Information

Is there a DNA analogue to ribozymes?


If not, is it impossible for DNA to have enzymatic activity?


Not very common, and not found so far in nature, but they exist and are called deoxyribozymes.

Additional information:

Deoxyribozymes are the equivalent of ribozymes in the DNA world and can function as catalysts for different biochemical reactions, such as DNA cleavage. While DNAzymes (short name) were synthesized in a laboratory context (In-vitro) and proved to be active, no observations were made of DNA molecules having an enzymatic activity In-vivo which is of course not a definitive proof they don't exist in nature.

The latter point is a major difference compared to ribozymes which were proved to be active and functional in living cells.


Ribozyme

Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems. [1] The most common activities of natural or in vitro-evolved ribozymes are the cleavage or ligation of RNA and DNA and peptide bond formation. [2] Within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link amino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.

Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis from activated monomers under very specific conditions, such as an RNA polymerase ribozyme. [3] Mutagenesis and selection has been performed resulting in isolation of improved variants of the "Round-18" polymerase ribozyme from 2001. "B6.61" is able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds. [4] The "tC19Z" ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide. [5]

Attempts have been made to develop ribozymes as therapeutic agents, as enzymes which target defined RNA sequences for cleavage, as biosensors, and for applications in functional genomics and gene discovery. [6]


A DNA enzyme that cleaves RNA

Background: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology.

Results: Beginning with a population of 1014 DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb 2+ -dependent cleavage of a target ribonucleoside 3′-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min −1 . Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min −1 . This rate is about 105-fold increased compared to the uncatalyzed reaction.

Conclusions: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb 2+ -dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.


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Molecular Biology

The phosphate closest to the sugar is the alpha phosphate and next phosphate (middle) is the Beta phosphate and phosphate furthest from sugar is the gamma phosphate.

There are 4 nitrogenous bases, adenine and guanine (purines) which have 2 rings. Cytosine and thymine (pyramidines) have 1 ring. These bases attach to the deoxyribose sugar using a B-N-glycosidic bond.

Purines pair with pyramidines.
Adenine and Thymine have 2 H bonds, Guanine and Cytosine have 3 bonds. (GC content is 40.3% in humans)

Many genes are discontinuous, and they split into
Þ Exons - contain information to code for a protein
Þ Introns - contain information not needed to code for a protein

Positional - tissue/cell specific - which tissues/cells are targeted

● In a simple multigene family all the genes are the same
○ Occur when when the gene product is needed in large quantities
○ E.g. the ribosomal RNA genes exists in a simple multi-gene family s lots of ribosomal RNA's are needed for ribosomes

● In complex multigene families the genes are not identical but have similar DNA sequences, so they code for similar proteins
○ The human globin genes are an example
■ 2 alpha and 2 beta globin proteins combine making haemoglobin
■ The human globin genes form 2 complex multigene families, the alpha-globin genes = chromosome 16
beta-globin genes = chromosome 11
■ They're expressed at different stages in human development: the fetal gene makes haemoglobin with a higher affinity for oxygen so the oxygen can be transferred from the mother to the child

Conservative:
Original parent strands stay together and make a copy, creating a paired daughter strand

Semi-conservative:
Each strand acts as a template for a new strand.
Free floating nucleotides form phosphodiester bonds with complimentary bases on the templates and purine binds with a pyrimidine. DNA polymerase catalyses formation of phosphodiester bonds between nucleotides and the sugar phosphate backbone forms. Each new molecule contains one old and one new strand.

Dispersive:
A hypothetical mode of DNA replication in which both polynucleotides of each daughter double helix are made up partly of parental DNA and partly of newly synthesized DNA.

DNA topoisomerases are enzymes that separate the two strands in a DNA molecule without actually rotating the double helix. They achieve this feat by causing transient breakages in the polynucleotide backbone.

2) DNA polymerases work in the 5' to 3' direction.
Leading strand uses continuous replication as it exists in a 5' to 3' direction.
Lagging strand copies in the 3' to 5' direction so it uses discontinuous replication (okazaki fragments)

3) Priming DNA: In bacteria the primer is made by primase enzyme (two primase enzymes form primosome),
In eukaryotes, the primer is extended by DNA polymerase alpha
The primer in all DNA replication is made of RNA.

4) Bacterial DNA replication
Primase enzyme [a RNA polymerase] makes the primer
DNA polymerase III = main replicating enzyme

Eukaryotic DNA replication
DNA polymerase alpha = initiates synthesis of DNA by extending primer
DNA polymerase delta = main replicating enzyme of synthesis


For as long as history has been recorded, humanity has tried to answer the ancient question of our origins. The ‘central dogma’ of molecular biology, first stated by Francis Crick in 1958, represented a major step forward in our efforts to answer this question (Figure 1A Crick, 1958). In this model, the genetic information stored in DNA is transcribed to produce RNA, which is then translated by the ribosome to produce chains of amino acids. These chains fold to make the proteins that are responsible for almost everything that happens in cells.

