase-b. AMP is also a component in the synthesis of  RNA . [ 3 ]  AMP is present in all known forms of life. [ 4 ]


AMP does not have the high energy  phosphoanhydride  bond associated with ADP and ATP. AMP can be produced from  ADP  by the  myokinase (adenylate kinase)  reaction when the ATP reservoir in the cell is low: [ 5 ] [ 6 ]


Or AMP may be produced by the  hydrolysis  of one  high energy phosphate  bond of ADP:


AMP can also be formed by hydrolysis of  ATP  into AMP and  pyrophosphate :


When RNA is broken down by living systems, nucleoside monophosphates, including adenosine monophosphate, are formed.


AMP can be regenerated to ATP as follows:


AMP can be converted into  inosine monophosphate  by the  enzyme   myoadenylate deaminase , freeing an  ammonia  group.


In a  catabolic  pathway, the  purine nucleotide cycle , adenosine monophosphate can be converted to  uric acid , which is excreted from the body in mammals. [ 7 ]


The eukaryotic cell enzyme  5' adenosine monophosphate-activated protein kinase , or AMPK, utilizes AMP for  homeostatic  energy processes during times of high cellular energy expenditure, such as exercise. [ 8 ]  Since ATP cleavage, and corresponding  phosphorylation  reactions, are utilized in various processes throughout the body as a source of energy, ATP production is necessary to further create energy for those mammalian cells. AMPK, as a cellular energy sensor, is activated by decreasing levels of ATP, which is naturally accompanied by increasing levels of ADP and AMP. [ 9 ]


Though phosphorylation appears to be the main  activator  for AMPK, some studies suggest that AMP is an  allosteric regulator  as well as a  direct agonist  for AMPK. [ 10 ]  Furthermore, other studies suggest that the high ratio of AMP:ATP levels in cells, rather than just AMP, activate AMPK. [ 11 ]  For example, the AMP-activated kinases of  Caenorhabditis elegans  and  Drosophila melanogaster  were found to have been activated by AMP, while  yeast  and plant kinases were not allosterically activated by AMP. [ 11 ]


AMP binds to the  γ -subunit of AMPK, leading to the activation of the kinase, and then eventually a  cascade  of other processes such as the activation of  catabolic  pathways and  inhibition  of  anabolic  pathways to regenerate ATP. Catabolic mechanisms, which generate ATP through the release of energy from breaking down molecules, are activated by the AMPK enzyme while anabolic mechanisms, which utilize energy from ATP to form products, are inhibited. [ 12 ]  Though the  γ- subunit can bind AMP/ADP/ATP, only the binding of AMP/ADP results in a conformational shift of the enzyme protein. This variance in AMP/ADP versus ATP binding leads to a shift in the  dephosphorylation  state for the enzyme. [ 13 ]  The dephosphorylation of AMPK through various protein  phosphatases  completely inactivates catalytic function. AMP/ADP protects AMPK from being inactivated by binding to the  γ -subunit and maintaining the dephosphorylation state. [ 14 ]


AMP can also exist as a cyclic structure known as  cyclic AMP  (or cAMP). Within certain cells the enzyme  adenylate cyclase  makes cAMP from ATP, and typically this reaction is regulated by hormones such as  adrenaline  or  glucagon . cAMP plays an important role in intracellular signaling. [ 15 ]  In skeletal muscle, cyclic AMP, triggered by adrenaline, starts a cascade ( cAMP-dependent pathway ) for the conversion of myophosphorylase-b into the phosphorylated form of  myophoshorylase -a for glycogenolysis. [ 16 ] [ 17 ]


Adenosine diphosphate  ( ADP ), also known as  adenosine pyrophosphate  ( APP ), is an important  organic compound  in  metabolism  and is essential to the flow of energy in living  cells . ADP consists of three important structural components: a  sugar  backbone attached to  adenine  and two  phosphate  groups bonded to the 5 carbon atom of  ribose . The diphosphate group of ADP is attached to the 5’ carbon of the sugar backbone, while the adenine attaches to the 1’ carbon. [ 1 ]


ADP can be interconverted to  adenosine triphosphate  (ATP) and  adenosine monophosphate  (AMP). ATP contains one more phosphate group than ADP, while AMP contains one fewer phosphate group. Energy transfer used by all living things is a result of  dephosphorylation  of ATP by enzymes known as  ATPases . The cleavage of a phosphate group from ATP results in the coupling of energy to metabolic reactions and a by-product of ADP. [ 1 ]  ATP is continually reformed from lower-energy species ADP and AMP. The biosynthesis of ATP is achieved throughout processes such as  substrate-level phosphorylation ,  oxidative phosphorylation , and  photophosphorylation , all of which facilitate the addition of a phosphate group to ADP.


ADP cycling supplies the  energy  needed to do work in a biological system, the  thermodynamic  process of transferring energy from one source to another. There are two types of energy:  potential energy  and  kinetic energy . Potential energy can be thought of as stored energy, or usable energy that is available to do work. Kinetic energy is the energy of an object as a result of its motion. The significance of ATP is in its ability to store potential energy within the phosphate bonds. The energy stored between these bonds can then be transferred to do work. For example, the transfer of energy from ATP to the protein  myosin  causes a conformational change when connecting to  actin  during  muscle contraction . [ 1 ]


It takes multiple reactions between myosin and actin to effectively produce one muscle contraction, and, therefore, the availability of large amounts of ATP is required to produce each muscle contraction. For this reason, biological processes have evolved to produce efficient ways to replenish the potential energy of ATP from ADP. [ 2 ]


Breaking one of ATP's phosphorus bonds generates approximately 30.5  kilojoules  per  mole  of ATP (7.3  kcal ). [ 3 ]  ADP can be converted, or powered back to ATP through the process of releasing the chemical energy available in food; in humans, this is constantly performed via  aerobic respiration  in the  mitochondria . [ 2 ]  Plants use  photosynthetic  pathways to convert and store energy from sunlight, also conversion of ADP to ATP. [ 3 ]  Animals use the energy released in the breakdown of glucose and other molecules to convert ADP to ATP, which can then be used to fuel necessary growth and cell maintenance. [ 2 ]


