Regardless of the details, an outstanding issue is that the physiological route of NAADP synthesis has still not been  unequivocally  identified — neither the reaction(s) nor the enzyme(s). Clearly, it is theoretically possible there may be multiple routes of synthesis, but this would be unprecedented in the second messenger world. To date, the most favoured hypothesis is the so-called  base-exchange reaction  ( nicotinic acid  +  NADP  → NAADP +  nicotinamide ; catalyzed by  ADP-ribosyl cyclases ) which are a family of enzymes that include  CD38  and  CD157  in mammals (and orthologs in sea urchin and  Aplysia  ovotestis). These were first discovered as the synthetic enzymes for  cADPR  but later revealed to be multifunctional, promiscuous enzymes that can also produce NAADP. Certainly NAADP production can occur  in vitro  but whether it occurs  in vivo  is another question (because genetic knockout or knock-down of ADP-ribosyl cyclases has no effect on NAADP production in some cell types), and there may be other routes which require different substrates and enzymes. [ 9 ]


The  SARM1  enzyme also catalyzes the formation of NAADP from NAD+. [ 10 ]


The first chemical synthesis of NAADP was achieved in 2004 using a chemoenzymatic approach: a total chemical synthesis of NADP and then conversion of this to NAADP enzymatically. [ 11 ]


Like any second messenger system, the signal must be terminated and there must be routes for NAADP removal but again, little is known with any degree of certainty. A 2'-3'-phosphatase stimulated by Ca 2+  has been proposed in brain [ 12 ]  and, possibly in pancreatic acinar cells, that catabolises NAADP to inactive NAAD.  CD38  has also been found to breakdown NAADP (to ADPRP — see inset). [ 10 ]  NAADP may also be reduced to NAADPH. [ 13 ]


Hardly surprisingly, the three major second messengers do not do the same thing and cannot always substitute for each other. The physiological consequences of Ca 2+  release by each messenger may be different i.e. NAADP couples to downstream responses that cannot be mimicked by  IP3  and  cADPR . For example, NAADP selectively stimulates neuronal differentiation, [ 14 ]  or exocytosis in cytotoxic T-cells. [ 15 ]


In contrast to  IP3  and  cyclic ADP-ribose  which predominantly mobilize Ca 2+  from the neutral and abundant  endoplasmic reticulum  (ER) store, NAADP selectively targets acidic Ca 2+  stores [ 16 ]  — usually less abundant than the ER but with a pivotal role that belies their size. This paradigm shift away from the ER derives from seminal studies, again in sea urchin egg, that showed NAADP-mediated Ca 2+  release was sensitive to agents that target acidic organelles (e.g.  bafilomycin A1 ) but was less sensitive to ones that interfere with ER Ca 2+  storage (e.g.  thapsigargin ). [ 16 ]


This is a blanket term that encompasses a spectrum of acidic vesicles that include  endosomes ,  lysosomes , and lysosome-related organelles and  secretory vesicles  and  acidocalcisomes . [ 17 ]  They are a highly dynamic continuum of vesicles with a rich variety of established biochemical roles in cells, to which Ca 2+  storage can now be added. Their luminal pH is one characteristic that distinguishes a given vesicle class from another: where endosomes are weakly acidic (pH 6-6.5), lysosomes are typically the most acidic (pH 4.5-5.0) and secretory vesicles are typically pH 5.5. Ca 2+  is seen to be increasingly important for endo-lysosomal function, e.g. trafficking and  autophagy . Aberrations in Ca 2+  signals can have pathophysiological consequences, including lysosomal storage diseases such as  Niemann–Pick disease, type C  and  Mucolipidosis IV . [ 18 ]


When NAADP mobilizes Ca 2+  from these stores, the pH of the stores concomitantly increases (becomes more alkaline), as testified by studies in sea urchin egg, [ 19 ]  mammalian heart and pancreas. Whether this has consequences for vesicle (or NAADP) function remains to be seen, but luminal pH is usually crucial for resident protein activity. [ citation needed ]


In other Ca 2+ -storing organelles such as the  endoplasmic reticulum  or  Golgi , stores are filled by  calcium ATPase  pumps, typified by the ubiquitous members of the  SERCA  or the SPCA (secretory pathway Ca 2+ -ATPase) families respectively. Ca 2+  uptake by acidic stores occurs via other proteins: in yeast and plants (the best understood systems) the acidic vacuoles host two uptake pathways: a high affinity Ca 2+ -ATPase and a low affinity Ca 2+ /H +  antiporter (or exchanger, generically denoted as CHX). The pumps are different from the SERCA family (and, importantly, are insensitive to their inhibitor,  thapsigargin ) whereas the exchanger exploit the H +  gradient to drive Ca 2+  uptake against its concentration gradient. The genes encoding these proteins are well-defined. [ citation needed ]


In higher organisms, the situation is less clear. Ca 2+  uptake usually occurs via a thapsigargin-insensitive pathway (therefore precluding SERCA involvement) and appears to be dependent upon the H +  gradient; whether this occurs via a single (unknown) CHX or via exchangers in series (e.g. Na + /H +  exchanger coupled to a Na + /Ca 2+  exchanger) is unproven. Acidic vesicles in some cell types may well take a leaf out of the yeasts'/plants' book and host two uptake pathways, but whether this is a widespread template is unclear. [ citation needed ]


In the absence of selective Ca 2+  uptake inhibitors (often because we do not even know the protein/route), it is common to  indirectly  inhibit Ca 2+  uptake by collapsing the thermodynamic drive (the H +  gradient). The H +  gradient can be eliminated either with H +  ionophores (protonophores) such as  nigericin  or  monensin  or by inhibiting the  V-ATPase  that generates the H +  gradient with compounds such as  bafilomycin A1  or  concanamycin . [ citation needed ]


Even from the early pioneering work in sea urchin egg, it was clear from the pharmacological profile that NAADP acted upon a different channel from the  IP3 receptor  and  ryanodine receptor  and this has recently been borne out by the molecular identification of the NAADP receptor as members of the TPC ( two-pore channel ) family. [ 20 ] [ 21 ]  As structural intermediates between single domain  TRP  and four-domain  voltage-dependent calcium channel , the TPCs form oligomers (possibly dimers) to form the functional Ca 2+  channel. [ 22 ]  Appropriately, these channels reside on acidic organelles (including different classes of  endosomes  and  lysosomes ) likely due to the presence of endolysomal targeting sequences. [ 23 ]


The effect of genetic manipulation of TPC levels (i.e. over-expression, knock-down or knock-out) is consistent with TPCs being the NAADP-gated channel. Moreover, TPCs recapitulate many of the characteristics of NAADP-induced Ca 2+  release i.e. they promote Ca 2+  release from acidic stores, correlate with NAADP-binding sites, exhibit a bell-shaped NAADP concentration-response curve, sensitivity to the NAADP antagonist, Ned-19, and provide trigger Ca 2+  that is subsequently amplified by ER Ca 2+  channels.


There are 3 genes that encode three isoforms of TPC1-3 that differ substantially from each other in their primary sequence (but these differences are preserved across species, such that human and sea urchin TPC1 are more closely related than are human TPC1 and human TPC2). Moreover, the TPC isoforms exhibit different organellar distributions, with TPC1 being found throughout the endo-lysosomal system (although predominantly in recycling and early endosomes) whereas TPC2 shows a more restricted late-endosomal/lysosomal localization. [ 24 ]


In spite of a burgeoning literature supporting TPCs as the NAADP-regulated channel, this was challenged in 2012/13 by reports that TPCs are, instead, Na +  channels regulated by the endo-lysosomal lipid,  Phosphatidylinositol 3,5-bisphosphate , PI(3,5)P 2 [ 25 ]  and also by metabolic state (via ATP and  mTOR ). [ 26 ]  This controversy ultimately evolved into a new model of how TPCs work.


The challenge raised two different, but interrelated issues: (a) TPCs are insensitive to NAADP; (b) TPCs are Na + - (and not Ca 2+ -) permeable.


The surprising conclusion in 2012 that TPCs are not involved in NAADP signalling was due to two technical difficulties that have since been overcome or explained.


(i) The transgenic mice (designed to knockout both TPC1 and TPC2; double-knockout, DKO) retained sensitivity to NAADP. However, others have questioned whether these mice are true DKO when they are predicted to retain >90% of the TPC protein sequences (i.e. they express only mildly truncated TPCs which are still functional and NAADP-sensitive  [ 27 ] ). In a different DKO mouse that is demonstrably TPC-null, NAADP responses are completely abolished, confirming TPCs are an NAADP target. [ 27 ]


(ii) The authors could not observe NAADP-stimulated currents in patched lysosomes. These technically challenging protocols  were overcome by others who have successfully observed NAADP-dependent lysosomal currents that are dependent on TPCs.


Several groups reinvestigated the permeability properties of TPCs and their role in NAADP-induced Ca 2+  release, and they agreed that TPCs are indeed permeable to Na +  but they could not necessarily recapitulate the Na +  selectivity shown in the 2012/13 studies. [ 28 ] [ 29 ] [ 30 ] [ 31 ]  It was therefore initially proposed that TPCs may conduct both Ca 2+  and Na +  (analogous to the  NMDA receptor  of the plasma membrane).


As more studies were published, why some groups observe a Na +  selectivity while others see a mixed Na + /Ca 2+  permeability was unclear until the important realization that the TPC2  ionic selectivity wholly depended on the activating ligand.  Currents activated by PI(3,5)P 2  were predominantly carried by Na +  whereas NAADP-activated currents showed an eight-fold increase in the Ca 2+  permeability. This conveniently explained the discrepancies between groups as well as revealing that TPC2 is extraordinarily plastic in operating in different conductance modalities.


Since then, it appears that TPC2 can be synergistically activated by co-application of NAADP and PI(3,5)P 2 , although the molecular mechanisms are unclear.


Therefore, TPC2 can operate as either a  Ca 2+  or Na +  channel, depending on whether NAADP or lipid activate it.


IP3  binds directly to its cognate  IP3 receptor  which is therefore a true ligand-gated ion channel. In contrast, NAADP does not appear to bind directly to TPCs but requires an intermediate unknown accessory protein(s). In sea urchin egg homogenate and T-cells, the binding protein(s) may be smaller than TPCs themselves, judging by photoaffinity labelling with [ 32 P]azido-NAADP. Therefore, the NAADP receptor was believed to be a multi-protein complex on acidic vesicles. [ 32 ] [ 33 ] [ 34 ]


In spite of a decade of graft using conventional biochemical purification, these proteins remained elusive. Recently, two different NAADP-binding proteins have finally been identified that are essential for TPC activation: LSm12 and JPT2.


The 20kDa Jupiter microtubule-associated homolog 2 ( JPT2 ) (also known as HN1L) was the first accessory protein to be published as a mediator of NAADP-dependent Ca 2+  release. [ 35 ] [ 36 ]  Both studies exploited the same novel 'clickable' NAADP photoprobe, but regardless of the different blood cell types, the authors converged on the same molecular partner (although the studies differ in the channel that is activated). JPT2 binds [ 32 P]NAADP with selectivity over a spectrum of different nucleotides. [ 36 ]  In Ca 2+ -release studies, NAADP-dependent signals were inhibited by siRNA knockdown of JPT2, but not of its homolog, JPT1, attesting to isoform specificity. Interestingly, JPT2 exhibited a preferential interaction with TPC1 over TPC2. [ 36 ]  Given that TPCs are known to drive pathogen uptake into cells, it was striking that uptake of a  Severe acute respiratory syndrome coronavirus 2  (SARS-CoV-2) pseudovirus was likewise reduced by JPT2 gene silencing.