The emergence of DNA genomes in the RNA world.

(A) In the central dogma of molecular biology, information flows from DNA (red oval) to RNA (green oval) to protein (blue box). DNA is formed of building blocks called deoxynucleoside triphosphates (dNTPs) and can be replicated (solid looping red arrow) RNA is formed of nucleoside triphosphates (NTPs). Enzymes called reverse transcriptases (RT) enable complementary DNA to be made from the building blocks of RNA (dashed arrow). Blue rectangles represent processes catalyzed by proteins green rectangles show processes catalyzed by RNA translation is mediated by an RNA catalyst (green inner rectangle) that has proteins that modulate its activity (blue outline). (B) In the RNA world, ribozymes (RdRp) replicate RNA genomes (solid looping red arrow). Based on the work of Joyce and Samanta, if dNTPs were present in the RNA world, reverse transcriptase ribozymes could have constructed DNA genomes using RNA genomes as a template (straight red arrow). Ribozymes could also have potentially replicated DNA genomes (dashed red arrow).

The flow of information from DNA to RNA to protein is thought to have evolved out of a simpler evolutionary period when genetic information was stored and transmitted solely by RNA molecules. This theory, known as the ‘RNA world hypothesis’, posits that an RNA enzyme or ‘ribozyme’ capable of copying RNA molecules existed early in evolution, and that protein synthesis by the ribosome (which is also an RNA enzyme) evolved out of this system (Figure 1B Gilbert, 1986 Atkins et al., 2011). The theory, however, is largely silent on how DNA genomes evolved.

In modern metabolism, protein-based enzymes called reverse transcriptases can copy RNA to produce molecules of complementary DNA. Other enzymes can promote the production of DNA nucleotides (the building blocks of DNA molecules) from RNA nucleotides via challenging chemical reactions. So how did the first DNA genomes come to be? There are two possibilities within the framework of the RNA world. In the first, protein enzymes evolved before DNA genomes. In the second, the RNA world contained RNA polymerase ribozymes that were able to produce single-stranded complementary DNA and then convert it into stable double-stranded DNA genomes.

A number of laboratories around the world are trying to build ribozymes that can sustain RNA replication (Wang et al., 2011 Attwater et al., 2013). Recently, David Horning and Gerald Joyce artificially evolved a ribozyme that is capable of copying complex RNAs and amplifying short RNA templates (Horning and Joyce, 2016). Now, in eLife, Joyce and Biswajit Samanta at the Salk Institute demonstrate that this ribozyme is also a reverse transcriptase (Samanta and Joyce, 2017). Feeding DNA nucleotides to this ribozyme enabled it to copy short segments of RNA templates into complementary DNA. This suggests that if an RNA world contained DNA nucleotides, DNA genomes could have been assembled and then presumably replicated by ribozymes.

Whether DNA genomes existed very early in evolution fundamentally rests on whether DNA nucleotides were available in the RNA world. There are plausible routes by which RNA and DNA nucleotides could have been synthesized before life emerged, meaning that they are likely to have been available at the dawn of an RNA world (Ritson and Sutherland, 2014 Becker et al., 2016 Kim and Benner, 2017). Likewise, artificially selected ribozymes have been used to synthesize the two types of bases found in RNA nucleotides from simpler precursors, suggesting RNA nucleotides could have been built by early RNA systems (Martin et al., 2015). If DNA precursors were also available early in evolution, then the synthesis of DNA nucleotides by an RNA system appears likely. While this area is currently underexplored experimentally, there appears to be no fundamental reason why DNA nucleotides could not have been abundant quite early in evolution.

Demonstrating that DNA polymerase ribozymes are able to rapidly use such DNA nucleotides would represent a major step forward for the early DNA genome model. While the field of artificial RNA polymerase ribozymes has made rapid strides, their ability to add multiple nucleotides rapidly is still very limited. Current ribozymes are significantly longer and more complex than the sequences that they are able to copy, but to make self-evolving systems, ribozymes need to be able to copy sequences that are longer and more complex than themselves. It will therefore be exciting to see if the techniques that have created such RNA polymerases are also able to evolve DNA polymerase ribozymes that have the potential to make self-replicating systems using DNA and not RNA as a source of genetic material. Such a system would bring us closer to understanding the transition from an RNA world to a type of life that respects the rules of the central dogma of modern biology.


Acknowledgements

We thank A. Roth and other members of the Breaker laboratory for helpful discussions, and N. Carriero and R. Bjornson for assistance with the Yale Life Sciences High Performance Computing Center funded by the US National Institutes of Health (NIH RR19895-02). P.B.K. was supported by an NIH Genetics Training Grant (5T32GM007499). C.E.L. was supported by the Deutsche Forschungsgemeinschaft (LU1889/1-1). This work was also supported by an NIH grant (GM022778) to R.R.B. and by the Howard Hughes Medical Institute.