The ten-step  catabolic  pathway of  glycolysis  is the initial phase of free-energy release in the breakdown of   glucose  and can be split into two phases, the preparatory phase and payoff phase. ADP and  phosphate  are needed as precursors to synthesize ATP in the payoff reactions of the  TCA cycle  and  oxidative phosphorylation  mechanism. [ 4 ]   During the payoff phase of glycolysis, the enzymes phosphoglycerate kinase and pyruvate kinase facilitate the addition of a phosphate group to ADP by way of  substrate-level phosphorylation . [ 5 ]


Glycolysis is performed by all living organisms and consists of 10 steps.  The net reaction for the overall process of  glycolysis  is: [ 6 ]


Steps 1 and 3 require the input of energy derived from the hydrolysis of ATP to ADP and P i  (inorganic phosphate), whereas steps 7 and 10 require the input of ADP, each yielding ATP. [ 7 ]   The  enzymes  necessary to break down glucose are found in the  cytoplasm , the viscous fluid that fills living cells, where the glycolytic reactions take place. [ 1 ]


The  citric acid cycle , also known as the Krebs cycle or the TCA (tricarboxylic acid) cycle is an 8-step process that takes the pyruvate generated by glycolysis and generates 4 NADH, FADH2, and GTP, which is further converted to ATP. [ 8 ]  It is only in step 5, where GTP is generated, by succinyl-CoA synthetase, and then converted to ATP, that ADP is used (GTP + ADP → GDP + ATP). [ 9 ]


Oxidative phosphorylation  produces 26 of the 30 equivalents of ATP generated in cellular respiration by transferring electrons from NADH or FADH2 to  O 2  through electron carriers. [ 10 ]  The energy released when electrons are passed from higher-energy NADH or FADH2 to the lower-energy O 2  is required to phosphorylate ADP and once again generate ATP. [ 11 ]  It is this energy coupling and phosphorylation of ADP to ATP that gives the electron transport chain the name oxidative phosphorylation. [ 1 ]


During the initial phases of  glycolysis  and the  TCA cycle ,  cofactors  such as  NAD+  donate and accept electrons [ 12 ]  that aid in the  electron transport chain 's ability to produce a proton gradient across the inner mitochondrial membrane. [ 13 ]  The ATP synthase complex exists within the mitochondrial membrane (F O  portion) and protrudes into the matrix (F 1  portion).  The energy derived as a result of the chemical gradient is then used to synthesize ATP by coupling the reaction of inorganic phosphate to ADP in the active site of the  ATP synthase  enzyme; the equation for this can be written as ADP + P i  → ATP. [ citation needed ]


Under normal conditions, small disk-shape  platelets  circulate in the blood freely and without interaction with one another. ADP is stored in  dense bodies  inside  blood   platelets  and is released upon platelet activation. ADP interacts with a family of ADP receptors found on platelets (P2Y1,  P2Y12 , and P2X1), which leads to platelet activation. [ 14 ]


ADP in the blood is converted to  adenosine  by the action of  ecto-ADPases , inhibiting further platelet activation via  adenosine receptors . [ citation needed ]


Adenosine triphosphate  ( ATP ) is a  nucleoside triphosphate [ 2 ]  that provides  energy  to drive and support many processes in living  cells , such as  muscle contraction ,  nerve impulse  propagation, and  chemical synthesis . Found in all known forms of  life , it is often referred to as the "molecular unit of  currency " for intracellular  energy transfer . [ 3 ]


When consumed in a  metabolic  process, ATP converts either to  adenosine diphosphate  (ADP) or to  adenosine monophosphate  (AMP). Other processes regenerate ATP. It is also a  precursor  to  DNA  and  RNA , and is used as a  coenzyme . An average adult human processes around 50 kilograms (about 100  moles ) daily. [ 4 ]


From the perspective of  biochemistry , ATP is classified as a  nucleoside triphosphate , which indicates that it consists of three components: a nitrogenous base ( adenine ), the sugar  ribose , and the  triphosphate .


ATP consists of an  adenine  attached by the #9-nitrogen atom to the 1′  carbon   atom  of a sugar ( ribose ), which in turn is attached at the 5' carbon atom of the sugar to a triphosphate group. In its many reactions related to metabolism, the adenine and sugar groups remain unchanged, but the triphosphate is converted to di- and monophosphate, giving respectively the derivatives  ADP  and  AMP . The three phosphoryl groups are labeled as alpha (α), beta (β), and, for the terminal phosphate, gamma (γ). [ 5 ]


In neutral solution, ionized ATP exists mostly as ATP 4− , with a small proportion of ATP 3− . [ 6 ]


Polyanionic and featuring a potentially  chelating  polyphosphate group, ATP binds metal cations with high affinity. The  binding constant  for  Mg 2+  is ( 9 554 ). [ 7 ]   The binding of a  divalent   cation , almost always  magnesium , strongly affects the interaction of ATP with various proteins. Due to the strength of the ATP-Mg 2+  interaction, ATP exists in the cell mostly as a complex with  Mg 2+  bonded to the phosphate oxygen centers. [ 6 ] [ 8 ]


A second magnesium ion is critical for ATP binding in the kinase domain. [ 9 ]  The presence of Mg 2+  regulates kinase activity. [ 10 ]    It is interesting from an RNA world perspective that ATP can carry a Mg ion which catalyzes RNA polymerization. [ citation needed ]


Salts of ATP can be isolated as colorless solids. [ 11 ]


ATP is stable in aqueous solutions between  pH  6.8 and 7.4 (in the absence of catalysts).  At more extreme pH levels, it rapidly  hydrolyses  to ADP and phosphate. Living cells maintain the ratio of ATP to ADP at a point ten orders of magnitude from equilibrium, with ATP concentrations fivefold higher than the concentration of ADP. [ 12 ] [ 13 ]  In the context of biochemical reactions, the P-O-P bonds are frequently referred to as  high-energy bonds . [ 14 ]