Using entirely different affinity techniques, other workers unexpectedly identified a member of an RNA-binding protein  LSm  family (LSm12) as an NAADP-dependent accessory protein for TPCs. [ 37 ]  Composed of two domains (an N-terminus LSm domain and a C-terminus anticodon-binding [AD] domain), LSm12 mediates NAADP activation of TPCs via its LSm domain. [ 37 ]  This domain binds NAADP with appropriate nanomolar affinity and selectivity over NADP, and appears to be needed for activation of either TPC1 or TPC2 (contrasting with the isoform-selectivity of JPT2). Other LSm family members (5 and 11) were not required.


The fact that two entirely different proteins potentially converge on the activation of TPCs raises future questions of whether both NAADP-binding proteins are part of a common complex or pathway.


In addition to NAADP gating the channel, there is evidence that the luminal pH also affects TPC channel activity, either TPC1  [1]  or TPC2  [2] [3] . However, a clear consensus on the effect of pH has not been reached with some suggesting that acidic pH favours TPC1 or TPC2 opening, whereas others report that a more alkaline pH favours TPC2 opening. [ 38 ]


Furthermore, luminal Ca 2+  also promotes TPC1 and TPC2 opening (in the latter case, luminal Ca 2+  also sensitizes TPCs to NAADP (analogous to luminal Ca 2+  regulation of IP3Rs and RyRs), but this demands wider study across isoforms and species. [ citation needed ]  This is one way by which cross-talk can occur between acidic Ca 2+  stores and the ER i.e. Ca 2+  release from the ER can 'prime' acidic Ca 2+  stores and promote further NAADP-dependent Ca 2+  responses  [4] .


Early evidence was  against  the NAADP receptor being regulated by either cytosolic Ca 2+  or pH. [ 39 ]  Since then, Ca 2+  has been shown to stimulate human TPC1 on both its cytosolic and luminal faces. [ 40 ] [ 41 ]


Back in 2009, a selective cell-permeant NAADP antagonist, trans- Ned-19  was discovered [ 42 ]  which blocks Ca 2+  signals and downstream Ca 2+ -dependent processes such as differentiation. [ 43 ]  Prior to that, only high concentrations of blockers of L-type Ca 2+  channels (e.g.  diltiazem ,  dihydropyridines ) could be used (with obvious concerns over non-NAADP effects). [ 44 ]  A minor modification of Ned-19 produced another, more soluble antagonist, Ned-K. [ 45 ]


Although not true antagonism, the NAADP 'receptor' can self-inactivate when bound to non-releasing concentrations of NAADP itself. [ 46 ] [ 47 ]  Such inactivating pre-pulses of NAADP were the first strategy for implicating NAADP in subsequent physiological pathways. [ citation needed ]


NAADP is charged and cannot cross cell membranes. Therefore, an inactive, lipophilic ester precursor (NAADP/AM) has been synthesised which crosses membranes and rapidly regenerates NAADP in the cytosol following the action of endogenous esterases. [ 48 ]


Caged NAADP is an inactive, membrane-impermeant analog of NAADP that can be introduced into cells by microinjection or a patch pipette.  Flash photolysis  with a UV light source rapidly converts this into NAADP, allowing the experimenter to precisely manipulate NAADP levels in time and space. [ 49 ]


An indirect means of inhibiting NAADP action is to deplete its target Ca 2+  stores. As noted above, this usually entails collapsing the H +  gradient with either V-ATPase inhibitors (e.g.  Bafilomycin A1 ) or protonophores (e.g.  nigericin  or  monensin ). In platelets it has been suggested that SERCA3 inhibition with  tBHQ  can also abrogate NAADP-dependent signals. [ citation needed ]


The two paralogous enzymes- transmembrane  CD38  and GPI anchored  CD157 , that produce NAADP (and cADPR) in humans both have their active synthesis site in the ectodomain. Though this may involve vesicular synthesis but it has been shown that it is produced at the extracellular sites, and also can act when produced by a different cell or added artificially from outside. So the NAADP has to enter the cell either by diffusion or by transport. Considering the fact that the substrate of NAADP synthesis (NADP) itself is very sparse in the extracellular medium, a purse diffusion based mechanism has been suspected to be less likely than a transporter mediated path. This is compatible with recent data which indicate a carrier mediated transport partially blockable by  dipyridamole  and cold temperature. [ 50 ]


The ER and acidic Ca 2+  stores have similarities as well as key differences. Both transport Ca 2+  into their lumina where it is stored, and is subsequently released in response to stimuli by opening resident Ca 2+  channels. Indeed, the free [Ca 2+ ] of each is broadly similar (~0.5-1.0 mM). However, they differ in the cohort of transporters, their luminal pH and their total volume per cell. The total amount of Ca 2+  that is stored in each is a product of the volume and concentration; since the [Ca 2+ ] is the same for each, the total amount of releasable Ca 2+  is directly proportional to the organellar volume and therefore lysosomes can release only a small amount Ca 2+  of when compared to the ER.


This is important because the maximal Ca 2+  release from lysosomes is so small that it is frequently 'invisible' in global Ca 2+  recordings e.g. using cytosolic fluorescent reporters. In contrast, ER-derived Ca 2+  is globally substantial and the predominant intracellular signal visible in global recordings.


If lysosomal Ca 2+  release is so small, how then can it affect cellular physiology? The answer is that it can exert its effects in two different signalling modalities: local and global, as shall be described.


In bacterial infection, however, NAADP induction of lysosomal Ca 2+  efflux and  TFEB  activation leads to enhanced expression of  inflammatory   cytokines . [ 51 ]


Since the diffusion of Ca 2+  in the cell is spatially restricted, Ca 2+  released by a channel does not travel far from its source (the channel mouth) — model estimates are in the 50 nm range. Therefore, the small total amount of Ca 2+  released from lysosomal channels will form locally high [Ca 2+ ] domains around the cytosolic face of lysosomal Ca 2+  channels. By strategic placement of Ca 2+ -sensitive decoding proteins within these domains (e.g. in a complex with the channel), local Ca 2+  signals can stimulate Ca 2+ -dependent events — crucially, even in the absence of a global cytosolic Ca 2+  signal. This is the modality when lysosomes are acting on their own.


The NAADP/TPC axis has been reported to exhibit such signal compartmentation, such local Ca 2+  signalling, in different physiological settings. In other words, this local modality can explain why some processes are driven uniquely by NAADP/TPCs rather than other Ca 2+  signalling pathways. For example, NAADP/TPCs are unique drivers of cell killing by  Cytotoxic T cells . [ 52 ]  Similarly,  phagocytosis  via the  Fc receptor  in the  macrophage  is driven only by highly local Ca 2+  domains generated by NAADP/TPCs (whereas the global ER Ca 2+  signals play no role). [ 53 ]  This unique dependence is not restricted to immune cells, but is also observed in neurons during  long-term potentiation , [ 54 ]  and neuronal differentiation. [ 55 ] [ 56 ]   Angiogenesis  is another NAADP/TPC-dependent pathway. [ 57 ]


A different modality is when lysosomes do not just act alone, but in concert with the ER. In this scenario, lysosomes first supply a local, 'trigger' Ca 2+  release that then secondarily recruits the IP3Rs or RyRs on the ER by  Calcium-induced calcium release  (the 'amplifier'). In this mode, lysosomes indirectly evoke a global cytosolic Ca 2+  signal (which is actually mediated by the ER). In this way, lysosomal Ca 2+  release is amplified, in what is referred to as the 'trigger hypothesis'.


An  NTP binding site  is a type of  binding site  found in  nucleoside monophosphate  (NMP)  kinases , N can be  adenosine  or  guanosine . A  P-loop  is one of the  structural motifs  common for  nucleoside triphosphate  (NTP) binding sites, it interacts with the bound nucleotide's  phosphoryl  groups. For the binding site to be able to bind a nucleotide, the nucleotide must be complex bound to Mg 2+  or Mn 2+ .  Nucleotide  binding will cause conformational changes in the protein because the P-loop will bend.


NTP binding sites play a role in  poliovirus   RNA  replication. [ 1 ]   On the poliovirus RNA-dependent polymerase, also known as 3D pol , there are two binding sites. Both binding sites contain lysine residues; however, only the lysine at position 61 is essential for RNA sequence elongation as part of the replication process. [ 2 ]  Additionally, a method known as computational alanine mutagenesis, has been used to find the key portions within the amino acid sequences that majorly characterize a particular NTP binding site; these regions within the sequence are conserved. [ 3 ]


This  enzyme -related article is a  stub . You can help Wikipedia by  expanding it .


The  nucleic acid notation  currently in use was first formalized by the  International Union of Pure and Applied Chemistry  (IUPAC) in 1970. [ 1 ]  This universally accepted notation uses the Roman characters G, C, A, and T, to represent the four nucleotides commonly found in  deoxyribonucleic acids  (DNA).


Given the rapidly expanding role for genetic sequencing, synthesis, and analysis in biology, some researchers have developed alternate notations to further support the analysis and manipulation of genetic data. These notations generally exploit size, shape, and symmetry to accomplish these objectives.


Degenerate base symbols in  biochemistry  are an  IUPAC [ 2 ] [ 3 ]  representation for a position on a  DNA sequence  that can have multiple possible alternatives. These should not be confused with  non-canonical bases  because each particular sequence will have in fact one of the regular bases. These are used to encode the consensus sequence of a population of aligned sequences and are used for example in  phylogenetic analysis  to summarise into one multiple sequences or for  BLAST  searches, even though IUPAC degenerate symbols are masked (as they are not coded).


Under the commonly used IUPAC system,  nucleobases  are represented by the first letters of their chemical names: guanine, cytosine, adenine, and thymine. [ 1 ]  This shorthand also includes eleven "ambiguity" characters associated with every possible combination of the four DNA bases. [ 4 ]  The ambiguity characters were designed to encode positional variations in order to report  DNA sequencing  errors,  consensus sequences , or  single-nucleotide polymorphisms . The IUPAC notation, including ambiguity characters and suggested mnemonics, is shown in Table 1.


Despite its broad and nearly universal acceptance, the IUPAC system has a number of limitations, which stem from its reliance on the Roman alphabet. The poor legibility of upper-case Roman characters, which are generally used when displaying genetic data, may be chief among these limitations. The value of external projections in distinguishing letters has been well documented. [ 5 ]  However, these projections are absent from upper case letters, which in some cases are only distinguishable by subtle internal cues. Take for example the upper case C and G used to represent cytosine and guanine. These characters generally comprise half the characters in a genetic sequence but are differentiated by a small internal tick (depending on the typeface). Nevertheless, these Roman characters are available in the  ASCII  character set most commonly used in textual communications, which reinforces this system's ubiquity.


Another shortcoming of the IUPAC notation arises from the fact that its eleven ambiguity characters have been selected from the remaining characters of the Roman alphabet. The authors of the notation endeavored to select ambiguity characters with logical mnemonics. For example, S is used to represent the possibility of finding cytosine or guanine at genetic loci, both of which form  strong  cross-strand binding interactions. Conversely, the  weaker  interactions of thymine and adenine are represented by a W.  However, convenient mnemonics are not as readily available for the other ambiguity characters displayed in Table 1. This has made ambiguity characters difficult to use and may account for their limited application.


The positions of the  carbons  in the  ribose  sugar that forms the backbone of the nucleic acid chain are numbered, and are used to indicate the direction of nucleic acids (5'->3' versus 3'->5'). This is referred to as  directionality . [ 3 ]


Legibility issues associated with IUPAC-encoded genetic data have led biologists to consider alternative strategies for displaying genetic data. These creative approaches to visualizing DNA sequences have generally relied on the use of spatially distributed symbols and/or visually distinct shapes to encode lengthy nucleic acid sequences. Alternative notations for nucleotide sequences have been attempted, however general uptake has been low.  Several of these approaches are summarized below.