OVERCOMING THE TRANSFORMED PHENOTYPE

The mRNAs from oncogenes have also been targeted by ribozyme technology. H-ras is activated by a mutation at codon 12 (G G U → G U U), with the activated form being a substrate for hammerhead ribozyme cleavage. When transformed NIH3T3 cells that displayed a neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo were transfected by a ribozyme designed to cleave the activated H-ras mRNA, the transformed phenotype was abrogated.72 A reduction in H-rasmRNA was observed. A mutant ribozyme resulted in cells with an intermediate phenotype, probably due to an antisense effect of the ribozyme hybridizing arms. This group also addressed the question of specificity by looking at the K-ras mRNA levels in control and ribozyme-treated cells and showed no cross-reactivity. Ohta et al73 demonstrated that a tissue-specific promoter for the expression of an anti–H-ras ribozyme produced better results than using a viral promoter. This result suggests that, depending on the target mRNA, the choice of viral promoter for ribozyme expression is not always the best option. The transfected ribozyme appeared to affect not only proliferation but also the differentiation process of the melanoma cells in vitro.89

BCR-ABL in chronic myeloid leukemia.

Chromosome translocations and the resulting chimaeric genes are good targets for sequence-specific strategies. The hybrid mRNA will only be present in the cells with the translocation, and antisense ODNs or ribozymes targeted to the hybrid's junction should be specific for the hybrid and not affect the wild-type mRNA sequences. One such translocation results in the Philadelphia chromosome (Ph + ) of chronic myeloid leukemia, and the bcr-abl oncogene. The expression of the bcr-abl protein tyrosine kinase is thought to be responsible for the malignant phenotype of Ph + cells. Both the wild-type abl and bcr proteins are thought to be important for normal cell proliferation. This makes Ph + cells the ideal system to address the question of ribozyme sequence specificity. We have looked at the specificity of three different hammerhead ribozymes designed to cleave two splice variants of thebcr-abl mRNA90 in an attempt to clear up some of the contradictory results published.91-93 We showed the specific nature of one ribozyme (cleaved only bcr-abl RNA) but a lack of specificity of two others (cleaved both bcr-abl andabl RNAs). In a cell line system, the question of specificity has not really been addressed, but a decrease in cell proliferation,74 a decrease in bcr-abl mRNA and protein levels,75,76 and a decrease in bcr-ablkinase activity93 have been observed (Table 3). These studies all used slightly different hammerhead ribozymes: varying lengths of the flanking arms and various modifications to improve nuclease resistance or expression from vectors. Some groups observed quite substantial effects,75,76,93 whereas others have seen more modest91 or shown very few effects (our unpublished data). This is probably due to differences in the ribozymes' structures. Until the structure-function relationship is understood, such effects will not be predictable.

Effects of bcr-abl Ribozymes in Cell Lines

Reference . Cell Line . Transfection Method . Robozyme . Effects .
74,91 K562 DOTAP Unmodified RNA Uptake followed by fluorescence, ribozyme stable up to 12 hours Decrease in proliferation
75 EM-2, HL60 Transfectam DNA-RNA hybrid Decrease inbcr-abl mRNA, protein expression, growth
(lipopolyamine) No observable effects on p160bcr levels, β-actin mRNA unaffected Unrelated ribozyme had very limited effect
93 K562 Various vectors Decrease inbcr-abl kinase activity, cell number
Vector and ribozyme expression level dependent (RNA polymerase III tRNA vector best)
76 32Dbcr-abl Lipofectin Folic acid-poly lysine Unmodified RNA Slight effect on mRNA levels Decrease in bcr-abl mRNA levels Inactive ribozyme had no effect on mRNA levels
77 32Db3a2 in SCID mice Lipofectamine DNA-RNA hybrid Increase in survival of SCID mice after injection with ribozyme treated 32Db3a2 cells
Reference . Cell Line . Transfection Method . Robozyme . Effects .
74,91 K562 DOTAP Unmodified RNA Uptake followed by fluorescence, ribozyme stable up to 12 hours Decrease in proliferation
75 EM-2, HL60 Transfectam DNA-RNA hybrid Decrease inbcr-abl mRNA, protein expression, growth
(lipopolyamine) No observable effects on p160bcr levels, β-actin mRNA unaffected Unrelated ribozyme had very limited effect
93 K562 Various vectors Decrease inbcr-abl kinase activity, cell number
Vector and ribozyme expression level dependent (RNA polymerase III tRNA vector best)
76 32Dbcr-abl Lipofectin Folic acid-poly lysine Unmodified RNA Slight effect on mRNA levels Decrease in bcr-abl mRNA levels Inactive ribozyme had no effect on mRNA levels
77 32Db3a2 in SCID mice Lipofectamine DNA-RNA hybrid Increase in survival of SCID mice after injection with ribozyme treated 32Db3a2 cells

The hybrid gene AML1/MTG8 mRNA that results from a translocation between chromosomes 8 and 21 (associated with acute myeloid leukemia) is also being targeted by hammerhead ribozymes.94


Contents

Most of the antiviral drugs now available are designed to help deal with HIV, herpes viruses, SARS-CoV-2, the hepatitis B and C viruses, and influenza A and B viruses. [ citation needed ] Researchers are working to extend the range of antivirals to other families of pathogens.