The hydrolysis of ATP into ADP and inorganic phosphate


releases 20.5 kilojoules per mole (4.9 kcal/mol) of  enthalpy . This may differ under physiological conditions if the reactant and products are not exactly in these ionization states. [ 15 ]  The values of the free energy released by cleaving either a phosphate (P i ) or a pyrophosphate (PP i ) unit from ATP at  standard state  concentrations of 1 mol/L at pH 7 are: [ 16 ]


These abbreviated equations at a pH near 7 can be written more explicitly (R =  adenosyl ):


At cytoplasmic conditions, where the ADP/ATP ratio is 10 orders of magnitude from equilibrium, the Δ G  is around −57 kJ/mol. [ 12 ]


Along with pH, the free energy change of ATP hydrolysis is also associated with Mg 2+  concentration, from ΔG°' = −35.7 kJ/mol at a Mg 2+  concentration of zero, to ΔG°' = −31 kJ/mol at [Mg 2+ ] = 5 mM. Higher concentrations of Mg 2+  decrease free energy released in the reaction due to binding of Mg 2+  ions to negatively charged oxygen atoms of ATP at pH 7. [ 17 ]


A typical intracellular  concentration  of ATP may be 1–10 μmol per gram of tissue in a variety of eukaryotes. [ 18 ]  The dephosphorylation of ATP and rephosphorylation of ADP and AMP occur repeatedly in the course of aerobic metabolism. [ 19 ]


ATP can be produced by a number of distinct cellular processes; the three main pathways in  eukaryotes  are (1)  glycolysis , (2) the  citric acid cycle / oxidative phosphorylation , and (3)  beta-oxidation . The overall process of oxidizing  glucose  to  carbon dioxide , the combination of pathways 1 and 2, known as  cellular respiration , produces about 30 equivalents of ATP from each molecule of glucose. [ 20 ]


ATP production by a non- photosynthetic  aerobic eukaryote occurs mainly in the  mitochondria , which comprise nearly 25% of the volume of a typical cell. [ 21 ]


In glycolysis, glucose and glycerol are metabolized to  pyruvate . Glycolysis generates two equivalents of ATP through  substrate phosphorylation  catalyzed by two enzymes,   phosphoglycerate kinase  (PGK) and  pyruvate kinase . Two equivalents of  nicotinamide adenine dinucleotide  (NADH) are also produced, which can be oxidized via the  electron transport chain  and result in the generation of additional ATP by  ATP synthase . The pyruvate generated as an end-product of glycolysis is a substrate for the  Krebs Cycle . [ 22 ]


Glycolysis is viewed as consisting of two phases with five steps each. In phase 1, "the preparatory phase", glucose is converted to 2 d-glyceraldehyde-3-phosphate (g3p). One ATP is invested in Step 1, and another ATP is invested in Step 3. Steps 1 and 3 of glycolysis are referred to as "Priming Steps". In Phase 2, two equivalents of g3p are converted to two pyruvates. In Step 7, two ATP are produced. Also, in Step 10, two further equivalents of ATP are produced. In Steps 7 and 10, ATP is generated from ADP. A net of two ATPs is formed in the glycolysis cycle. The glycolysis pathway is later associated with the Citric Acid Cycle which produces additional equivalents of ATP. [ citation needed ]


In glycolysis,  hexokinase  is directly inhibited by its product, glucose-6-phosphate, and  pyruvate kinase  is inhibited by ATP itself. The main control point for the glycolytic pathway is  phosphofructokinase  (PFK), which is allosterically inhibited by high concentrations of ATP and activated by high concentrations of AMP. The inhibition of PFK by ATP is unusual since ATP is also a substrate in the reaction catalyzed by PFK; the active form of the enzyme is a  tetramer  that exists in two conformations, only one of which binds the second substrate fructose-6-phosphate (F6P). The protein has two  binding sites  for ATP – the  active site  is accessible in either protein conformation, but ATP binding to the inhibitor site stabilizes the conformation that binds F6P poorly. [ 22 ]  A number of other small molecules can compensate for the ATP-induced shift in equilibrium conformation and reactivate PFK, including  cyclic AMP ,  ammonium  ions, inorganic phosphate, and fructose-1,6- and -2,6-biphosphate. [ 22 ]


In the  mitochondrion , pyruvate is oxidized by the  pyruvate dehydrogenase complex  to the  acetyl  group, which is fully oxidized to carbon dioxide by the  citric acid cycle  (also known as the  Krebs  cycle). Every "turn" of the citric acid cycle produces two molecules of carbon dioxide, one equivalent of ATP  guanosine triphosphate  (GTP) through  substrate-level phosphorylation  catalyzed by  succinyl-CoA synthetase , as succinyl-CoA is converted to succinate, three equivalents of NADH, and one equivalent of  FADH 2 . NADH and FADH 2  are recycled (to NAD +  and  FAD , respectively) by  oxidative phosphorylation , generating additional ATP. The oxidation of NADH results in the synthesis of 2–3 equivalents of ATP, and the oxidation of one FADH 2  yields between 1–2 equivalents of ATP. [ 20 ]  The majority of cellular ATP is generated by this process. Although the citric acid cycle itself does not involve molecular  oxygen , it is an obligately  aerobic  process because O 2  is used to recycle the NADH and FADH 2 . In the absence of oxygen, the citric acid cycle ceases. [ 21 ]


The generation of ATP by the mitochondrion from cytosolic NADH relies on the  malate-aspartate shuttle  (and to a lesser extent, the  glycerol-phosphate shuttle ) because the inner mitochondrial membrane is impermeable to NADH and NAD + . Instead of transferring the generated NADH, a  malate dehydrogenase  enzyme converts  oxaloacetate  to  malate , which is translocated to the mitochondrial matrix. Another malate dehydrogenase-catalyzed reaction occurs in the opposite direction, producing oxaloacetate and NADH from the newly transported malate and the mitochondrion's interior store of NAD + . A  transaminase  converts the oxaloacetate to  aspartate  for transport back across the membrane and into the intermembrane space. [ 21 ]


In oxidative phosphorylation, the passage of electrons from NADH and FADH 2  through the electron transport chain releases the energy to pump  protons  out of the mitochondrial matrix and into the intermembrane space. This pumping generates a  proton motive force  that is the net effect of a pH gradient and an  electric potential  gradient across the inner mitochondrial membrane. Flow of protons down this potential gradient – that is, from the intermembrane space to the matrix – yields ATP by ATP synthase. [ 23 ]  Three ATP are produced per turn.