In 1986, Cowin et al. described a novel method for visualizing DNA sequence known as the Stave Projection. [ 6 ]  Their strategy was to encode nucleotides as circles on series of horizontal bars akin to notes on musical stave. As illustrated in Figure 1, each gap on the five-line staff corresponded to one of the four DNA bases. The spatial distribution of the circles made it far easier to distinguish individual bases and compare genetic sequences than IUPAC-encoded data.


The order of the bases (from top to bottom, G, A, T, C) is chosen so that the complementary strand can be read by turning the projection upside down.


Zimmerman et al. took a different approach to visualizing genetic data. [ 7 ]  Rather than relying on spatially distributed circles to highlight genetic features, they exploited four geometrically diverse symbols found in a standard computer font to distinguish the four bases. The authors developed a simple WordPerfect macro to translate IUPAC characters into the more visually distinct symbols.


With the growing availability of font editors, Jarvius and Landegren devised a novel set of genetic symbols, known as the DNA Skyline font, which uses increasingly taller blocks to represent the different DNA bases. [ 8 ]  While reminiscent of Cowin  et al .'s spatially distributed Stave Projection, the DNA Skyline font is easy to download and permits translation to and from the IUPAC notation by simply changing the font in most standard word processing applications.


Ambigrams  (symbols that convey different meaning when viewed in a different orientation) have been designed to mirror structural symmetries found in the DNA double helix. [ 9 ]  By assigning ambigraphic characters to complementary bases (i.e. guanine: b, cytosine: q, adenine: n, and thymine: u), it is possible to complement DNA sequences by simply rotating the text 180 degrees. [ 10 ]  An ambigraphic nucleic acid notation also makes it easy to identify genetic palindromes, such as endonuclease restriction sites, as sections of text that can be rotated 180 degrees without changing the sequence.


One example of an  ambigraphic  nucleic acid notation is AmbiScript, a rationally designed nucleic acid notations that combined many of the visual and functional features of its predecessors. [ 11 ]  Its notation also uses spatially offset characters to facilitate the visual review and analysis of genetic data. AmbiScript was also designed to indicate ambiguous nucleotide positions via compound symbols. This strategy aimed to offer a more intuitive solution to the use of ambiguity characters first proposed by the IUPAC. [ 4 ]  As with Jarvius and Landegren's DNA Skyline fonts, AmbiScript fonts can be downloaded and applied to IUPAC-encoded sequence data.


Watson and Crick base pairs are indicated by a "•" or a "-" or a "." (example: A•T, or poly(rC)•2poly(rC)).


Hoogsteen  triple helix  base pairs are indicated by a "*" or a ":" (example: C•G*G+, or T•A*T, or C•G*G, or T•A*A).


A  nucleoside triphosphate  is a  nucleoside  containing a  nitrogenous base  bound to a 5-carbon sugar (either  ribose  or  deoxyribose ), with three  phosphate groups  bound to the sugar. [ 1 ]  They are the molecular precursors of both  DNA  and  RNA , which are chains of  nucleotides  made through the processes of  DNA replication  and  transcription . [ 2 ]  Nucleoside triphosphates also serve as a source of energy for cellular reactions [ 3 ]  and are involved in signalling pathways. [ 4 ]


Nucleoside triphosphates cannot easily cross the cell membrane, so they are typically synthesized within the cell. [ 5 ]  Synthesis pathways differ depending on the specific nucleoside triphosphate being made, but given the many important roles of nucleoside triphosphates, synthesis is tightly regulated in all cases. [ 6 ]   Nucleoside analogues  may also be used to treat viral infections. [ 7 ]  For example,  azidothymidine  (AZT) is a nucleoside analogue used to prevent and treat  HIV/AIDS . [ 8 ]


The term  nucleoside  refers to a  nitrogenous base  linked to a 5-carbon sugar (either  ribose  or  deoxyribose ). [ 1 ]   Nucleotides  are nucleosides  covalently  linked to one or more  phosphate groups . [ 9 ]  To provide information about the number of phosphates, nucleotides may instead be referred to as nucleoside (mono, di, or tri) phosphates. [ 10 ]  Thus, nucleoside triphosphates are a type of nucleotide. [ 10 ]


Nucleotides are commonly abbreviated with 3 letters (4 or 5 in case of deoxy- or dideoxy-nucleotides). The first letter indicates the identity of the nitrogenous base (e.g., A for  adenine , G for  guanine ), the second letter indicates the number of phosphates (mono, di, tri), and the third letter is P, standing for phosphate. [ 11 ]  Nucleoside triphosphates that contain  ribose  as the sugar are conventionally abbreviated as NTPs, while nucleoside triphosphates containing  d eoxyribose  as the sugar are abbreviated as dNTPs. For example, dATP stands for deoxyribose adenosine triphosphate. NTPs are the building blocks of  RNA , and dNTPs are the building blocks of  DNA . [ 12 ]


The carbons of the sugar in a nucleoside triphosphate are numbered around the carbon ring starting from the original  carbonyl  of the sugar. Conventionally, the carbon numbers in a sugar are followed by the prime symbol (‘) to distinguish them from the carbons of the nitrogenous base. The nitrogenous base is linked to the 1’ carbon through a  glycosidic bond , and the phosphate groups are covalently linked to the 5’ carbon. [ 13 ]  The first phosphate group linked to the sugar is termed the α-phosphate, the second is the β-phosphate, and the third is the γ-phosphate; these are linked to one another by two  phosphoanhydride  bonds. [ 14 ]


The cellular processes of  DNA replication  and  transcription  involve DNA and RNA synthesis, respectively. DNA synthesis uses dNTPs as substrates, while RNA synthesis uses rNTPs as substrates. [ 2 ]  NTPs cannot be converted directly to dNTPs. DNA contains four different nitrogenous bases:  adenine ,  guanine ,  cytosine  and  thymine . RNA also contains adenine, guanine, and cytosine, but replaces thymine with  uracil . [ 15 ]  Thus, DNA synthesis requires dATP, dGTP, dCTP, and dTTP as substrates, while RNA synthesis requires ATP, GTP, CTP, and UTP.


Nucleic acid synthesis is catalyzed by either  DNA polymerase  or  RNA polymerase  for DNA and RNA synthesis respectively. [ 16 ]  These enzymes  covalently  link the free  -OH  group on the 3’ carbon of a growing chain of nucleotides to the α-phosphate on the 5’ carbon of the next (d)NTP, releasing the β- and γ-phosphate groups as  pyrophosphate  (PP i ). [ 17 ]  This results in a  phosphodiester  linkage between the two (d)NTPs. The release of PP i  provides the energy necessary for the reaction to occur. [ 17 ]  It is important to note that nucleic acid synthesis occurs exclusively in the  5’ to 3’ direction .


Given their importance in the cell, the synthesis and degradation of nucleoside triphosphates is under tight control. [ 6 ]  This section focuses on nucleoside triphosphate metabolism in humans, but the process is fairly conserved among species. [ 18 ]  Nucleoside triphosphates cannot be absorbed well, so all nucleoside triphosphates are typically made  de novo . [ 19 ]  The synthesis of ATP and GTP ( purines ) differs from the synthesis of CTP, TTP, and UTP ( pyrimidines ). Both purine and pyrimidine synthesis use  phosphoribosyl pyrophosphate  (PRPP) as a starting molecule. [ 20 ]


The conversion of NTPs to dNTPs can only be done in the diphosphate form. Typically a NTP has one phosphate removed to become a NDP, then is converted to a dNDP by an enzyme called  ribonucleotide reductase , then a phosphate is added back to give a dNTP. [ 21 ]


A nitrogenous base called  hypoxanthine  is assembled directly onto PRPP. [ 22 ]  This results in a nucleotide called  inosine monophosphate  (IMP). IMP is then converted to either a precursor to AMP or GMP. Once AMP or GMP are formed, they can be phosphorylated by ATP to their diphosphate and triphosphate forms. [ 23 ]


Purine synthesis is regulated by the  allosteric inhibition  of IMP formation by the adenine or guanine nucleotides. [ 24 ]  AMP and GMP also  competitively inhibit  the formation of their precursors from IMP. [ 25 ]


A nitrogenous base called  orotate  is synthesized independently of PRPP. [ 25 ]  After orotate is made it is covalently attached to PRPP. This results in a nucleotide called orotate monophosphate (OMP). [ 26 ]  OMP is converted to UMP, which can then be phosphorylated by ATP to UDP and UTP. UTP can then be converted to CTP by a  deamination  reaction. [ 27 ]  TTP is not a substrate for nucleic acid synthesis, so it is not synthesized in the cell. Instead, dTTP is made indirectly from either dUDP or dCDP after conversion to their respective deoxyribose forms. [ 20 ]


Pyrimidine synthesis is regulated by the allosteric inhibition of orotate synthesis by UDP and UTP. PRPP and ATP are also allosteric activators of orotate synthesis. [ 28 ]


Ribonucleotide reductase  (RNR) is the enzyme responsible for converting NTPs to dNTPs. Given that dNTPs are used in DNA replication, the activity of RNR is tightly regulated. [ 6 ]  It is important to note that RNR can only process NDPs, so NTPs are first dephosphorylated to NDPs before conversion to dNDPs. [ 29 ]  dNDPs are then typically re-phosphorylated. RNR has 2 subunits and 3 sites: the catalytic site, activity (A) site, and specificity (S) site. [ 29 ]  The catalytic site is where the NDP to dNDP reaction takes place, the activity site determines whether or not the enzyme is active, and the specificity site determines which reaction takes place in the catalytic site.


The activity site can bind either ATP or dATP. [ 30 ]  When bound to ATP, RNR is active. When ATP or dATP is bound to the S site, RNR will catalyze synthesis of dCDP and dUDP from CDP and UDP. dCDP and dUDP can go on to indirectly make dTTP. dTTP bound to the S site will catalyze synthesis of dGDP from GDP, and binding of dGDP to the S site will promote synthesis of dADP from ADP. [ 31 ]  dADP is then phosphorylated to give dATP, which can bind to the A site and turn RNR off. [ 30 ]


ATP  is the primary energy currency of the cell. [ 32 ]  Despite being synthesized through the metabolic pathway described above, it is primarily synthesized during both  cellular respiration [ 33 ]  and  photosynthesis [ 34 ]  by  ATP synthase . ATP synthase couples the synthesis of ATP from ADP and phosphate with an  electrochemical gradient  generated by the pumping of protons through either the  inner mitochondrial membrane  (cellular respiration) or the  thylakoid membrane  (photosynthesis). [ 35 ]  This electrochemical gradient is necessary because the formation of ATP is  energetically unfavourable .


The  hydrolysis of ATP  to ADP and P i  proceeds as follows: [ 36 ]


This reaction is  energetically favourable  and releases 30.5 kJ/mol of energy. [ 3 ]  In the cell, this reaction is often coupled with unfavourable reactions to provide the energy for them to proceed. [ 37 ]   GTP  is occasionally used for energy-coupling in a similar manner. [ 38 ]


GTP is essential for  signal transduction , especially with  G proteins . G proteins are coupled with a cell membrane bound receptor. [ 4 ]  This whole complex is called a  G protein-coupled receptor  (GPCR). G proteins can bind either GDP or GTP. When bound to GDP, G proteins are inactive. When a  ligand  binds a GPCR, an  allosteric  change in the G protein is triggered, causing GDP to leave and be replaced by GTP. [ 39 ]  GTP activates the alpha subunit of the G protein, causing it to dissociate from the G protein and act as a downstream effector. [ 39 ]


Nucleoside analogues  can be used to treat  viral infections . [ 40 ]  Nucleoside analogues are nucleosides that are structurally similar (analogous) to the nucleosides used in DNA and RNA synthesis. [ 41 ]  Once these nucleoside analogues enter a cell, they can become  phosphorylated  by a viral enzyme. The resulting nucleotides are similar enough to the nucleotides used in DNA or RNA synthesis to be incorporated into growing DNA or RNA strands, but they do not have an available 3' OH group to attach the next nucleotide, causing  chain termination . [ 42 ]  This can be exploited for therapeutic uses in viral infections because viral DNA polymerase recognizes certain nucleotide analogues more readily than eukaryotic DNA polymerase. [ 40 ]  For example,  azidothymidine  is used in the treatment of  HIV/AIDS . [ 8 ]  Some less selective nucleoside analogues can be used as  chemotherapy  agents to treat cancer, [ 43 ]  such as  cytosine arabinose  (ara-C) in the treatment of certain forms of  leukemia . [ 7 ]


Resistance to nucleoside analogues is common, and is frequently due to a mutation in the enzyme that phosphorylates the nucleoside after entry into the cell. [ 7 ]  This is common in nucleoside analogues used to treat HIV/AIDS. [ 44 ]


A  salvage pathway  is a  pathway  in which a biological product is produced from intermediates in the degradative pathway of its own or a similar substance. The term often refers to  nucleotide salvage  in particular, in which  nucleotides  ( purine  and  pyrimidine ) are synthesized from intermediates in their degradative pathway.