Designing safe and effective antiviral drugs is difficult because viruses use the host's cells to replicate. This makes it difficult to find targets for the drug that would interfere with the virus without also harming the host organism's cells. Moreover, the major difficulty in developing vaccines and anti-viral drugs is due to viral variation.

The emergence of antivirals is the product of a greatly expanded knowledge of the genetic and molecular function of organisms, allowing biomedical researchers to understand the structure and function of viruses, major advances in the techniques for finding new drugs, and the pressure placed on the medical profession to deal with the human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS).

The first experimental antivirals were developed in the 1960s, mostly to deal with herpes viruses, and were found using traditional trial-and-error drug discovery methods. Researchers grew cultures of cells and infected them with the target virus. They then introduced into the cultures chemicals which they thought might inhibit viral activity and observed whether the level of virus in the cultures rose or fell. Chemicals that seemed to have an effect were selected for closer study.

This was a very time-consuming, hit-or-miss procedure, and in the absence of a good knowledge of how the target virus worked, it was not efficient in discovering effective antivirals which had few side effects. Only in the 1980s, when the full genetic sequences of viruses began to be unraveled, did researchers begin to learn how viruses worked in detail, and exactly what chemicals were needed to thwart their reproductive cycle.

Anti-viral targeting Edit

The general idea behind modern antiviral drug design is to identify viral proteins, or parts of proteins, that can be disabled. These "targets" should generally be as unlike any proteins or parts of proteins in humans as possible, to reduce the likelihood of side effects. The targets should also be common across many strains of a virus, or even among different species of virus in the same family, so a single drug will have broad effectiveness. For example, a researcher might target a critical enzyme synthesized by the virus, but not by the patient, that is common across strains, and see what can be done to interfere with its operation.

Once targets are identified, candidate drugs can be selected, either from drugs already known to have appropriate effects or by actually designing the candidate at the molecular level with a computer-aided design program.

The target proteins can be manufactured in the lab for testing with candidate treatments by inserting the gene that synthesizes the target protein into bacteria or other kinds of cells. The cells are then cultured for mass production of the protein, which can then be exposed to various treatment candidates and evaluated with "rapid screening" technologies.

Approaches by virus life cycle stage Edit

Viruses consist of a genome and sometimes a few enzymes stored in a capsule made of protein (called a capsid), and sometimes covered with a lipid layer (sometimes called an 'envelope'). Viruses cannot reproduce on their own and instead propagate by subjugating a host cell to produce copies of themselves, thus producing the next generation.

Researchers working on such "rational drug design" strategies for developing antivirals have tried to attack viruses at every stage of their life cycles. Some species of mushrooms have been found to contain multiple antiviral chemicals with similar synergistic effects. [6] Compounds isolated from fruiting bodies and filtrates of various mushrooms have broad-spectrum antiviral activities, but successful production and availability of such compounds as frontline antiviral is a long way away. [7] Viral life cycles vary in their precise details depending on the type of virus, but they all share a general pattern:

  1. Attachment to a host cell.
  2. Release of viral genes and possibly enzymes into the host cell.
  3. Replication of viral components using host-cell machinery.
  4. Assembly of viral components into complete viral particles.
  5. Release of viral particles to infect new host cells.

Before cell entry Edit

One anti-viral strategy is to interfere with the ability of a virus to infiltrate a target cell. The virus must go through a sequence of steps to do this, beginning with binding to a specific "receptor" molecule on the surface of the host cell and ending with the virus "uncoating" inside the cell and releasing its contents. Viruses that have a lipid envelope must also fuse their envelope with the target cell, or with a vesicle that transports them into the cell before they can uncoat.

This stage of viral replication can be inhibited in two ways:

  1. Using agents which mimic the virus-associated protein (VAP) and bind to the cellular receptors. This may include VAP anti-idiotypicantibodies, natural ligands of the receptor and anti-receptor antibodies. [clarification needed]
  2. Using agents which mimic the cellular receptor and bind to the VAP. This includes anti-VAP antibodies, receptor anti-idiotypic antibodies, extraneous receptor and synthetic receptor mimics.

This strategy of designing drugs can be very expensive, and since the process of generating anti-idiotypic antibodies is partly trial and error, it can be a relatively slow process until an adequate molecule is produced.

Entry inhibitor Edit

A very early stage of viral infection is viral entry, when the virus attaches to and enters the host cell. A number of "entry-inhibiting" or "entry-blocking" drugs are being developed to fight HIV. HIV most heavily targets the immune system's white blood cells known as "helper T cells", and identifies these target cells through T-cell surface receptors designated "CD4" and "CCR5". Attempts to interfere with the binding of HIV with the CD4 receptor have failed to stop HIV from infecting helper T cells, but research continues on trying to interfere with the binding of HIV to the CCR5 receptor in hopes that it will be more effective.