Although oxygen consumption appears fundamental for the maintenance of the proton motive force, in the event of oxygen shortage ( hypoxia ), intracellular acidosis (mediated by enhanced glycolytic rates and  ATP hydrolysis ), contributes to mitochondrial membrane potential and directly drives ATP synthesis. [ 24 ]


Most of the ATP synthesized in the mitochondria will be used for cellular processes in the cytosol; thus it must be exported from its site of synthesis in the mitochondrial matrix. ATP outward movement is favored by the membrane's electrochemical potential because the cytosol has a relatively positive charge compared to the relatively negative matrix. For every ATP transported out, it costs 1 H + . Producing one ATP costs about 3  H + . Therefore, making and exporting one ATP requires  4H +.  The inner membrane contains an  antiporter , the ADP/ATP translocase, which is an  integral membrane protein  used to exchange newly synthesized ATP in the matrix for ADP in the intermembrane space. [ 25 ]


The citric acid cycle is regulated mainly by the availability of key substrates, particularly the ratio of NAD +  to NADH and the concentrations of  calcium , inorganic phosphate, ATP, ADP, and AMP.  Citrate  – the ion that gives its name to the cycle – is a feedback inhibitor of  citrate synthase  and also inhibits PFK, providing a direct link between the regulation of the citric acid cycle and glycolysis. [ 22 ]


In the presence of air and various cofactors and enzymes, fatty acids are converted to  acetyl-CoA . The pathway is called  beta-oxidation . Each cycle of beta-oxidation shortens the fatty acid chain by two carbon atoms and produces one equivalent each of acetyl-CoA, NADH, and FADH 2 . The acetyl-CoA is metabolized by the citric acid cycle to generate ATP, while the NADH and FADH 2  are used  by oxidative phosphorylation to generate ATP.  Dozens of ATP equivalents are generated by the beta-oxidation of a single long acyl chain. [ 26 ]


In oxidative phosphorylation, the key control point is the reaction catalyzed by  cytochrome c oxidase , which is regulated by the availability of its substrate – the reduced form of  cytochrome c . The amount of reduced cytochrome c available is directly related to the amounts of other substrates:


which directly implies this equation:


Thus, a high ratio of [NADH] to [NAD + ] or a high ratio of [ADP] [P i ] to [ATP] imply a high amount of reduced cytochrome c and a high level of cytochrome c oxidase activity. [ 22 ]  An additional level of regulation is introduced by the transport rates of ATP and NADH between the mitochondrial matrix and the cytoplasm. [ 25 ]


Ketone bodies can be used as fuels, yielding 22 ATP and 2  GTP  molecules per acetoacetate molecule when oxidized in the mitochondria. Ketone bodies are transported from the  liver  to other tissues, where  acetoacetate  and  beta -hydroxybutyrate  can be reconverted to acetyl-CoA to produce reducing equivalents (NADH and FADH 2 ), via the citric acid cycle. Ketone bodies cannot be used as fuel by the liver, because the liver lacks the enzyme β-ketoacyl-CoA transferase, also called  thiolase .  Acetoacetate  in low concentrations is taken up by the liver and undergoes detoxification through the methylglyoxal pathway which ends with lactate. Acetoacetate in high concentrations is absorbed by cells other than those in the liver and enters a different pathway via  1,2-propanediol .  Though the pathway follows a different series of steps requiring ATP, 1,2-propanediol can be turned into pyruvate. [ 27 ]


Fermentation  is the metabolism of organic compounds in the absence of air. It involves  substrate-level phosphorylation  in the absence of a respiratory  electron transport chain . The equation for the reaction of glucose to form  lactic acid  is:


Anaerobic respiration  is respiration in the absence of  O 2 . Prokaryotes can utilize a variety of electron acceptors. These include  nitrate ,  sulfate , and carbon dioxide.


ATP can also be synthesized through several so-called "replenishment" reactions catalyzed by the enzyme families of  nucleoside diphosphate kinases  (NDKs), which use other nucleoside triphosphates as a high-energy phosphate donor, and the  ATP:guanido-phosphotransferase  family. [ citation needed ]


In plants, ATP is synthesized in the  thylakoid membrane  of the  chloroplast . The process is called  photophosphorylation . The "machinery" is similar to that in mitochondria except that light energy is used to pump protons across a membrane to produce a proton-motive force. ATP synthase then ensues exactly as in oxidative phosphorylation. [ 28 ]  Some of the ATP produced in the chloroplasts is consumed in the  Calvin cycle , which produces  triose  sugars.


The total quantity of ATP in the human body is about 0.1  mol/L . [ 29 ]  The majority of ATP is recycled from ADP by the aforementioned processes. Thus, at any given time, the total amount of ATP + ADP remains fairly constant.


The energy used by human cells in an adult requires the hydrolysis of 100 to 150 mol/L of ATP daily, which means a human will typically use their body weight worth of ATP over the course of the day. [ 30 ]  Each equivalent of ATP is recycled 1000–1500 times during a single day ( 150 / 0.1 = 1500 ), [ 29 ]  at approximately 9×10 20  molecules/s. [ 29 ]


ATP is involved in  signal transduction  by serving as substrate for kinases, enzymes that transfer phosphate groups.  Kinases are the most common ATP-binding proteins.  They share a small number of common folds. [ 31 ]   Phosphorylation  of a protein by a kinase can activate a cascade such as the  mitogen-activated protein kinase  cascade. [ 32 ]


ATP is also a substrate of  adenylate cyclase , most commonly in  G protein-coupled receptor  signal transduction pathways and is transformed to  second messenger , cyclic AMP, which is involved in triggering calcium signals by the release of calcium from intracellular stores. [ 33 ]  This form of signal transduction is particularly important in brain function, although it is involved in the regulation of a multitude of other cellular processes. [ 34 ]


ATP is one of four monomers required in the synthesis of  RNA . The process is promoted by  RNA polymerases . [ 35 ]  A similar process occurs in the formation of DNA, except that ATP is first converted to the  deoxyribonucleotide  dATP.  Like many condensation reactions in nature,  DNA replication  and  DNA transcription  also consume ATP.