Nucleotide salvage pathways are used to recover  bases  and  nucleosides  that are formed during  degradation  of  RNA  and  DNA . This is important in some organs because some tissues cannot undergo  de novo synthesis . The salvaged products can then be converted back into nucleotides. Salvage pathways are targets for  drug development , one family being called  antifolates . [ 1 ]


A number of other biologically-important substances, like  methionine  and  nicotinate , have their own salvage pathways to recycle parts of the molecule.


The nucleotide salvage pathway requires distinct substrates:


Uridine phosphorylase  or  pyrimidine-nucleoside phosphorylase  substitutes the anomeric-carbon-bonded  phosphate of ribose 1-phosphate for the free base  uracil , forming the nucleoside  uridine .  Uridine kinase  (aka  uridine–cytidine kinase ) can then  phosphorylate  the 5’-carbon of this nucleoside into  uridine monophosphate  (UMP). UMP/CMP kinase ( EC   2.7.4.14 ) can phosphorylate UMP into  uridine diphosphate , which  nucleoside diphosphate kinase  can phosphorylate into  uridine triphosphate .


Thymidine phosphorylase  or pyrimidine-nucleoside phosphorylase adds 2-deoxy-alpha-D-ribose 1-phosphate to  thymine , with thymine bonding at the anomeric carbon of the deoxyribose, forming the deoxynucleoside  thymidine .  Thymidine kinase  can then phosphorylate the 5’-carbon of this compound into  thymidine monophosphate  (TMP).  Thymidylate kinase  can phosphorylate TMP into  thymidine diphosphate , which nucleoside diphosphate kinase can phosphorylate into  thymidine triphosphate .


The nucleosides  cytidine  and  deoxycytidine  can be salvaged along the uracil pathway by  cytidine deaminase , which converts them to uridine and  deoxyuridine , respectively. Alternatively, uridine–cytidine kinase can phosphorylate them into  cytidine monophosphate  (CMP) or  deoxycytidine monophosphate  (dCMP). UMP/CMP kinase can phosphorylate (d)CMP into  cytidine diphosphate  or  deoxycytidine diphosphate , which nucleoside diphosphate kinase can phosphorylate into  cytidine triphosphate  or  deoxycytidine triphosphate .


Phosphoribosyltransferases add activated ribose-5-phosphate ( Phosphoribosyl pyrophosphate , PRPP) to bases, creating nucleoside monophosphates. There are two types of phosphoribosyltransferases: adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). HGPRT is an important enzyme in Purine pathway metabolism and [ 2 ]  its deficiency is implicated in  Lesch–Nyhan syndrome .


Tetrahydrofolic acid  and its derivatives are produced by salvage pathways from GTP. [ 1 ]


L-methionine salvage  is the pathway that regenerates  methionine  from its downstream products. A version of the pathway uses  methylthioadenosine  (MTA), forming the so-called MTA cycle with its synthesizing reaction. This sulphur-recycling action is found in humans, and seems to be universal among aerobic life. [ 3 ] [ 4 ]


Nicotinate salvage  is the process of regenerating  nicotinamide adenine dinucleotide  from  nicotinic acid . This pathway is important for controlling the level of  oxidative stress  in cells. The human gene  NAPRT  encodes the main enzyme in the pathway. [ 5 ]  Cancer cells, which have increased NAD requirements, tend to upregulate the pathway. [ 6 ]


Salvage pathways also exist for  ceramide ,  cobalamin ,  cell wall  components, and  tetrahydrobiopterin  in various organisms.


Nucleotide sugars  are the activated forms of  monosaccharides .  Nucleotide  sugars act as glycosyl donors in  glycosylation  reactions. Those reactions are catalyzed by a group of enzymes called  glycosyltransferases .


The anabolism of oligosaccharides - and, hence, the role of nucleotide sugars - was not clear until the 1950s when  Leloir  and his coworkers found that the key enzymes in this process are the glycosyltransferases.  These enzymes transfer a glycosyl group from a sugar nucleotide to an acceptor. [ 1 ]


To act as glycosyl donors, those monosaccharides should exist in a highly energetic form. This occurs as a result of a reaction between nucleoside triphosphate (NTP) and glycosyl monophosphate (phosphate at  anomeric carbon ). The recent discovery of the reversibility of many  glycosyltransferase -catalyzed reactions calls into question the designation of sugar nucleotides as 'activated' donors. [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ]


There are nine sugar nucleotides in humans which act as glycosyl donors and they can be classified depending on the type of the nucleoside forming them: [ 7 ]


In other forms of life many other sugars are used and various donors are utilized for them. All five of the common nucleosides are used as a base for a nucleotide sugar donor somewhere in nature.  As examples,  CDP-glucose  and  TDP-glucose   give rise to various other forms of CDP and TDP-sugar donor nucleotides. [ 9 ] [ 10 ]


Listed below are the structures of some nucleotide sugars (one example from each type).


Normal metabolism of nucleotide sugars is very important. Any malfunction in any contributing enzyme will lead to a certain disease  [ 11 ]  for example:


The development of chemoenzymatic strategies to generate large libraries of non-native sugar nucleotides has enabled a process referred to as  glycorandomization  where these sugar nucleotide libraries serve as donors for permissive  glycosyltransferases  to afford differential glycosylation of a wide range of  pharmaceuticals  and complex  natural product -based leads. [ 12 ] [ 13 ]


Orotidine 5'-monophosphate  ( OMP ), also known as  orotidylic acid , is a  pyrimidine   nucleotide [ 1 ]  which is the last intermediate in the biosynthesis of  uridine monophosphate . [ 2 ]  OMP is formed from  orotate  and  phosphoribosyl pyrophosphate  by the enzyme  orotate phosphoribosyltransferase .


In humans, the enzyme  UMP synthase  converts OMP into  uridine 5'- monophosphate . [ 2 ]  If UMP synthase is defective,  orotic aciduria  can result. [ 2 ]


3′-Phosphoadenosine-5′-phosphosulfate  ( PAPS ) is a derivative of  adenosine monophosphate  (AMP) that is  phosphorylated  at the 3′ position and has a  sulfate  group attached to the 5′  phosphate .  It is the most common  coenzyme  in  sulfotransferase  reactions and hence part of  sulfation pathways . [ 1 ]  It is endogenously synthesized by organisms via the phosphorylation of adenosine 5′-phosphosulfate (APS), an intermediary metabolite. [ 2 ]  In humans such reaction is performed by bifunctional 3′-phosphoadenosine 5′-phosphosulfate synthases ( PAPSS1  and  PAPSS2 ) using  ATP  as the phosphate donor. [ 3 ] [ 4 ]


APS and PAPS are intermediates in the reduction of sulfate to  sulfite , an exothermic conversion that is carried out by  sulfate-reducing bacteria . In these organisms, sulfate serves as an electron acceptor, akin to the use of O 2  as an electron acceptor by aerobic organisms. Sulfate is not reduced directly but must be activated by the formation of APS or PAPS. These carriers of activated sulfate are produced by reaction with ATP.  The first reaction is catalysed by  ATP sulfurylase :


The conversion of APS to PAPS is catalysed by  APS kinase :


Reduction of APS leads to sulfite, which is further reduced to  hydrogen sulfide , which is excreted.  This process is called dissimilatory sulfate reduction.  Reduction of PAPS, a more elaborated  sulfate ester , leads also to hydrogen sulfide.  But in this case, the product is used in biosynthesis, e.g. for the production of  cysteine .  The latter process is called assimilatory sulfate reduction because the sulfate sulfur is assimilated. [ 5 ]


5′-Phosphoribosyl-4-carboxy-5-aminoimidazole  (or  CAIR ) is an  intermediate  in the formation of  purines .


It is formed by  phosphoribosylaminoimidazole carboxylase .


Phosphoribosyl- N -formylglycineamide  (or  FormylGlycinAmideRibotide , FGAR) 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 ] [ 2 ]  The vitamins  thiamine [ 3 ]  and  cobalamin [ 4 ]  also contain fragments derived from FGAR. [ 5 ]


FGAR is formed when the enzyme  phosphoribosylglycinamide formyltransferase  adds a  formyl  group from  10-formyltetrahydrofolate  to  glycineamide ribonucleotide  (GAR) in reaction  EC   2.1.2.2 : [ 6 ]


The biosynthesis pathway next converts FGAR to an  amidine  by the action of  phosphoribosylformylglycinamidine synthase  ( EC   6.3.5.3 ), transferring an amino group from glutamine and giving  5'-phosphoribosylformylglycinamidine  (FGAM) in a reaction that also requires ATP: [ 6 ]


Phosphoribosylaminoimidazolesuccinocarboxamide  ( SAICAR ) is an intermediate in the formation of  purines . The conversion of ATP, L- aspartate , and  5-aminoimidazole-4-carboxyribonucleotide  (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide, ADP, and  phosphate  by  phosphoribosylaminoimidazolesuccinocarboxamide synthetase  ( SAICAR synthetase ) represents the eighth step of de novo  purine nucleotide  biosynthesis. [ 1 ]


A  phosphorimidazolide  is a chemical compound in which a  phosphoryl  mono-ester is  covalently  bound to a nitrogen atom in an  imidazole  ring. They are a type of  phosphoramidate . These  phosphorus  (V) compounds are encountered as reagents used for making new  phosphoanhydride  bonds with  phosphate  mono-esters, and as reactive intermediates in phosphoryl transfer reactions in some enzyme-catalyzed transformations. They are also being studied as critical chemical intermediates for the polymerization of nucleotides in pre-biotic settings. [ 1 ]  They are sometimes referred to as phosphorimidazolidates, imidazole-activated phosphoryl groups, and P-imidazolides.