HIV infects a cell through fusion with the cell membrane, which requires two different cellular molecular participants, CD4 and a chemokine receptor (differing depending on the cell type). Approaches to blocking this virus/cell fusion have shown some promise in preventing entry of the virus into a cell. At least one of these entry inhibitors—a biomimetic peptide called Enfuvirtide, or the brand name Fuzeon—has received FDA approval and has been in use for some time. Potentially, one of the benefits from the use of an effective entry-blocking or entry-inhibiting agent is that it potentially may not only prevent the spread of the virus within an infected individual but also the spread from an infected to an uninfected individual.

One possible advantage of the therapeutic approach of blocking viral entry (as opposed to the currently dominant approach of viral enzyme inhibition) is that it may prove more difficult for the virus to develop resistance to this therapy than for the virus to mutate or evolve its enzymatic protocols.

Uncoating inhibitor Edit

Inhibitors of uncoating have also been investigated. [8] [9]

Amantadine and rimantadine have been introduced to combat influenza. These agents act on penetration and uncoating. [10]

Pleconaril works against rhinoviruses, which cause the common cold, by blocking a pocket on the surface of the virus that controls the uncoating process. This pocket is similar in most strains of rhinoviruses and enteroviruses, which can cause diarrhea, meningitis, conjunctivitis, and encephalitis.

Some scientists are making the case that a vaccine against rhinoviruses, the predominant cause of the common cold, is achievable. Vaccines that combine dozens of varieties of rhinovirus at once are effective in stimulating antiviral antibodies in mice and monkeys, researchers have reported in Nature Communications in 2016.

Rhinoviruses are the most common cause of the common cold other viruses such as respiratory syncytial virus, parainfluenza virus and adenoviruses can cause them too. Rhinoviruses also exacerbate asthma attacks. Although rhinoviruses come in many varieties, they do not drift to the same degree that influenza viruses do. A mixture of 50 inactivated rhinovirus types should be able to stimulate neutralizing antibodies against all of them to some degree.

During viral synthesis Edit

A second approach is to target the processes that synthesize virus components after a virus invades a cell.

Reverse transcription Edit

One way of doing this is to develop nucleotide or nucleoside analogues that look like the building blocks of RNA or DNA, but deactivate the enzymes that synthesize the RNA or DNA once the analogue is incorporated. This approach is more commonly associated with the inhibition of reverse transcriptase (RNA to DNA) than with "normal" transcriptase (DNA to RNA).

The first successful antiviral, aciclovir, is a nucleoside analogue, and is effective against herpesvirus infections. The first antiviral drug to be approved for treating HIV, zidovudine (AZT), is also a nucleoside analogue.

An improved knowledge of the action of reverse transcriptase has led to better nucleoside analogues to treat HIV infections. One of these drugs, lamivudine, has been approved to treat hepatitis B, which uses reverse transcriptase as part of its replication process. Researchers have gone further and developed inhibitors that do not look like nucleosides, but can still block reverse transcriptase.

Another target being considered for HIV antivirals include RNase H—which is a component of reverse transcriptase that splits the synthesized DNA from the original viral RNA.

Integrase Edit

Another target is integrase, which integrate the synthesized DNA into the host cell genome.

Transcription Edit

Once a virus genome becomes operational in a host cell, it then generates messenger RNA (mRNA) molecules that direct the synthesis of viral proteins. Production of mRNA is initiated by proteins known as transcription factors. Several antivirals are now being designed to block attachment of transcription factors to viral DNA.

Translation/antisense Edit

Genomics has not only helped find targets for many antivirals, it has provided the basis for an entirely new type of drug, based on "antisense" molecules. These are segments of DNA or RNA that are designed as complementary molecule to critical sections of viral genomes, and the binding of these antisense segments to these target sections blocks the operation of those genomes. A phosphorothioate antisense drug named fomivirsen has been introduced, used to treat opportunistic eye infections in AIDS patients caused by cytomegalovirus, and other antisense antivirals are in development. An antisense structural type that has proven especially valuable in research is morpholino antisense.

Morpholino oligos have been used to experimentally suppress many viral types:

Translation/ribozymes Edit

Yet another antiviral technique inspired by genomics is a set of drugs based on ribozymes, which are enzymes that will cut apart viral RNA or DNA at selected sites. In their natural course, ribozymes are used as part of the viral manufacturing sequence, but these synthetic ribozymes are designed to cut RNA and DNA at sites that will disable them.

A ribozyme antiviral to deal with hepatitis C has been suggested, [16] and ribozyme antivirals are being developed to deal with HIV. [17] An interesting variation of this idea is the use of genetically modified cells that can produce custom-tailored ribozymes. This is part of a broader effort to create genetically modified cells that can be injected into a host to attack pathogens by generating specialized proteins that block viral replication at various phases of the viral life cycle.