Aminoacyl-tRNA synthetase  enzymes consume ATP in the attachment tRNA to amino acids, forming aminoacyl-tRNA complexes. Aminoacyl transferase binds AMP-amino acid to tRNA. The coupling reaction proceeds in two steps:


The amino acid is coupled to the penultimate nucleotide at the 3′-end of the tRNA (the A in the sequence CCA) via an ester bond (roll over in illustration).


Transporting chemicals out of a cell against a gradient is often associated with ATP hydrolysis.  Transport is mediated by  ATP binding cassette transporters .  The human genome encodes 48 ABC transporters, that are used for exporting drugs, lipids, and other compounds. [ 36 ]


Cells secrete ATP to communicate with other cells in a process called  purinergic signalling . ATP serves as a  neurotransmitter  in many parts of the nervous system, modulates ciliary beating, affects vascular oxygen supply etc.  ATP is either secreted directly across the cell membrane through channel proteins [ 37 ] [ 38 ]  or is pumped into vesicles [ 39 ]  which then  fuse  with the membrane. Cells detect ATP using the  purinergic receptor  proteins  P2X  and  P2Y . [ 40 ]  ATP has been shown to be a critically important signalling molecule for  microglia  -  neuron  interactions in the adult brain, [ 41 ]  as well as during brain development. [ 42 ]  Furthermore, tissue-injury induced ATP-signalling is a major factor in rapid microglial phenotype changes. [ 43 ]


ATP fuels  muscle contractions . [ 44 ]  Muscle contractions are regulated by signaling pathways, although different  muscle  types being regulated by specific pathways and stimuli based on their particular function. However, in all muscle types, contraction is performed by the proteins  actin  and  myosin . [ 45 ]


ATP is initially bound to myosin. When  ATPase  hydrolyzes the bound ATP into  ADP  and inorganic  phosphate , myosin is positioned in a way that it can bind to actin. Myosin bound by ADP and P i  forms cross-bridges with actin and the subsequent release of ADP and P i  releases energy as the power stroke. The power stroke causes actin filament to slide past the myosin filament, shortening the muscle and causing a contraction. Another ATP molecule can then bind to myosin, releasing it from actin and allowing this process to repeat. [ 45 ] [ 46 ]


ATP has recently been proposed to act as a biological  hydrotrope [ 47 ]  and has been shown to affect proteome-wide solubility. [ 48 ]


Acetyl phosphate (AcP), a precursor to ATP, can readily be synthesized at modest yields from thioacetate in pH 7 and 20 °C and pH 8 and 50 °C, although acetyl phosphate is less stable in warmer temperatures and alkaline conditions than in cooler and acidic to neutral conditions. It is unable to promote polymerization of ribonucleotides and amino acids and was only capable of phosphorylation of organic compounds. It was shown that it can promote aggregation and stabilization of AMP in the presence of Na + , aggregation of nucleotides could promote polymerization above 75 °C in the absence of Na + . It is possible that polymerization promoted by AcP could occur at mineral surfaces. [ 49 ]  It was shown that ADP can only be phosphorylated to ATP by AcP and other nucleoside triphosphates were not phosphorylated by AcP. This might explain why all lifeforms use ATP to drive biochemical reactions. [ 50 ]


Biochemistry laboratories often use  in vitro  studies to explore ATP-dependent molecular processes. ATP analogs are also used in  X-ray crystallography  to determine a  protein structure  in complex with ATP, often together with other substrates. [ citation needed ]


Enzyme inhibitors  of ATP-dependent enzymes such as  kinases  are needed to examine the  binding sites  and  transition states  involved in ATP-dependent reactions. [ citation needed ]


Most useful ATP analogs cannot be hydrolyzed as ATP would be; instead, they trap the enzyme in a structure closely related to the ATP-bound state. Adenosine 5′-(γ-thiotriphosphate) is an extremely common ATP analog in which one of the gamma-phosphate oxygens is replaced by a  sulfur  atom; this anion is hydrolyzed at a dramatically slower rate than ATP itself and functions as an inhibitor of ATP-dependent processes. In crystallographic studies, hydrolysis transition states are modeled by the bound  vanadate  ion.


Caution is warranted in interpreting the results of experiments using ATP analogs, since some enzymes can hydrolyze them at appreciable rates at high concentration. [ 51 ]


ATP is used intravenously for some heart related conditions. [ 52 ]


ATP was discovered in 1929 by  Karl Lohmann  [ de ] [ 53 ]  and Jendrassik [ 54 ]  and, independently, by Cyrus Fiske and  Yellapragada Subba Rao  of  Harvard Medical School , [ 55 ]  both teams competing against each other to find an assay for phosphorus.


It was proposed to be the intermediary between energy-yielding and energy-requiring reactions in cells by  Fritz Albert Lipmann  in 1941. [ 56 ]


It was first synthesized in the laboratory by  Alexander Todd  in 1948, [ 57 ]  and he was awarded the  Nobel Prize in Chemistry  in 1957 partly for this work.


The 1978  Nobel Prize in Chemistry  was awarded to  Peter Dennis Mitchell  for the discovery of the  chemiosmotic  mechanism of ATP synthesis.