Phosphorimidazolides have been investigated for their mechanistic role in  abiogenesis  (the natural process by which life arose from non-living matter). Specifically, they have been proposed as the active  electrophilic  species which may have mediated the formation of inter-nucleotide  phosphodiester bonds , thereby enabling template-directed oligonucleotide replication before the advent of enzymes. Phosphorimidazolides were originally proposed as mediators of this process by  Leslie Orgel  in 1968. [ 2 ]  Early studies showed that divalent metal cations such as Mg 2+ , Zn 2+ , and Pb 2+  and a complementary template were required for the formation of short oligonucleotides, although nucleotides exhibited 5'-2' connectivity instead of 5'-3' connectivity of present-day life forms. It was also shown that Montmorillonite clay could provide a surface for phosphorimidazolide-mediated oligonucleotide formation with lengths of 20-50 bases. [ 3 ]


The research group of  Jack W. Szostak  has continued to investigate the role of phosphorimidazolides in pre-biotic nucleotide polymerization. The group has investigated a number of imidazole derivatives in the search for chemical moieties which provide longer oligonucleotides necessary for propagating genetic information. [ 1 ]  Significantly, they discovered that phosphorimidazolides promote template-directed oligonucleotide formation via imidazolium-bridged dinucleotide intermediates. [ 4 ]


John D. Sutherland and colleagues have proposed that phosphorimidazolides may have formed in the chemical environment of early Earth via the activation of ribonucleotide phosphates by  methyl isocyanaide  and  acetaldehyde  followed by substitution with imidazole. [ 5 ]


While early studies of phosphorimidazolide derivatives of nucleotides found that oligonucleotides could form in the presence of a complementary template, pyrophosphate-linked dimers formed predominantly in the absence of a template. [ 2 ]  This proclivity for forming new phosphoanhydride bonds has been used in the synthesis of several pyrophosphate-containing organic compounds. A variety of modified nucleotide triphosphates were synthesized using a cyanoethyl-protected phosphorimidazolide reagent. [ 6 ]  Phosphoanhydride bond forming reactions were found to proceed most rapidly in amide-based organic solvents such as  N,N -dimethylformamide  and particularly in  N,N -dimethylacetamide  with Mg 2+  or Zn 2+  catalysts. [ 7 ]


Phosphorimidazolide reagents have been synthesized from phosphate mono-esters.


In one method, a phosphate mono-ester is dissolved in anhydrous pyridine [ 8 ]  or  N,N -dimethylformamide  (DMF) and activated using  triphenylphosphine  (PPh 3 ) and 2,2’-Dithiodipyridine (2,2’-DTDP) in the presence of  triethylamine  (TEA) base and excess imidazole. In another method using fewer reagents, a phosphate mono-ester is dissolved in DMF and  carbonyldiimidazole  (CDI) is used to both remove an oxygen atom from the phosphate group and supply the imidazole substituent. The product of either reaction may be collected by precipitation using  acetonitrile  or  acetone  as antisolvent with sodium or lithium  perchlorate  to supply the sodium or lithium salt of the phosphorimidazolide respectively. Alternatively, the phosphorimidazolide may be isolated by reverse-phase flash column chromatography with TEAB buffer and acetonitrile. [ 9 ]


In  molecular biology , a  polynucleotide   (from  Ancient Greek     πολυς  (polys)  'many') is a  biopolymer  composed of  nucleotide   monomers  that are  covalently bonded  in a chain. [ 1 ]   DNA  (deoxyribonucleic acid) and  RNA  (ribonucleic acid) are examples of polynucleotides with distinct biological functions. DNA consists of two chains of polynucleotides, with each chain in the form of a  helix  (like a spiral staircase). [ citation needed ]


Although DNA and RNA do not generally occur in the same polynucleotide, the four species of nucleotides may occur in any order in the chain. The sequence of DNA or RNA species for a given polynucleotide is the main factor determining its function  in a living  organism  or a scientific  experiment . [ citation needed ]


Polynucleotides occur naturally in all living organisms. The  genome  of an organism consists of complementary pairs of enormously long polynucleotides wound around each other in the form of a  double helix . Polynucleotides have a variety of other roles in organisms. [ citation needed ]


Polynucleotides are used in biochemical experiments such as  polymerase chain reaction  (PCR)  or DNA sequencing. Polynucleotides are made artificially from  oligonucleotides , smaller nucleotide chains with generally fewer than 30 subunits. A  polymerase   enzyme  is used to extend the chain by adding nucleotides according to a pattern specified by the scientist. [ citation needed ]


In order to understand how  life  arose, knowledge is required of the chemical pathways that permit formation of the key building blocks of life under plausible  prebiotic conditions .  According to the  RNA world  hypothesis free-floating  ribonucleotides  were present in the primitive soup.  These were the fundamental molecules that combined in series to form  RNA .  Molecules as complex as 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 natural selection and  evolution . Nam et al. [ 2 ]  demonstrated the direct condensation of nucleobases with ribose to give ribonucleosides in aqueous microdroplets, a key step leading to RNA formation. Similar results were obtained using wet-dry cycles by Becker et al. [ 3 ]


Fish derived polynucleotide is used in aesthetic medicine as a dermal filler. [ 1 ]  It has also been found effective in alleviating knee osteoarthritic pain. [ 2 ] [ failed verification ]


The  Pseudo K-tuple nucleotide composition  or  PseKNC , is a method for converting a  nucleotide sequence  (DNA or RNA) into a numerical vector so as to be used in pattern recognition techniques. Generally, the K-tuple can refer to a dinucleotide (when K=2) or a trinucleotide (when K=3). Depending on the instance, the technique can also be called  PseDNC  or  PseTNC . [ 1 ]


The method was derived from an analogous method in proteomics known as  PseAAC  (Pseudo Amino Acid Composition) that is applied to  protein sequence s. [ 2 ]


PseKNC was derived from an analogous method in proteomics known as  PseAAC  (Pseudo Amino Acid Composition). [ 2 ]  Previously, investigations either relied on sequential models for making predictions of certain protein properties (which, in its simplest case, just refers to the amino acid composition of the protein), or a discrete model which represents a vector of twenty elements, each of which represent the frequency of each amino acid in the protein sample. The discrete model, however, fails to account for sequence-order information. The PseACC model extends the 20-length vector in the discrete model with λ components, each of which in some way captures sequence-order information, and this vector becomes the basis for making predictions. [ 3 ]


Analogously, a discrete model of a nucleotide sequence based on its dinucleotide composition would lay involve a vector of 16 elements, the value of which one representing the frequency of each dinucleotide in the sequence: [ 1 ]


D

 =

 [

 f
 (
 A
 A
 )
 f
 (
 A
 C
 )
 ⋯
 f
 (
 T
 T
 )

 ]

 T

 {\displaystyle \mathbf {D} ={\begin{bmatrix}f(AA)f(AC)\cdots f(TT)\end{bmatrix}}^{\mathbf {T} }}


Where  D  is the DNA sequence, T is the transpose operator, and f(AA) is the normalized occurrence frequency of AA in the DNA sequence. A trinucleotide representation can be denoted as: [ 1 ]


D

 =

 [

 f
 (
 A
 A
 A
 )
 f
 (
 A
 A
 C
 )
 ⋯
 f
 (
 T
 T
 T
 )

 ]

 T

 {\displaystyle \mathbf {D} ={\begin{bmatrix}f(AAA)f(AAC)\cdots f(TTT)\end{bmatrix}}^{\mathbf {T} }}


As can be seen, these discrete models fail to consider any global or long-range sequence-order information. To address this for both DNA and RNA sequences, the pseudo K-tuple  nucleotide  composition or PseKNC was proposed. [ 4 ] [ 5 ] [ 6 ]


PseKNC extends the discrete model by adding λ components to represent sequence-order and physico-chemical properties of the nucleotide sequence. The original KNC model will involve 4 K  components. In a dinucleotide situation where K = 2, 4 2  = 16 components will be included. The extension by PseKNC results in (4 K  + λ) components. [ 1 ]


A wide diversity of applications have been developed with respect to the PseKNC method. [ 7 ]  For example, it has become an integral component of many algorithms designed to predict the locations of recombination hotspots and coldspots from sequence information. [ 8 ] [ 9 ]


For the convenience scientific community, a freely available web server called PseKNC [ 4 ]  and an open source package called PseKNC-General [ 5 ]  were developed in 2013 and 2014, respectively, that could convert large-scale sequence datasets to pseudo nucleotide compositions with numerous choices of physicochemical property combinations. PseKNC-General can generate several modes of pseudo nucleotide compositions, including conventional k-tuple nucleotide compositions, Moreau–Broto autocorrelation coefficient, Moran autocorrelation coefficient, Geary autocorrelation coefficient, Type I PseKNC and Type II PseKNC.


Another web server, Pse-in-One, allows users to hand-select all pre-existing PseAAC and PseKNC methods for protein, RNA, and DNA sequences, along with any selection of the existing availability of physicochemical property combinations for these options. [ 10 ]


In  chemistry ,  pyrophosphates  are  phosphorus   oxyanions  that contain two phosphorus atoms in a  P−O−P  linkage. A number of pyrophosphate salts exist, such as  disodium pyrophosphate  ( Na 2 H 2 P 2 O 7 ) and  tetrasodium pyrophosphate  ( Na 4 P 2 O 7 ), among others. Often pyrophosphates are called  diphosphates . The parent pyrophosphates are derived from partial or complete neutralization of  pyrophosphoric acid . The  pyrophosphate bond  is also sometimes referred to as a phosphoanhydride bond, a naming convention which emphasizes the loss of water that occurs when two phosphates form a new  P−O−P  bond, and which mirrors the nomenclature for  anhydrides of carboxylic acids . Pyrophosphates are found in  ATP  and other  nucleotide  triphosphates, which are important in biochemistry. The term pyrophosphate is also the name of  esters  formed by the condensation of a phosphorylated biological compound with  inorganic phosphate , as for  dimethylallyl pyrophosphate . This bond is also referred to as a  high-energy phosphate  bond.


Pyrophosphoric acid is a tetraprotic acid, with four distinct  p K a 's: [ 1 ]


The pKa's occur in two distinct ranges because deprotonations occur on separate phosphate groups. For comparison with the p K a 's for  phosphoric acid  are 2.14, 7.20, and 12.37.


At physiological  pH 's, pyrophosphate exists as a mixture of doubly and singly protonated forms.


Disodium pyrophosphate is prepared by thermal condensation of  sodium dihydrogen phosphate  or by partial deprotonation of pyrophosphoric acid. [ 2 ]


Pyrophosphates are generally white or colorless. The  alkali metal  salts are water-soluble. [ 3 ]  They are good complexing agents for metal ions (such as calcium and many transition metals) and have many uses in industrial chemistry. Pyrophosphate is the first member of an entire series of  polyphosphates . [ 4 ]


The anion  P 2 O 4− 7  is abbreviated  PP i , standing for  i norganic  p yro p hosphate . It is formed by the  hydrolysis  of  ATP  into  AMP  in  cells .


For example, when a nucleotide is incorporated into a growing  DNA  or  RNA  strand by a  polymerase , pyrophosphate (PP i ) is released. Pyrophosphorolysis is the reverse of the  polymerization  reaction in which pyrophosphate reacts with the 3′-nucleosidemonophosphate ( NMP  or dNMP), which is removed from the  oligonucleotide  to release the corresponding triphosphate ( dNTP  from DNA, or  NTP  from RNA).


The pyrophosphate anion has the structure  P 2 O 4− 7 , and is an  acid anhydride  of  phosphate . It is unstable in  aqueous solution  and  hydrolyzes  into inorganic phosphate:


or in biologists' shorthand notation:


In the absence of enzymic catalysis, hydrolysis reactions of simple polyphosphates such as pyrophosphate, linear triphosphate,  ADP , and ATP normally proceed extremely slowly in all but highly acidic media. [ 5 ]


(The reverse of this reaction is a method of preparing pyrophosphates by heating phosphates.)


This hydrolysis to inorganic phosphate effectively renders the cleavage of ATP to AMP and PP i   irreversible , and biochemical reactions coupled to this hydrolysis are irreversible as well.


PP i  occurs in  synovial fluid ,  blood plasma , and  urine  at levels sufficient to block  calcification  and may be a natural inhibitor of  hydroxyapatite  formation in  extracellular fluid  (ECF). [ 6 ]  Cells may channel intracellular PP i  into ECF. [ 7 ]   ANK  is a nonenzymatic plasma-membrane PP i  channel that supports extracellular PP i  levels. [ 7 ]  Defective function of the membrane PP i  channel ANK is associated with low extracellular PP i  and elevated intracellular PP i . [ 6 ]   Ectonucleotide pyrophosphatase/phosphodiesterase  (ENPP) may function to raise extracellular PP i . [ 7 ]


From the standpoint of  high energy phosphate  accounting, the hydrolysis of ATP to AMP and PP i  requires two high-energy phosphates, as to reconstitute AMP into ATP requires two  phosphorylation  reactions.