Protein processing and targeting Edit

Interference with post translational modifications or with targeting of viral proteins in the cell is also possible. [18]

Protease inhibitors Edit

Some viruses include an enzyme known as a protease that cuts viral protein chains apart so they can be assembled into their final configuration. HIV includes a protease, and so considerable research has been performed to find "protease inhibitors" to attack HIV at that phase of its life cycle. [19] Protease inhibitors became available in the 1990s and have proven effective, though they can have unusual side effects, for example causing fat to build up in unusual places. [20] Improved protease inhibitors are now in development.

Protease inhibitors have also been seen in nature. A protease inhibitor was isolated from the Shiitake mushroom (Lentinus edodes). [21] The presence of this may explain the Shiitake mushroom's noted antiviral activity in vitro. [22]

Long dsRNA helix targeting Edit

Most viruses produce long dsRNA helices during transcription and replication. In contrast, uninfected mammalian cells generally produce dsRNA helices of fewer than 24 base pairs during transcription. DRACO (double-stranded RNA activated caspase oligomerizer) is a group of experimental antiviral drugs initially developed at the Massachusetts Institute of Technology. In cell culture, DRACO was reported to have broad-spectrum efficacy against many infectious viruses, including dengue flavivirus, Amapari and Tacaribe arenavirus, Guama bunyavirus, H1N1 influenza and rhinovirus, and was additionally found effective against influenza in vivo in weanling mice. It was reported to induce rapid apoptosis selectively in virus-infected mammalian cells, while leaving uninfected cells unharmed. [23] DRACO effects cell death via one of the last steps in the apoptosis pathway in which complexes containing intracellular apoptosis signalling molecules simultaneously bind multiple procaspases. The procaspases transactivate via cleavage, activate additional caspases in the cascade, and cleave a variety of cellular proteins, thereby killing the cell.

Assembly Edit

Rifampicin acts at the assembly phase. [24]

Release phase Edit

The final stage in the life cycle of a virus is the release of completed viruses from the host cell, and this step has also been targeted by antiviral drug developers. Two drugs named zanamivir (Relenza) and oseltamivir (Tamiflu) that have been recently introduced to treat influenza prevent the release of viral particles by blocking a molecule named neuraminidase that is found on the surface of flu viruses, and also seems to be constant across a wide range of flu strains.

Immune system stimulation Edit

Rather than attacking viruses directly, a second category of tactics for fighting viruses involves encouraging the body's immune system to attack them. Some antivirals of this sort do not focus on a specific pathogen, instead stimulating the immune system to attack a range of pathogens.

One of the best-known of this class of drugs are interferons, which inhibit viral synthesis in infected cells. [25] One form of human interferon named "interferon alpha" is well-established as part of the standard treatment for hepatitis B and C, [26] and other interferons are also being investigated as treatments for various diseases.

A more specific approach is to synthesize antibodies, protein molecules that can bind to a pathogen and mark it for attack by other elements of the immune system. Once researchers identify a particular target on the pathogen, they can synthesize quantities of identical "monoclonal" antibodies to link up that target. A monoclonal drug is now being sold to help fight respiratory syncytial virus in babies, [27] and antibodies purified from infected individuals are also used as a treatment for hepatitis B. [28]

Antiviral resistance can be defined by a decreased susceptibility to a drug caused by changes in viral genotypes. In cases of antiviral resistance, drugs have either diminished or no effectiveness against their target virus. [29] The issue inevitably remains a major obstacle to antiviral therapy as it has developed to almost all specific and effective antimicrobials, including antiviral agents. [30]

The Centers for Disease Control and Prevention (CDC) inclusively recommends anyone six months and older to get a yearly vaccination to protect them from influenza A viruses (H1N1) and (H3N2) and up to two influenza B viruses (depending on the vaccination). [29] Comprehensive protection starts by ensuring vaccinations are current and complete. However, vaccines are preventative and are not generally used once a patient has been infected with a virus. Additionally, the availability of these vaccines can be limited based on financial or locational reasons which can prevent the effectiveness of herd immunity, making effective antivirals a necessity. [29]

The three FDA-approved neuraminidase antiviral flu drugs available in the United States, recommended by the CDC, include: oseltamivir (Tamiflu), zanamivir (Relenza), and peramivir (Rapivab). [29] Influenza antiviral resistance often results from changes occurring in neuraminidase and hemagglutinin proteins on the viral surface. Currently, neuraminidase inhibitors (NAIs) are the most frequently prescribed antivirals because they are effective against both influenza A and B. However, antiviral resistance is known to develop if mutations to the neuraminidase proteins prevent NAI binding. [31] This was seen in the H257Y mutation, which was responsible for oseltamivir resistance to H1N1 strains in 2009. [29] The inability of NA inhibitors to bind to the virus allowed this strain of virus with the resistance mutation to spread due to natural selection. Furthermore, a study published in 2009 in Nature Biotechnology emphasized the urgent need for augmentation of oseltamivir (Tamiflu) stockpiles with additional antiviral drugs including zanamivir (Relenza). This finding was based on a performance evaluation of these drugs supposing the 2009 H1N1 'Swine Flu' neuraminidase (NA) were to acquire the Tamiflu-resistance (His274Tyr) mutation which is currently widespread in seasonal H1N1 strains. [32]