The 1997 Nobel Prize in Chemistry was divided, one half jointly to  Paul D. Boyer  and  John E. Walker  "for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)" and the other half to  Jens C. Skou  "for the first discovery of an ion-transporting enzyme, Na + , K +  -ATPase." [ 58 ]


Adenosine thiamine diphosphate  (AThDP), or thiaminylated adenosine diphosphate (ADP) is a naturally occurring thiamine adenine  nucleotide . It was chemically synthesized and exists in small amounts in vertebrate liver. Its biological significance remains unknown. [ 1 ] [ 2 ]


Adenosine thiamine triphosphate  ( AThTP ), or  thiaminylated adenosine triphosphate , is a natural  thiamine   adenine   nucleotide . [ 1 ]   It was discovered in  Escherichia coli  where it may account for up to 15 - 20% of total thiamine under carbon starvation. AThTP also exists in  eukaryotic  organisms such as yeast, roots of higher plants and animal tissues, albeit at a much lower concentration. It was found to exist in small amounts in the muscle, heart, brain, kidneys and liver of mice. [ 2 ]


In  E. coli  AThTP is synthesized from  thiamine diphosphate  (ThDP) according to the following reaction catalyzed by  thiamine diphosphate adenylyl transferase : [ 3 ]


The molecule is made up of thiamine and adenosine joined together with phosphate groups. It is similar in structure to NAD+. The function of AThTP is not currently known but it has been shown to inhibit the activity of PARP-1. [ 2 ]


Adenylosuccinate , also known as  succinyl-adenosine monophosphate  ( S-AMP ), is an intermediate in the interconversion of  purine   nucleotides   inosine monophosphate  (IMP) and  adenosine monophosphate  (AMP). The enzyme  adenylosuccinate synthase  carries out the reaction of IMP to S-AMP in a 2 step mechanism, [ 1 ]  requiring the input of energy from a  phosphoanhydride  bond from the  hydrolysis  of  guanosine triphosphate  (GTP) [ 2 ]  first, followed by the addition of  aspartate . This reaction needs Mg 2+ , [ 3 ]  and is  competitively inhibited  by the subsequent product AMP [ 3 ]  in a  negative feedback  mechanism. [ 4 ]  GTP, the product of another pathway from IMP, is used instead of  adenosine triphosphate  (ATP) as the phosphate source. The enzyme  adenylosuccinate lyase  carries out the reaction removing the carbon skeleton from S-AMP attached from aspartate, forming AMP and  fumarate . [ 4 ]  The pathway from IMP to AMP is present across various prokaryotes and eukaryotes, and is linked to various diseases. [ 5 ]  S-AMP has been observed to stimulate insulin production. [ 6 ]


5-Aminoimidazole-4-carboxamide ribonucleotide  ( AICAR ) is an intermediate in the generation of  inosine monophosphate . AICAR is an  analog  of  adenosine monophosphate  (AMP) that is capable of stimulating  AMP-dependent protein kinase  (AMPK) activity. The drug has also been shown as a potential treatment for diabetes by increasing the metabolic activity of tissues by changing the physical composition of muscle. [ 1 ]


The  nucleoside  form of AICAR,  acadesine , is an analog of  adenosine  that enters cardiac cells to inhibit adenosine kinase and adenosine deaminase. It enhances the rate of  nucleotide  re-synthesis increasing adenosine generation from adenosine monophosphate only during conditions of myocardial ischemia. [ 2 ]  In cardiac  myocytes , acadesine is phosphorylated to AICAR to activate AMPK without changing the levels of the nucleotides. [ 3 ]  AICAR is able to enter the  de novo synthesis  pathway for adenosine synthesis to inhibit adenosine deaminase causing an increase in ATP levels and adenosine levels. [ 4 ]


In 2009, the  French Anti-Doping Agency , suspected that AICAR had been used in the  2009 Tour de France  for its supposed performance enhancing properties. [ 5 ] [ 6 ]  Although a detection method was reportedly given to the  World Anti-Doping Agency , it was unknown if this method was implemented. [ 7 ]  As of January 2011, AICAR was officially a banned substance in the World Anti Doping Code, [ 8 ]  and the standard levels in elite athletes have been determined, to interpret test results. [ 9 ] [ 10 ]


Aminoallyl nucleotide  is a nucleotide with a modified base containing an  allylamine . They are used in post-labeling of  nucleic acids  by  fluorescence  detection in  microarray . They are reactive with  N-Hydroxysuccinimide  ester group which helps attach a  fluorescent dye  to the primary amino group on the nucleotide. These nucleotides are known as 5-(3-amino allyl )-nucleotides since the aminoallyl group is usually attached to carbon 5 of the  pyrimidine  ring of  uracil  or  cytosine . The primary  amine  group in the aminoallyl moiety is  aliphatic  and thus more reactive compared to the amine groups that are directly attached to the rings ( aromatic ) of the bases. Common names of aminoallyl  nucleosides  are initially abbreviated with aa- or AA- to indicate aminoallyl. The 5-carbon sugar is indicated with or without the lowercase "d" indicating  deoxyribose  if included or  ribose  if not. Finally the  nitrogenous base  and number of  phosphates  are indicated (i.e. aa-UTP = aminoallyl uridine  triphosphate ).


The goal of combining fluorescence and nucleic acids has been to provide a non- isotopic  tag that is detectable to study  DNA  or  RNA . This type of labeling allows scientists to study DNA or RNA in their structure, function, or formation with other nucleic acids. [ 2 ]  The first base modification for fluorescent labeling occurred in 1971 with a  4-thiouridine  and  4-thiouracil . [ 3 ]  This research along with others, which included various types of direct and non-direct labeling via:  analogs , addition via enzymes, or other methods made labeling of nucleotides much safer for scientist to study DNA. [ 2 ]


As instrumentation and technologies become more advanced in the field of  DNA microarray , better reagents and techniques will be needed to further scientific studies. Fluorescent labeling with  Cy3  was shown to be more insufficient and skew results; the method of aminoallyl nucleotide incorporation was opted instead. Using aminoallyl nucleotides as indirect fluorescent labeling seemed to nullify the sensitivity issues seen in  cyanine-labeling . [ 4 ]


Aminoallyl  nucleosides  can be  synthesized  via  Heck coupling  as shown in the image below. [ 5 ]