The plasma concentration of inorganic pyrophosphate has a reference range of 0.58–3.78  μM  (95% prediction interval). [ 8 ]


Isopentenyl pyrophosphate  converts to  geranyl pyrophosphate , the precursor to tens of thousands of  terpeness  and  terpenoids . [ 9 ] [ 10 ]


Various diphosphates are used as  emulsifiers ,  stabilisers ,  acidity regulators ,  raising agents ,  sequestrants , and  water retention agents  in food processing. [ 11 ]  They are classified in the  E number  scheme under E450: [ 12 ]


In particular, various formulations of diphosphates are used to stabilize  whipped cream . [ 13 ]


Remdesivir , sold under the brand name  Veklury , [ 13 ] [ 15 ]  is a  broad-spectrum antiviral medication  developed by the  biopharmaceutical  company  Gilead Sciences . [ 16 ]  It is administered via  injection into a vein . [ 17 ] [ 18 ]  During the  COVID‑19 pandemic , remdesivir was approved or authorized for emergency use to treat  COVID‑19  in numerous countries. [ 19 ]


Remdesivir was originally developed to treat  hepatitis C , [ 20 ]  and was subsequently investigated for  Ebola virus disease  and  Marburg virus  infections [ 21 ]  before being studied as a post-infection treatment for COVID‑19. [ 22 ]


Remdesivir is a  prodrug  that is intended to allow intracellular delivery of  GS-441524  monophosphate and subsequent biotransformation into  GS-441524   triphosphate , a ribo nucleotide analogue  inhibitor of viral  RNA polymerase . [ 23 ]


The most common side effect in healthy volunteers is raised blood levels of  liver enzymes . [ 13 ]  The most common side effect in people with COVID‑19 is  nausea . [ 13 ]  Side effects may include liver inflammation and an  infusion -related reaction with nausea,  low blood pressure , and sweating. [ 24 ]


The US  Food and Drug Administration  (FDA) considers it to be a  first-in-class medication . [ 25 ]


In the European Union, remdesivir is  indicated  for the treatment of COVID‑19 in adults and adolescents (aged twelve years and older with body weight at least 40 kilograms (88 lb)) with  pneumonia  requiring supplemental oxygen and for adults who do not require supplemental oxygen and who are at increased risk of progressing to severe COVID‑19. [ 10 ] [ 13 ]


In the United States, remdesivir is indicated for the treatment of COVID‑19 in people 28 days of age and older and weighing at least 3 kilograms (6.6 lb) who are hospitalized; or not hospitalized and have mild-to-moderate COVID‑19, and are at high risk for progression to severe COVID‑19, including hospitalization or death. [ 12 ] [ 26 ]


In November 2020, the FDA issued an emergency use authorization (EUA) for the combination of  baricitinib  with remdesivir, for the treatment of suspected or laboratory confirmed COVID‑19 in hospitalized people two years of age or older requiring supplemental oxygen, invasive mechanical ventilation, or  extracorporeal membrane oxygenation  (ECMO). [ 27 ]


In Australia, it is approved for those aged four weeks of age and older with a body weight at least 3 kilograms (6.6 lb) with  pneumonia  requiring supplemental oxygen or those aged four weeks of age and older with body weight at least 40 kilograms (88 lb) who do not require supplemental oxygen and who are at high risk of progressing to severe COVID‑19. [ 28 ] [ 2 ]


In 2024, a retrospective study found treatment with the antiviral remdesivir plus dexamethasone was associated with fewer deaths in hospitalized COVID-19 patients compared to dexamethasone alone. [ 29 ]  The combination led to a 26% reduction in mortality at 14 days and a 24% reduction at 28 days. [ 30 ]


The most common adverse effects in people treated with remdesivir were  respiratory failure  and blood  biomarkers  of  organ  impairment, including  low albumin ,  low potassium ,  low count of red blood cells ,  low count of thrombocytes , and  elevated bilirubin (jaundice) . [ 31 ]  Other reported adverse effects include  gastrointestinal distress , elevated  transaminase  levels in the blood ( liver enzymes ),  infusion  site reactions, and  electrocardiogram abnormalities . [ 18 ]  Remdesivir may cause  infusion-related  reactions, including low blood pressure, nausea, vomiting, sweating or shivering. [ 32 ]


Other possible side effects of remdesivir include:


Remdesivir is a  protide  ( prodrug  of nucleotide) able to diffuse into cells, where it is converted to  GS-441524  monophosphate via the actions of  esterases  ( CES1  and  CTSA ) and a  phosphoamidase  ( HINT1 ); this in turn is further phosphorylated to its  active metabolite  triphosphate by  nucleoside-phosphate kinases . [ 34 ] [ 35 ]  This pathway of bioactivation is meant to occur  intracellularly , but a substantial amount of remdesivir is prematurely  hydrolyzed  in plasma, with GS-441524 being the major metabolite in plasma, and the only metabolite remaining two hours after dosing. [ 23 ]


As an  adenosine  nucleoside triphosphate analog (GS-443902), [ 36 ]  the  active metabolite  of remdesivir interferes with the action of viral  RNA-dependent RNA polymerase  and evades  proofreading  by viral  exoribonuclease  (ExoN), causing a decrease in viral RNA production. [ 16 ] [ 37 ]  In some viruses, such as the  respiratory syncytial virus , it causes the RNA-dependent RNA polymerases to pause, but its predominant effect (as in Ebola) is to induce an irreversible  chain termination . Unlike with many other chain terminators, this is not mediated by preventing addition of the immediately subsequent nucleotide, but is instead delayed, occurring after five additional bases have been added to the growing RNA chain. [ 38 ]  For the RNA-dependent RNA polymerases of  MERS-CoV ,  SARS-CoV-1 , and  SARS-CoV-2 , arrest of RNA synthesis occurs after incorporation of three additional nucleotides. [ 39 ] [ 35 ]  Hence, remdesivir is classified as a direct-acting antiviral agent that works as a delayed chain terminator. [ 33 ] [ 35 ]


In non-human primates, the plasma  half-life  of the prodrug is 20 minutes, with the main metabolite being the  nucleoside ,  GS-441524 . Two hours post injection, the main metabolite GS-441524 is present at micromolar concentrations, whilst intact Remdesivir is no longer detectable. Because of this rapid extracellular conversion to the nucleoside GS-441524, some researchers have questioned whether the active nucleotide triphosphate is truly derived from Remdesivir pro-drug removal or whether it occurs by GS-441524 phosphorylation, and whether direct administration of GS-441524 would constitute a cheaper and easier to administer COVID‑19 drug compared to Remdesivir. [ 40 ] [ 23 ]  The activated nucleotide triphosphate form has sustained intracellular levels in  PBMC  and presumably in other cells as well. [ 33 ]


Mutations in the  mouse hepatitis virus   RNA replicase  that cause partial resistance to remdesivir were identified in 2018. These mutations make the viruses less effective in nature, and the researchers believe they will likely not persist where the drug is not being used. [ 41 ]


Remdesivir is at least partially metabolized by the  cytochrome P450  enzymes  CYP2C8 ,  CYP2D6 , and  CYP3A4 . [ 42 ] [ 24 ]   Blood plasma  concentrations of remdesivir are expected to decrease if it is administered together with cytochrome P450  inducers  such as  rifampicin ,  carbamazepine ,  phenobarbital ,  phenytoin ,  primidone , and  St John's wort . [ 43 ]


Using chloroquine or  hydroxychloroquine  with remdesivir may reduce the antiviral activity of remdesivir. [ 12 ] [ 10 ] [ 44 ]  Coadministration of remdesivir and chloroquine phosphate or hydroxychloroquine sulfate is not recommended based on  in vitro  data demonstrating an antagonistic effect of chloroquine on the intracellular metabolic activation and antiviral activity of remdesivir. [ 12 ]


Remdesivir can be synthesized in multiple steps from  ribose  derivatives. The figure to the right is one of the synthesis routes of remdesivir invented by Chun and coauthors from Gilead Sciences. [ 45 ] [ 46 ]


An  in vitro  study of remdesivir assessing antiviral activity against SARS-CoV-2 was performed. [ 47 ]  Cells were pre-treated with the different doses of remdesivir for 1 hour, and the virus (MOI of 0.05) was subsequently added to allow infection for 2 hours. [ 47 ]  The results found that remdesivir functioned well as an inhibitor of the infection. [ 47 ]  The study was published as a letter to the editor, and as such did not undergo peer review.


Remdesivir requires "70 raw materials, reagents, and catalysts" to make, and approximately "25 chemical steps." [ 48 ]  Some of the ingredients are extremely dangerous to humans, especially  trimethylsilyl cyanide . [ 48 ]  The original end-to-end manufacturing process required 9 to 12 months to go from raw materials at contract manufacturers to finished product, but after restarting production in January 2020, Gilead Sciences was able to find ways to reduce the production time to six months. [ 48 ]


In January 2020, Gilead began working on restarting remdesivir production in glass-lined steel  chemical reactors  at its manufacturing plant in  Edmonton, Alberta . [ 48 ]  On 2 February 2020, the company flew its entire stock of remdesivir, 100 kilograms in powder form (left over from Ebola research), to its filling plant in  La Verne, California  to start filling vials. [ 48 ]  The Edmonton plant finished its first new batch of remdesivir in April 2020. [ 48 ]  Around the same time, fresh raw materials began to arrive from contract manufacturers reactivated by Gilead in January. [ 48 ]


Another challenge is getting remdesivir into patients despite the drug's "poor predicted solubility and poor stability." [ 49 ]  In June 2020,  Ligand Pharmaceuticals  revealed that Gilead has been managing those issues by mixing Ligand's proprietary  excipient  Captisol (based on  University of Kansas  research into  cyclodextrin ) with remdesivir at a 30:1 ratio. [ 49 ]  Since that implies an enormous amount of Captisol is needed to stabilize and deliver remdesivir (on top of amounts needed for several other drugs for which the excipient is already in regular use), Ligand announced that it is trying to boost Captisol annual manufacturing capacity to as much as 500 metric tons. [ 49 ]


On 12 May 2020, Gilead announced that it had granted non-exclusive voluntary licenses to five  generic drug  companies in India and Pakistan to manufacture remdesivir for distribution to 127 countries. [ 50 ] [ 51 ] [ 52 ]  The agreements were structured so that the licensees can set their own prices and will not have to pay royalties to Gilead until the WHO declares an end to the COVID‑19 emergency or another medicine or vaccine is approved for COVID‑19, whichever comes first. [ 50 ]  On 23 June 2020, India granted emergency marketing approval of generic remdesivir manufactured by two Gilead licensees,  Cipla  and  Hetero Drugs . [ 53 ]


Remdesivir is approved, or authorized for emergency use, to treat COVID‑19 in many countries. [ 19 ]  Remdesivir has been authorized for emergency use in India, [ 54 ]  Singapore, [ 55 ]  and approved for use in Japan, [ 56 ]  the European Union, the United States, and Australia for people with severe symptoms. [ 13 ] [ 15 ] [ 57 ] [ 58 ] [ 59 ] [ 60 ] [ 61 ] [ 62 ] [ 63 ]


Remdesivir is the first treatment for COVID‑19 to be approved by the US  Food and Drug Administration  (FDA). [ 63 ]  The approval by the FDA does not include the entire population that had been authorized to use remdesivir under an Emergency Use Authorization (EUA) originally issued in May 2020. [ 63 ]  In order to ensure continued access to the pediatric population previously covered under the EUA, the FDA revised the EUA for remdesivir to authorize the drug's use for treatment of suspected or laboratory-confirmed COVID‑19 in hospitalized pediatric patients weighing 3.5 kilograms (7.7 lb) to less than 40 kilograms (88 lb) or hospitalized pediatric patients less than twelve years of age weighing at least 3.5 kilograms (7.7 lb). [ 63 ]