Origin of antiviral resistance Edit

The genetic makeup of viruses is constantly changing, which can cause a virus to become resistant to currently available treatments. [33] Viruses can become resistant through spontaneous or intermittent mechanisms throughout the course of an antiviral treatment. [29] Immunocompromised patients, more often than immunocompetent patients, hospitalized with pneumonia are at the highest risk of developing oseltamivir resistance during treatment. [29] Subsequent to exposure to someone else with the flu, those who received oseltamivir for "post-exposure prophylaxis" are also at higher risk of resistance. [34]

The mechanisms for antiviral resistance development depend on the type of virus in question. RNA viruses such as hepatitis C and influenza A have high error rates during genome replication because RNA polymerases lack proofreading activity. [35] RNA viruses also have small genome sizes that are typically less than 30 kb, which allow them to sustain a high frequency of mutations. [36] DNA viruses, such as HPV and herpesvirus, hijack host cell replication machinery, which gives them proofreading capabilities during replication. DNA viruses are therefore less error prone, are generally less diverse, and are more slowly evolving than RNA viruses. [35] In both cases, the likelihood of mutations is exacerbated by the speed with which viruses reproduce, which provides more opportunities for mutations to occur in successive replications. Billions of viruses are produced every day during the course of an infection, with each replication giving another chance for mutations that encode for resistance to occur. [37]

Multiple strains of one virus can be present in the body at one time, and some of these strains may contain mutations that cause antiviral resistance. [30] This effect, called the quasispecies model, results in immense variation in any given sample of virus, and gives the opportunity for natural selection to favor viral strains with the highest fitness every time the virus is spread to a new host. [38] Also, recombination, the joining of two different viral variants, and reassortment, the swapping of viral gene segments among viruses in the same cell, play a role in resistance, especially in influenza. [36]

Antiviral resistance has been reported in antivirals for herpes, HIV, hepatitis B and C, and influenza, but antiviral resistance is a possibility for all viruses. [30] Mechanisms of antiviral resistance vary between virus types.

Detection of antiviral resistance Edit

National and international surveillance is performed by the CDC to determine effectiveness of the current FDA-approved antiviral flu drugs. [29] Public health officials use this information to make current recommendations about the use of flu antiviral medications. WHO further recommends in-depth epidemiological investigations to control potential transmission of the resistant virus and prevent future progression. [39] As novel treatments and detection techniques to antiviral resistance are enhanced so can the establishment of strategies to combat the inevitable emergence of antiviral resistance. [40]

Treatment options for antiviral resistant pathogens Edit

If a virus is not fully wiped out during a regimen of antivirals, treatment creates a bottleneck in the viral population that selects for resistance, and there is a chance that a resistant strain may repopulate the host. [41] Viral treatment mechanisms must therefore account for the selection of resistant viruses.

The most commonly used method for treating resistant viruses is combination therapy, which uses multiple antivirals in one treatment regimen. This is thought to decrease the likelihood that one mutation could cause antiviral resistance, as the antivirals in the cocktail target different stages of the viral life cycle. [42] This is frequently used in retroviruses like HIV, but a number of studies have demonstrated its effectiveness against influenza A, as well. [43] Viruses can also be screened for resistance to drugs before treatment is started. This minimizes exposure to unnecessary antivirals and ensures that an effective medication is being used. This may improve patient outcomes and could help detect new resistance mutations during routine scanning for known mutants. [41] However, this has not been consistently implemented in treatment facilities at this time.

While most antivirals treat viral infection, vaccines are a preemptive first line of defense against pathogens. Vaccination involves the introduction (i.e. via injection) of a small amount of typically inactivated or attenuated antigenic material to stimulate an individual's immune system. The immune system responds by developing white blood cells to specifically combat the introduced pathogen, resulting in adaptive immunity. [44] Vaccination in a population results in herd immunity and greatly improved population health, with significant reductions in viral infection and disease. [45]

Vaccination policy Edit

Vaccination policy in the United States consists of public and private vaccination requirements. For instance, public schools require students to receive vaccinations (termed "vaccination schedule") for viruses and bacteria such as diphtheria, pertussis, and tetanus (DTaP), measles, mumps, rubella (MMR), varicella (chickenpox), hepatitis B, rotavirus, polio, and more. Private institutions might require annual influenza vaccination. The Center for Disease Control and Prevention has estimated that routine immunization of newborns prevents about 42,000 deaths and 20 million cases of disease each year, saving about $13.6 billion. [46]