In the image above, on the left is a modified nucleoside with an  iodine  (the iodine is added via  electrophilic halogenation ) in the fifth carbon in the pyrimidine ring. Its formation can be associated with a reaction with an allylamine and various reagents via heck coupling are able to remove the  halogen  group from the base and add the allylamine to become the aminoallyl nucleoside shown on the right. [ 5 ]  The product on the right is then used to in  molecular biology  in RNA synthesis. [ 4 ] [ 6 ] [ 7 ]


Other reactions include using a  single pot synthesis  with other  halogens . [ 8 ]


The primary amine on the aminoallyl nucleotide reacts with amino-reactive dyes  [ 9 ]  such as a  cyanine  and patented dyes [ 10 ] [ 11 ]  which contain a reactive leaving group, such as a succinimidyl ester ( NHS ).The amine groups directly attached to the ring of the base are not affected. These nucleotides are used for labeling DNA. [ 4 ] [ 6 ] [ 10 ] [ 11 ] [ 12 ]


Aminoallyl NTPs are used for indirect DNA labeling in  PCR ,  nick translation ,  primer extensions  and  cDNA  synthesis. [ 13 ]  These labeled NTPs are helpful because of their application in molecular biology labs where they do not have the capacity to handle radioactive material. For example, 5-(3-Aminoallyl)-Uridine(AA-UTPs) are more effective for high density labeling of DNA than pre-labeling the DNA. After the enzymatic addition of the NTPs, amine reactant fluorescent dyes can be added for detection of the DNA molecule. [ 7 ]  When incorporated into DNA or RNA molecules by DNA/RNA  polymerase , 5-(3-aminoallyl)-UTP provide a reactive group for the addition of other chemical groups. Thus aminoallyl modified DNA or RNA can be labeled with any compound which has an amine-reactive group.
aa-NTPs incorporated into DNA/RNA in combination with a secondary dye coupling reagents can probe for an array analysis. [ 6 ]


cDNA relies on aminoallyl labeling for detection purposes. Although direct labeling of dNTP is the quickest and cheapest method of fluorescent labeling, it is disadvantageous as the sequence allows for only one modified nucleotide for use. Another disadvantage of direct labeling is the bulky nucleotides, however this can be overcome by indirect labeling using aminoallyl modified nucleotides. [ 14 ]  An easy way to check for labeling success is the color;Good labeling will result in visible blue (Cy5) or red (Cy3) color in the final material. [ 15 ]


Another process which uses aminoallyl labeling is  NASBA  ( Nucleic Acid Sequence Based Amplification), a highly sensitive technique for amplifying RNA. In this specific case, the aaUTP modified RNAs were tagged with fluorescent market Cy3. NASBA combined with aminoallyl-UTP labeling is very useful for many different areas of microbial diagnostics including  environmental monitoring , bio threat detection, industrial process monitoring and clinical microbiology. [ 16 ]  DNA microarray is another method which utilizes specifically AA-NTP's making DNA microarray testing quicker and cheaply. [ 12 ]


Post-synthesis labeling avoids the problems found in direct enzymatic incorporation of Cy-labeled dNTPs by generating probes with equal labeling effectiveness. With indirect labeling, amine-modified NTPs are incorporated during  reverse transcription , RNA amplification, or  PCR . Amino allyl-NTPs are incorporated with similar efficiency as unmodified NTPs during polymerization. [ 17 ] [ 18 ]


Concerns with labeling:
The amine group, in aminoallyl-modified nucleotide, is reactive with dyes such as the cyanine series, or other patented dyes. A problem arises when the dyes react with  buffering agents  which are necessary for the proper storage of the nucleotides. However, a carbonate buffer can be used to overcome this problem. [ 19 ]


5′-Phosphoribosyl-5-aminoimidazole  (or  aminoimidazole ribotide ,  AIR ) is a biochemical intermediate in the formation of  purine   nucleotides  via  inosine -5-monophosphate, and hence is a building block for  DNA  and  RNA . [ 1 ]  The vitamins  thiamine [ 2 ] [ 3 ]  and  cobalamin [ 4 ]  also contain fragments derived from AIR. [ 5 ]  It is an intermediate in the  adenine pathway  and is synthesized from  5′-phosphoribosylformylglycinamidine  by  AIR synthetase . [ 6 ]


5-aminoimidazole derivatives were considered unstable and therefore difficult to synthesize. The first non-enzymatic synthesis of 5-aminoimidazole ribotide (AIR) was only published in 1988 [ 7 ]  and general methodology for other examples was developed in the 1990s. [ 8 ] [ 9 ]


The  furanose  (5- carbon )  sugar  in AIR comes from the  pentose phosphate pathway , which converts  glucose  (as its  6-phosphate derivative ) into  ribose 5-phosphate   (R5P). [ 10 ]  The subsequent reactions which attach the  amino imidazole  portion of the molecule begin when R5P is activated as its  pyrophosphate  derivative,  phosphoribosyl pyrophosphate  (PRPP). This reaction is catalysed by  ribose-phosphate diphosphokinase . [ 11 ]


Five biosynthetic steps complete the transformation. [ 1 ] [ 12 ]  The first enzyme,  amidophosphoribosyltransferase , attaches  ammonia  from  glutamine  to the ribotide at its  anomeric carbon , forming  phosphoribosylamine  (PRA):


Next, PRA is converted to  glycineamide ribonucleotide  (GAR) by the action of  phosphoribosylamine—glycine ligase , forming an amide bond with  glycine  in a process driven by  ATP :


A third enzyme,  phosphoribosylglycinamide formyltransferase , adds a  formyl  group from  10-formyltetrahydrofolate  to GAR, giving  phosphoribosyl-N-formylglycineamide  (FGAR):


The penultimate step converts FGAR to an  amidine  by the action of  phosphoribosylformylglycinamidine synthase , transferring an amino group from glutamine and giving  5′-phosphoribosylformylglycinamidine  (FGAM) in a reaction that also requires ATP:


FGAM is finally converted to AIR by the action of  AIR synthetase  which uses ATP to activate the terminal  carbonyl group  to attack by the nitrogen atom at the anomeric centre:


The  purine  ring system of the  nucleotide   inosine monophosphate  is formed in a pathway from AIR [ 13 ]  that begins when  phosphoribosylaminoimidazole carboxylase  converts it to the  carboxylated  derivative in the imidazole ring,  5′-phosphoribosyl-4-carboxy-5-aminoimidazole  (CAIR). [ 14 ]


The same compound can be formed in a two-step pathway when the enzymes involved are  5-(carboxyamino)imidazole ribonucleotide synthase  and  5-(carboxyamino)imidazole ribonucleotide mutase . [ 14 ]


Rearrangement reactions starting from AIR incorporate portions of the molecule into additional biochemical pathways. The enzymes involved are in the  radical SAM  superfamily of   iron–sulfur proteins , which use  S -adenosyl methionine  as a  cofactor  to initiate the conversions via  radical  intermediates. [ 15 ] [ 5 ]


The vitamin  thiamine  contains a  pyrimidine  ring system which is formed from AIR in a reaction catalysed by  phosphomethylpyrimidine synthase . [ 2 ] [ 16 ]


This reaction incorporates the blue, green and red fragments shown into the product, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate. [ 3 ] [ 17 ]


In some  anaerobes , AIR is a precursor to  5,6-dimethylbenzimidazole , which is incorporated into  vitamin B 12  in later steps of  cobalamin biosynthesis . [ 5 ] [ 18 ]  The initial reaction is catalysed by  5-hydroxybenzimidazole synthase ,  EC   4.1.99.23 , and forms 5-hydroxybenzimidazole:


All the carbon atoms of the product are transferred from AIR, as shown. [ 4 ] [ 5 ]


Antagomirs , also known as  anti-miRs , are a class of chemically engineered  oligonucleotides  designed to silence endogenous  microRNAs  (also known as miRNAs or miRs). [ 1 ] [ 2 ] [ 3 ]


Antagomirs are a kind of  antisense oligonucleotide , as their sequence is complementary to their specific miRNA target. Their structure has modifications so as to make them more resistant to degradation. These include 2'- methoxy  groups on the  ribose  sugar, backbones with  phosphorothioate  bonds, and cholesterol conjugation on the 3' end. [ 4 ]


Antagomirs  are  microRNA  inhibitors that bind miRNAs and prevent them from binding to a target  mRNA  molecule, and the consequent degradation of that mRNA via the  RNA-induced silencing complex  (RISC). Due to the promiscuity of  microRNAs , each of which regulate multiple mRNAs, antagomirs can potentially affect the expression of many different mRNA molecules besides the desired target.


Blockmirs  are similarly engineered molecules which, on the other hand, are designed to have a sequence that is complementary to an  mRNA  sequence that is targeted by a  microRNA . Upon binding to an  untranslated region  of an mRNA, blockmirs sterically block microRNAs from binding to the same site. Because blockmirs bind individual mRNAs and not miRNAs, their activity is more predictable than antagomirs' and less likely to cause off-target effects. [ 5 ]


Antagomirs are used as a method to constitutively inhibit the activity of specific miRNAs associated with disease. For example, antagomirs against  miR-21  have been successfully used to inhibit fibrosis of heart [ 6 ]  and lung. [ 7 ]


The primary method for using  microRNA  technology to target the  Hepatitis C Virus  (HCV) is by knocking-out the liver-specific  microRNA .  miRNA-122  binds to the  5' untranslated region  of HCV's  mRNA  strand and, contrary to miRNA's normal function of repressing mRNA, actually upregulates the expression of the Hepatitis C Virus. Thus, the therapeutic goal in such a case would be to keep miRNA-122 from binding to HCV mRNA in order to prevent this mRNA from being expressed. However, miRNA-122 also regulates  cholesterol  ( HDL ) and the activity of tumor-suppressor genes ( oncogenes ).This means that not only will knocking out the microRNA-122 reduce the HCV infection, but it will also reduce the activity of  tumor suppressor genes , potentially leading to  liver cancer . In order to target HCV mRNA specifically (instead of miRNA-122 as a whole), Blockmir technology has been developed to solely target HCV mRNA, thus avoiding any sort of tampering with  oncogene  expression. This may be achieved by designing a Blockmir that matches seed 1. [ citation needed ]


MicroRNA-33a/b  inhibition in mice leads to increased blood  high-density lipoprotein  (HDL) levels.  Abca1  is essential for production of HDL precursors in liver cells. In  macrophages , Abca1 excretes cholesterol from oxidized cholesterol-carrying  lipoproteins  and thus counteracts  atherosclerotic  plaques. From this, it is hypothesized that microRNA-33 affects HDL via regulation of Abca1. Therefore, in order to target the regulation of Abca1, a blockmir can be developed that specifically binds to Abca1 mRNA molecules, thus blocking its miRNA site and upregulating its expression. Such an application of blockmir technology could lead to overall increased HDL levels. [ citation needed ]


MicroRNA-103/107 inhibition in mice leads to increased  insulin  sensitivity and signalling [ 8 ]  It has been previously shown that  caveolin-1 -deficient mice show insulin resistance. MicroRNA-103/107 inhibition in caveolin-1-deficient mice had no effect on insulin sensitivity and signalling. Thus, microRNA-103/107 may affect  insulin  sensitivity by targeting caveolin-1. [ 9 ]


The blockmir CD5-2 has been shown to inhibit the interaction between  miR-27  and  VE-cadherin , enhancing recovery from  ischemic injury  in mice. [ 10 ]  The drug has also been shown to enhance T cell infiltration in combination with  immunotherapy  in mouse models of  pancreatic cancer . [ 11 ]


Diadenosine tetraphosphate  or  Ap4A  is a putative  alarmone , ubiquitous in nature being common to everything from  bacteria  to  humans . It is made up of two adenosines joined together by a 5′-5′ linked chain of four phosphates. Adenosine polyphosphates are capable of inducing multiple  physiological  effects. [ 1 ]