In July 2020, remdesivir was provisionally approved for use in Australia for use in adults and adolescents with severe COVID‑19 symptoms who have been hospitalized. [ 60 ] [ 64 ]  Australia claims to have a sufficient supply of remdesivir in its national stockpile. [ 65 ]


As of 11 April 2020, access in Canada was available only through clinical trials. [ 66 ]   Health Canada  approved requests to treat twelve people with remdesivir under the department's special-access program (SAP). [ 67 ]  Additional doses of remdesivir are not available through the SAP except for pregnant women or children with confirmed COVID‑19 and severe illness. [ 66 ]


In June 2020, Health Canada received an application from Gilead for the use of remdesivir for treating COVID‑19. [ 66 ]  On 27 July 2020, Health Canada conditionally approved the application. [ 68 ] [ 7 ]


In September 2020,  Minister of Public Services and Procurement   Anita Anand  announced that Canada had entered into a deal to obtain up to 150,000 vials of remdesivir from Gilead starting in October. [ 69 ]  As of 8 October, remdesivir was still not widely available in  Alberta , because  Alberta Health Services  was undertaking a " formulary  review" to be completed by mid-November. [ 70 ]


In March 2020, the drug was provisionally approved for use for COVID‑19 patients in a serious condition as a result of  the outbreak in the Czech Republic . [ 71 ]


In February 2016,  orphan designation  (EU/3/16/1615) was granted by the European Commission to Gilead Sciences International Ltd, United Kingdom, for remdesivir for the treatment of Ebola virus disease. [ 72 ]


In April 2020, the  European Medicines Agency  (EMA) provided recommendations on compassionate use of remdesivir for COVID‑19 in the EU. [ 73 ]


In May 2020, the  Committee for Medicinal Products for Human Use  (CHMP) of the EMA recommended expanding the compassionate use of remdesivir to those not on mechanical ventilation. [ 74 ]  In addition to those undergoing invasive mechanical ventilation, the compassionate use recommendations cover the treatment of hospitalized individuals requiring supplemental oxygen, non-invasive ventilation, high-flow oxygen devices or ECMO (extracorporeal membrane oxygenation). [ 74 ]  The updated recommendations were based on preliminary results from the NIAID-ACTT study, [ 75 ]  which suggested a beneficial effect of remdesivir in the treatment of hospitalized individuals with severe COVID‑19. [ 74 ] [ 76 ]  In addition, a treatment duration of five days was introduced alongside the longer ten-day course, based on preliminary results from another study (GS-US-540-5773) suggesting that for those not requiring mechanical ventilation or ECMO, the treatment course may be shortened from ten to five days without any loss of efficacy. [ 74 ]  Individuals who receive a five-day treatment course but do not show clinical improvement will be eligible to continue receiving remdesivir for an additional five days. [ 74 ]


In July 2020, the European Union granted a conditional marketing authorization for remdesivir with an indication for the treatment of COVID‑19 in adults and adolescents (aged twelve years and older with body weight at least 40 kilograms [88 lb]) with pneumonia requiring supplemental oxygen. [ 13 ] [ 77 ]


In August 2022, the European Union granted a full marketing authorization for remdesivir. [ 13 ] [ 77 ] [ 78 ]


Remdesivir has been also produced in Iran by  Barakat ; Iran is planning to increase the productions of Remdesivir ampoules from 20,000 to 150,000 ampoules per month. It has also the permission of the "Food and Drug Administration" of  MOHME [ 79 ]


In May 2020,  Japan's Ministry of Health, Labour and Welfare  approved the drug for use in Japan, in a fast-tracked process, based on the US emergency authorization. [ 15 ] [ 59 ]


In October 2020, Deputy Secretary of Prevention and Health Promotion  Hugo López-Gatell Ramírez  stated at a news conference that Mexico would not necessarily follow the United States in approving the drug for use in Mexico. López-Gatell explained that the  Federal Commission for the Protection against Sanitary Risk  (Cofepris) had already twice denied the approval of remdesivir because, in that agency's view, the evidence does not suggest "sufficient efficacy". [ 80 ]  In March 2020, Cofepris authorized the drug for emergency cases, advising to give continuous surveillance of the integral health of the patient. [ 81 ]


In June 2020, Singapore's  Health Sciences Authority  conditionally approved the usage of remdesivir in Singapore. [ 55 ]


In March 2020, United States President  Donald Trump  announced that remdesivir was available for " compassionate use " for people with COVID‑19; FDA Commissioner  Stephen Hahn  confirmed the statement at the same press conference. [ 82 ]  It was later revealed that Gilead had been providing remdesivir in response to compassionate use requests since 25 January. [ 20 ] [ 83 ]  On 23 March 2020, Gilead voluntarily suspended access for compassionate use (excepting cases of critically ill children and pregnant women), for reasons related to supply, citing the need to continue to provide the agent for testing in clinical trials. [ 84 ] [ 85 ]


In May 2020, the US  Food and Drug Administration  granted Gilead  emergency use authorization  (EUA) for remdesivir to be distributed and used by licensed healthcare providers to treat adults and children hospitalized with severe COVID‑19. [ 58 ] [ 32 ]  Severe COVID‑19 is defined as patients with an oxygen saturation (SpO2) <= 94% on room air or requiring supplemental oxygen or requiring mechanical ventilation or requiring  extracorporeal membrane oxygenation  (ECMO), a heart–lung bypass machine. [ 24 ] [ 32 ] [ 86 ] [ 87 ]  Distribution of remdesivir under the EUA was controlled by the US government for use consistent with the terms and conditions of the EUA. [ 32 ]  Gilead supplied remdesivir to authorized distributors, or directly to a US government agency, who distributed it to hospitals and other healthcare facilities as directed by the US government, in collaboration with state and local government authorities, as needed. [ 32 ]  Gilead stated they were donating 1.5   million vials for emergency use [ 86 ]  and estimated, as of April 2020, they had enough remdesivir for 140,000 treatment courses and expect to have 500,000 courses by October 2020, and one million courses by the end of 2020. [ 88 ] [ 89 ]


The initial distribution of the drug in the US was tripped up by seemingly capricious decision-making and finger-pointing, resulting in over a week of confusion and frustration among healthcare providers and patients alike. [ 90 ] [ 91 ] [ 92 ]  On 9 May 2020, the  United States Department of Health and Human Services  (HHS) explained in a statement that it would be distributing remdesivir vials to  state health departments , then would allow each department to redistribute vials to hospitals in their respective states based upon each department's insight into "community-level needs." [ 93 ]  HHS also clarified that only 607,000 vials of Gilead's promised donation of 1.5   million vials would be going to American patients. [ 93 ]  However, HHS did not explain why several states with some of the highest caseloads had been omitted from the first two distribution rounds, including California, Florida, and Pennsylvania. [ 93 ]  In May 2020, Gilead indicated they would increase the number of doses donated to the US from 607,000 to around 940,000. [ 94 ] [ 92 ]  Some of the initial distribution was sent to the wrong hospitals, to hospitals with no intensive care units, and to facilities without the needed refrigeration to store it. [ 92 ]


In June 2020, HHS announced an unusual agreement with Gilead in which HHS agreed to Gilead's  wholesale acquisition price , HHS would continue to work together with state governments and drug wholesaler  AmerisourceBergen  to allocate shipments of remdesivir vials to American hospitals through the end of September 2020, and in exchange, during that three-month timeframe (July, August, and September), American patients would be allocated over 90% of Gilead's projected remdesivir output of more than 500,000 treatment courses. [ 95 ] [ 96 ]  Absent from these announcements was any discussion of allocation of remdesivir production to the approximately 70 countries omitted from Gilead's generic drug licensing agreements—including much of Europe [ 97 ]  and countries as populous as Brazil, China, and Mexico—or the 127 countries listed on those agreements (during the time it will take for Gilead's generic licensees to ramp up their own production). [ 98 ]  As the implications of this began to sink in, several countries publicly confirmed the next day that they already had adequate supplies of remdesivir to cover current needs, including Australia, [ 99 ]  Germany, [ 100 ]  and the United Kingdom. [ 101 ]


In August 2020, the FDA broadened the Emergency Use Authorization (EUA) for remdesivir to include all hospitalized patients with suspected or laboratory-confirmed COVID‑19, irrespective of the severity of their disease. [ 102 ] [ 103 ]  The Fact Sheet was updated to reflect the new guidance. [ 24 ]


In October 2020, Gilead and HHS announced that HHS was relinquishing control over remdesivir allocation because production of the drug had finally caught up with US domestic demand. [ 104 ] [ 105 ] [ 106 ]  AmerisourceBergen will remain the sole distributor of Veklury in the US through the end of 2020. [ 106 ]


On 22 October 2020, the FDA approved remdesivir and also revised the EUA to permit the use of remdesivir for treatment of suspected or laboratory confirmed COVID‑19 in hospitalized children weighing 3.5 kilograms (7.7 lb) to less than 40 kilograms (88 lb) or hospitalized children less than twelve years of age weighing at least 3.5 kilograms (7.7 lb). [ 63 ] [ 24 ] [ 107 ] [ 108 ]  This decision was criticized for an alleged lack of previous consultation on part of the FDA given the complications of antiviral drug issues. [ 109 ]


In November 2020, the FDA issued an EUA for the combination of  baricitinib  with remdesivir, for the treatment of suspected or laboratory-confirmed COVID‑19 in hospitalized people two years of age or older requiring supplemental oxygen, invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO). [ 27 ]  The data supporting the EUA for baricitinib combined with remdesivir are based on a randomized, double-blind, placebo-controlled clinical trial (ACTT-2), which was conducted by the National Institute of Allergy and Infectious Diseases (NIAID). [ 27 ]  The EUA was issued to Eli Lilly and Company. [ 27 ]


Remdesivir received approval from the US  Food and Drug Administration  (FDA) in October 2020, for use in adults and children twelve years and older requiring hospitalization for treatment of severe COVID‑19 infections. [ 63 ]  In January 2022, the FDA gave regulatory approval to remdesivir for use in adults and children (twelve years of age and older who weigh at least 40 kilograms (88 lb) and are positive for COVID‑19, not hospitalized, and are ill with COVID‑19 having high risk for developing severe COVID‑19, including hospitalization or death. [ 110 ]  In April 2022, the FDA expanded the approval of remdesivir to include people 28 days of age and older weighing at least 3 kilograms (6.6 lb). [ 26 ]


The FDA also provided  emergency use authorization  in 2022, for remdesivir treatment of children under age twelve who are COVID‑positive and not hospitalized, but have mild-to-moderate COVID‑19 with high risk of developing severe infection, including hospitalization or death. [ 110 ]


In June 2020, Gilead announced that it had set the price of remdesivir at  US$390  per vial for the governments of developed countries, including the United States, and  US$520  for US private health insurance companies. [ 111 ]  The expected course of treatment is six vials over five days for a total cost of  US$2,340 . [ 111 ]  Being a  repurposed drug , the minimum production cost for remdesivir is estimated at  US$0.93  per day of treatment. [ 112 ]


In July 2020, the European Union secured a  €63 million  ( US$74 million ) contract with Gilead, to make the drug available there in early August 2020. [ 113 ]  In October 2020, Gilead Sciences and the European Commission announced they had signed a joint procurement framework contract in which Gilead agreed to provide up to 500,000 remdesivir treatment courses over the next six months to 37 European countries. Among the contracting countries were all 27 EU member states plus the United Kingdom, "Albania, Bosnia & Herzegovina, Iceland, Kosovo, Montenegro, North Macedonia, Norway, and Serbia". [ 114 ]  At the time, the price per treatment course was not disclosed; Reuters reported the price was 2,070 euros, thereby implying the total value of the contract (if all 500,000 courses are ordered) is approximately €1.035 billion. [ 115 ]  Under the contract, each participating country will directly place orders with Gilead and pay Gilead directly for its own orders. [ 115 ]