Vaccination controversy Edit

Despite their successes, in the United States there exists plenty of stigma surrounding vaccines that cause people to be incompletely vaccinated. These "gaps" in vaccination result in unnecessary infection, death, and costs. [47] There are two major reasons for incomplete vaccination:

  1. Vaccines, like other medical treatments, have a risk of causing complications in some individuals (allergic reactions). Vaccines do not cause autism, as stated by national health agencies, such as the US Centers for Disease Control and Prevention, [48] the US Institute of Medicine, [49] and the UK National Health Service[50]
  2. Low rates of vaccine-preventable disease, as a result of herd immunity, also make vaccines seem unnecessary and leave many unvaccinated. [51][52]

Although the American Academy of Pediatrics endorses universal immunization, [53] they note that physicians should respect parents' refusal to vaccinate their children after sufficient advising and provided the child does not face a significant risk of infection. Parents can also cite religious reasons to avoid public school vaccination mandates, but this reduces herd immunity and increases risk of viral infection. [45]

Limitations of vaccines Edit

Vaccines boosts the body's immune system to better attack viruses in the "complete particle" stage, outside of the organism's cells. They traditionally consist of an attenuated (a live weakened) or inactivated (killed) version of the virus. These vaccines can, in very rare cases, harm the host by inadvertently infecting the host with a full-blown viral occupancy [ citation needed ] . Recently "subunit" vaccines have been devised that consist strictly of protein targets from the pathogen. They stimulate the immune system without doing serious harm to the host [ citation needed ] . In either case, when the real pathogen attacks the subject, the immune system responds to it quickly and blocks it.

Vaccines are very effective on stable viruses but are of limited use in treating a patient who has already been infected. They are also difficult to successfully deploy against rapidly mutating viruses, such as influenza (the vaccine for which is updated every year) and HIV. Antiviral drugs are particularly useful in these cases.

Antiretroviral therapy as HIV prevention Edit

Following the HPTN 052 study and PARTNER study, there is significant evidence to demonstrate that antiretroviral drugs inhibit transmission when the HIV virus in the person living with HIV has been undetectable for 6 months or longer. [54] [55]

Use and distribution Edit

Guidelines regarding viral diagnoses and treatments change frequently and limit quality care. [56] Even when physicians diagnose older patients with influenza, use of antiviral treatment can be low. [57] Provider knowledge of antiviral therapies can improve patient care, especially in geriatric medicine. Furthermore, in local health departments (LHDs) with access to antivirals, guidelines may be unclear, causing delays in treatment. [58] With time-sensitive therapies, delays could lead to lack of treatment. Overall, national guidelines, regarding infection control and management, standardize care and improve healthcare worker and patient safety. Guidelines, such as those provided by the Centers for Disease Control and Prevention (CDC) during the 2009 flu pandemic caused by the H1N1 virus, recommend, among other things, antiviral treatment regimens, clinical assessment algorithms for coordination of care, and antiviral chemoprophylaxis guidelines for exposed persons. [59] Roles of pharmacists and pharmacies have also expanded to meet the needs of public during public health emergencies. [60]

Stockpiling Edit

Public Health Emergency Preparedness initiatives are managed by the CDC via the Office of Public Health Preparedness and Response. [61] Funds aim to support communities in preparing for public health emergencies, including pandemic influenza. Also managed by the CDC, the Strategic National Stockpile (SNS) consists of bulk quantities of medicines and supplies for use during such emergencies. [62] Antiviral stockpiles prepare for shortages of antiviral medications in cases of public health emergencies. During the H1N1 pandemic in 2009–2010, guidelines for SNS use by local health departments was unclear, revealing gaps in antiviral planning. [58] For example, local health departments that received antivirals from the SNS did not have transparent guidance on the use of the treatments. The gap made it difficult to create plans and policies for their use and future availabilities, causing delays in treatment.


RiboaptDB: a comprehensive database of ribozymes and aptamers

Background: Catalytic RNA molecules are called ribozymes. The aptamers are DNA or RNA molecules that have been selected from vast populations of random sequences, through a combinatorial approach known as SELEX. The selected oligo-nucleotide sequences (

200 bp in length) have the ability to recognize broad range of specific ligands by forming binding pockets. These novel aptamer sequences can bind to nucleic acids, proteins or small organic and inorganic chemical compounds and have many potential uses in medicine and technology.

Results: The comprehensive sequence information on aptamers and ribozymes that have been generated by in vitro selection methods are included in this RiboaptDB database. Such types of unnatural data generated by in vitro methods are not available in the public 'natural' sequence databases such as GenBank and EMBL. The amount of sequence data generated by in vitro selection experiments has been accumulating exponentially. There are 370 artificial ribozyme sequences and 3842 aptamer sequences in the total 4212 sequences from 423 citations in this RiboaptDB. We included general search feature, and individual feature wise search, user submission form for new data through online and also local BLAST search.


Watch the video: rRNA. Ribosomal RNA. Ribozymes. Catalytic RNA. Biochemistry (December 2021).