Remdesivir is the  international nonproprietary name  (INN) [ 116 ]  while the development code name was GS-5734. [ 117 ]


Remdesivir was originally created and developed by  Gilead Sciences  in 2009, to treat  hepatitis C  and  respiratory syncytial virus  (RSV). [ 20 ] [ 118 ]  It did not work against hepatitis C or RSV, [ 20 ] [ 118 ]  but was then repurposed and studied as a potential treatment for  Ebola virus disease  and  Marburg virus  infections. [ 119 ] [ 118 ]  According to the  Czech News Agency , this new line of research was carried out under the direction of scientist  Tomáš Cihlář . [ 120 ]  A collaboration of researchers from the  Centers for Disease Control and Prevention  (CDC) and  Gilead Sciences  subsequently discovered that remdesivir had antiviral activity  in vitro  against multiple  filoviruses ,  pneumoviruses , paramyxoviruses, and  coronaviruses . [ 121 ]


Preclinical and clinical research and development was done in collaboration between Gilead Sciences and various US government agencies and academic institutions. [ 33 ] [ 122 ] [ 123 ] [ 124 ]


During the mid-2010s, the  Mintz Levin  law firm prosecuted various patent applications for remdesivir on behalf of Gilead Sciences before the  United States Patent and Trademark Office  (USPTO). The USPTO granted two patents on remdesivir to Gilead Sciences on 9 April 2019: one for filoviruses, [ 125 ]  and one which covered both  arenaviruses  and coronaviruses. [ 126 ]


In October 2015, the  United States Army Medical Research Institute of Infectious Diseases  (USAMRIID) announced preclinical results that remdesivir had blocked the  Ebola virus  in  Rhesus monkeys . Travis Warren, who has been a USAMRIID principal investigator since 2007, said that the "work is a result of the continuing collaboration between USAMRIID and Gilead Sciences". [ 127 ]  The "initial screening" of the "Gilead Sciences compound library to find molecules with promising antiviral activity" was performed by scientists at the  Centers for Disease Control and Prevention  (CDC). [ 127 ]  As a result of this work, it was recommended that remdesivir "should be further developed as a potential treatment." [ 119 ] [ unreliable medical source? ] [ 127 ]


Remdesivir was rapidly pushed through clinical trials due to the  West African Ebola virus epidemic  of 2013–2016, eventually being used in people with the disease. Preliminary results were promising; it was used in the emergency setting during the  Kivu Ebola epidemic  that started in 2018, along with further clinical trials, until August 2019, when Congolese health officials announced that it was significantly less effective than  monoclonal antibody  treatments such as  ansuvimab  and  atoltivimab/maftivimab/odesivimab . The trials, however, established its safety profile. [ 128 ]


Remdesivir was approved for medical use in the United States in October 2020. [ 61 ] [ 62 ] [ 63 ] [ 108 ]  The US  Food and Drug Administration  (FDA) approved remdesivir based on the agency's analysis of data from three randomized, controlled clinical trials that included participants hospitalized with mild-to-severe COVID‑19. [ 63 ] [ 108 ]  The FDA granted approval and reissued the revised EUA to Gilead Sciences Inc. [ 63 ]  The FDA approved remdesivir based primarily on evidence from three clinical trials (NCT04280705, NCT04292899, and NCT04292730) of 2043 hospitalized participants with COVID‑19. [ 108 ]  The trials were conducted at 226 sites in 17 countries including the United States. [ 108 ]


In November 2020, the World Health Organization (WHO) updated its guideline on therapeutics for COVID‑19 to include a conditional recommendation against the use of remdesivir, triggered by results from the WHO  Solidarity trial . [ 129 ] [ 130 ] [ 131 ] [ 132 ]  Meanwhile, the  Public Health Agency of Canada 's COVID‑19 Clinical Pharmacology Task Group recommended that remdesivir only be administered to hospitalized patients as part of a  randomized controlled trial  due to limited information on risks and benefits. [ 133 ]


In January 2022, the Canadian component of the WHO Solidarity Trial reported that in-hospital people with COVID‑19 treated with remdesivir had 17% lower relative risk of death (18.7% versus 22.6% death rates) and 47% reduced relative risk for needing oxygen and mechanical ventilation (8.0% versus 15.0%) compared to people receiving standard-of-care treatments. [ 134 ]


In September 2022, the WHO updated their guidelines to recommend use of remdesivir for both non-hospitalized and hospitalized patients. [ 135 ]  This was based on final results from the SOLIDARITY trial that showed a reduction in mortality or progression to mechanical ventilation for non-ventilated patients.


In January 2022, a study indicated that nonhospitalized people who were at high risk for COVID‑19 progression had an 87% lower risk of hospitalization or death after a 3-day course of intravenous remdesivir. [ 136 ]


In May 2020, the  National Institute of Allergy and Infectious Diseases  (NIAID) started the Adaptive COVID‑19 Treatment Trial 2 (ACTT-2) to evaluate the safety and efficacy of a treatment regimen consisting of remdesivir plus  baricitinib  for treating hospitalized adults who have a laboratory-confirmed SARS-CoV-2 infection with evidence of lung involvement, including a need for supplemental oxygen, abnormal chest X-rays, or illness requiring mechanical ventilation. [ 137 ] [ 138 ] [ 139 ]


In November 2020, the US  Food and Drug Administration  (FDA) issued an  emergency use authorization  (EUA) for the drug baricitinib, in combination with remdesivir, for the treatment of suspected or laboratory-confirmed COVID‑19 in hospitalized people two years of age or older requiring supplemental oxygen, invasive mechanical ventilation, or  extracorporeal membrane oxygenation  (ECMO). [ 27 ]  The data supporting the EUA for baricitinib combined with remdesivir are based on a randomized, double-blind, placebo-controlled clinical trial (ACTT-2), which was conducted by the National Institute of Allergy and Infectious Diseases (NIAID). [ 27 ]  The EUA was issued to Eli Lilly and Company. [ 27 ]


In August 2020, the NIAID started the Adaptive COVID‑19 Treatment Trial 3 (ACTT 3) to evaluate the safety and efficacy of a treatment regimen consisting of remdesivir plus  interferon beta-1a  for hospitalized adults who have a laboratory-confirmed SARS-CoV-2 infection with evidence of lung involvement, including a need for supplemental oxygen, abnormal chest X-rays, or illness requiring mechanical ventilation. [ 138 ] [ 140 ]


In 2019, GS-441524 was shown to have promise for treating  feline infectious peritonitis  caused by a  coronavirus . [ 141 ]  It has not been evaluated or approved by the US  Food and Drug Administration  (FDA) for the treatment of  feline coronavirus  or feline infectious peritonitis but has been available since 2019, through websites and social media as an unregulated black market substance. [ 142 ]  Because GS-441524 is the main circulating metabolite of remdesivir and because GS-441524 has similar potency against SARS-CoV-2  in vitro , some researchers have argued for the direct administration of GS-441524 as a COVID‑19 treatment. [ 143 ]


Sugar phosphates  ( sugars  that have added or substituted  phosphate  groups) are often used in biological systems to store or transfer  energy . They also form the backbone for  DNA  and  RNA . Sugar phosphate backbone geometry is altered in the vicinity of the modified nucleotides.


Examples include:


The sugar-phosphate backbone has  multiplex  electronic structure and the electron  delocalisation  complicates its  theoretical  description. Some part of the electronic density is delocalised over the whole backbone and the extent of the delocalisation is affected by backbone conformation due to  hyper-conjugation  effects. Hyper-conjugation arises from donor-acceptor interactions of localised orbitals in 1,3 positions.


The  phosphodiester backbone  of DNA and RNA consists of pairs of  deoxyribose  or  ribose  sugars linked by phosphates at the respective 3' and 5' positions. The backbone is negatively charged and  hydrophilic , which allows strong interactions with water. [ 1 ]  Sugar-phosphate backbone forms the structural framework of  nucleic acids , including  DNA  and  RNA . [ 2 ]


Sugar phosphates are defined as carbohydrates to which a phosphate group is bound by an ester or an either linkage, depending on whether it involves an alcoholic or a hemiacetalic hydroxyl, respectively.  Solubility , acid  hydrolysis  rates, acid strengths, and ability to act as sugar group donors are the knowledge of physical and chemical properties required for the analysis of both types of sugar phosphates. The  photosynthetic  carbon reduction cycle is closely associated with sugar phosphates, and sugar phosphates are one of the key molecules in metabolism ,(Sugar phosphates are major players in  metabolism  due to their task of storing and transferring energy. Not only ribose 5-phosphate but also fructose 6-phosphate are an intermediate of the pentose-phosphate pathway which generates  nicotinamide adenine dinucleotide phosphate  (NADPH) and pentoses from glucose polymers and their  degradation  products.)  oxidative pentose phosphate pathways,  gluconeogenesis , important intermediates in  glycolysis . Sugar phosphates are not only involved in metabolic regulation and signaling but also involved in the synthesis of other phosphate compounds. [ 3 ]


Peptide nucleic acid (PNA) is a nucleic acid in which natural nucleic acid has been replaced by a  synthetic peptide  backbone formed from  N -(2-amino-ethyl)-glycine units along with sugar phosphate backbone forming in an  achiral  and uncharged moiety that mimics RNA or DNA  oligonucleotides . PNA cannot be degraded inside living cells but it is chemically stable and resistant to  hydrolytic  (enzymatic)  cleavage . [ 4 ]


Sugar phosphates are major players in  metabolism  due to their task of storing and transferring energy. Not only ribose 5-phosphate but also fructose 6-phosphate are an intermediate of the pentose-phosphate pathway which generates  nicotinamide adenine dinucleotide phosphate  (NADPH) and pentoses from glucose polymers and their  degradation  products. The pathway is known as  glycolysis  where the same carbohydrates are degraded into  pyruvates  thus providing energy.  [ 5 ]  Enzymes are catalysed for the reactions of these pathways. Some  enzymes  contain metal centers in their  active site  which is important part of the enzymes and as well as for the catalysed reaction. The phosphate group can coordinate to the metal center for example, 1,6-bisphosphatase and ADP-ribose pyrophosphatase.


Phosphoglycerate and several sugar phosphates that are known intermediates of the Calvin photosynthetic  carbon cycle , stimulate light-dependent carbon dioxide fixation by isolated chloroplasts. This ability is shared by certain other metabolites (e.g. glucose 1-phosphate) from which the accepted Calvin-cycle intermediates could easily be derived by known metabolic routes.


Thioinosinic acid  (or  thioinosine monophosphate ,  TIMP ) is an intermediate  metabolite  of  azathioprine , an  immunosuppressive drug . [ 1 ] [ 2 ]


Thymidine diphosphate  ( TDP ) or  deoxythymidine diphosphate  ( dTDP ) (also  thymidine pyrophosphate ,  dTPP ) is a  nucleotide diphosphate . It is an  ester  of  pyrophosphoric acid  with the  nucleoside   thymidine . dTDP consists of the  pyrophosphate   group , the  pentose   sugar   ribose , and the  nucleobase   thymine .  Unlike the other  deoxyribonucleotides , thymidine diphosphate does not always contain the "deoxy" prefix in its name. [ 1 ]


Thymidine diphosphate glucose  (often abbreviated  dTDP-glucose  or  TDP-glucose ) is a nucleotide-linked sugar consisting of  deoxythymidine diphosphate  linked to  glucose . It is the starting compound for the syntheses of many  deoxysugars . [ 1 ]


DTDP-glucose is produced by the enzyme  glucose-1-phosphate thymidylyltransferase  and is synthesized from  dTTP  and  glucose-1-phosphate .  Pyrophosphate  is a byproduct of the reaction.