Nucleic acid analogue: Difference between revisions

Content deleted Content added

Inline

Revision as of 18:29, 6 April 2008

File:NA-comparedto-DNA thymineAndUracilCorrected.png

RNA with its nucleobases to the left and DNA to the right.

Nucleic acid analogues are compounds structurally similar (analog) to naturally occurring RNA and DNA, used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pucker-shaped pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these altered, typically the analogue nucleobases confer, among other things, different base pairing and base stacking proprieties such as universal bases, which can pair with all four canon bases, while the phosphate-sugar backbone analogues affect the properties of the chain, such as PNA which whose secondary structure differs significantly and may form a triplex (a triple stranded helix). ^[1]

Medicine

Several nucleoside analogues are used as antiviral or anticancer agents. The viral polymerase incorporates these compounds with non-canon bases. These compounds are activated in the cells by being converted into nucleotides, they are administered as nucleosides since charged nucleotides cannot easily cross cell membranes.

Molecular biology

Nucleic acid analogues are used in molecular biology for several purposes:

As a tool to detect particular sequences
As a tool with resistance to RNA hydrolysis)
As a tool for another purpose, such as sequencing
Naturally occurring, such as in tRNA
Investigation of the mechanisms used by enzyme, such as an Enzyme inhibitor)
Investigation of possible scenarios of the origin of life
Investigation of the structural features of nucleic acids
Investigation of the possible alternatives to the natural system in Synthetic biology

backbone analogues

Hydrolysis resistant RNA-analogues

To overcome the fact that ribose's 2' hydroxy group that reacts with the phosphate linked 3' hydroxy group (RNA is too unstable to be used or synthesised reliably), a ribose anologue is used. The most common RNA analogues are locked nucleic acid (LNA), morpholino, peptide nucleic acid (PNA). These oligonucleotides differ as they have a different backbone sugar but still bind according to Watson and Crick pairing with RNA or DNA, but are immune to nuclease activity (They generally cannot be enzymatically synthesised and can only be produced synthetically.

Other notable analogues used as tools

In sequencing dideoxynucleotides are used. These nucleotide triphosphates possess a non-canon sugar, dideoxyribose which lacks 3' hydroxyl group (which accepts the phosphate) and therefore cannot bond with the next base, terminating the chain as the DNA polymerases mistake it for a regular deoxyribonucleotide. The nucleoside analogue with a ribose lacking both 2' and 3' is called cordycepin, an anticancer drug. Another analogue in sequencing is a nucleobase analogue, 7-deaza-GTP and is used to sequence CG rich regions, instead 7-deaza-ATP is called tubercidin, an antibiotic.

precursors to the RNA-world

RNA may be too complex to be the first nucleic acid, so before the RNA world there may have been one of these several candidate original nucleic acids which differ in the backbone, such as TNA and GNA and PNA.

Base analogues

Nucleobase structure and nomeclature

Natural bases are divided into two classes depending on their structure: pyrimidine (an heterocyclic aromatic six-membered ring with nitrogen atoms in position 1 and 3) and purine (a pyrimidine (numeration inverted) fused with an imidazole ring, a five-membered ring with 2 nitrogen atoms separated by one carbon (meta), 7,9).


Purine	Pyrimidine

Several analogues have been made to study base stacking (caused by the attraction of the delocalized pi electron clouds).

See Simple aromatic ring for structures of the analogues that may be mentioned in the literature.

Fluorophores

Commonly fluorophores (such as rhodamine or fluorescein) are linked to the ring linked to the sugar (in para) via a flexible arm, presumably extruding form the major groove of the helix. Due to taq polymerases low processivity of the nucleotides linked to bulky adducts such as florophores, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling):

amine reactive: Aminoallyl nucleotide contain a primary amine group on a linker that reacts with the amino-reactive dye such as a cyanine or Alexa Fluor dyes, which contain a reactive leaving group, such as a succinimidyl ester (NHS). (base pairing amino groups are not affected).
thiol reactive: thiol containing nucleotides reacts with the fluorophore linked to a reactive leaving group, such as a a maleimide.
biotin linked nucleotides rely on the same indirect labelling principle (+ fluorescent streptavidin) and are used in Affymetrix DNAchips.

Fluorophores find a variety of uses in medicine and biochemistry.

Natural non-canon bases

In a cell, there are several noncanon bases present: CpG islands in DNA are often methylated, all eukaryotic mRNA are capped with a methyl-7-guanosine and several bases of the rRNAs are methylated. tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing in particular in/near the anticodon: inosine can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific that uracil (with a purine). Other common tRNA base modifications are pseudorindine (which gives its name to the TΨC loop), dihydrouridine (which does not stack as it is not aromatic), queosine, wyosine and so forth. Nevertheless these are all modifications to normal bases and are not placed by a polymerase.

base pairing

Canonical bases may have either a keto or amino group on the carbons surrounding the nitrogen atom furthest away from the glycosidic bond, which allows them to base pair (Watson-Crick base pairing) via hydrogen bonds (amine with keto, purine with pyrimidine). A and T are amine only and keto only, while C and G are mixed (inverted in respect to each other).

Natural bases pairs

A GC base pair: purine keto/amine forms three intermolecular hydrogen bonds with pyrimidine amine/keto	An AT base pair: purine amine/- forms two intermolecular hydrogen bonds with pyrimidine keto/keto

The precise reason why there are only four nucleotides is debated, but they are several unused possibilities. Furthermore adenine is not the most stable choice for base pairing: in Cyanophage S-2L diaminopurine (DAP) is used instead of adenine (host evasion). Diaminopurine basepairs perfectly with thymine as it is identical to adenine but has an amine group at position 2 forming 3 intramolecular hydrogen bonds, eliminating the major difference between the two types of basepairs (Weak:A-T and Strong:C-G). This improved stability affects protein binding ineractions which rely on those differences. Other combination include,

isoguanosine and isocytosine, which are have their amino and keto inverted, (not used probably as tautomers are problematic for base pairing, but isoG and isoG can be amplified correctly with PCR even in the presence of the 4 canon bases) ^[2]
diaminopyrimidine and a xanthine (not used as xanthine is a deamination product)

Unused base pair arrangements
	File:X-DAY DNA base pair.svg
A DAP-T base: purine amine/amine forms three intermolecular hydrogen bonds with pyrimidine keto/keto	An X-DAY base: purine keto/keto forms three intermolecular hydrogen bonds with pyrimidine amine/amine	A iG-iC base: purine amine/keto forms three intermolecular hydrogen bonds with pyrimidine keto/amine

However correct DNA structure can form even when the bases are not paired via hydrogen bonding, as studies have shown using DNA isosteres (analogues with same number of atoms), such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z). Other noteworthy base pairs are:

Several fluorescent bases have also been made, such as the 2-amino-6-(2-thienyl)purine and pyrrole-2-carbaldehyde base pair. ^[3]
Metal coordinated bases, such as two 2,6-bis(ethylthiomethyl)pyridine (SPy) with a silver ion or pyridine-2,6-dicarboxamide (Dipam) and a mondentate pyridine (Py) with a copper ion ^[4].
universal bases may pair indiscriminately with any other base, but generally lower the melting temperature of the sequence considerably, examples include 2'-deoxyinosine derivatives, nitroazole analogues and hydrophobic aromatic non-hydrogen bonding bases (strong stacking effects). These are used as proof of concept and are not generally utilised in degenerate primers (which are a mixture of primers).
The numbers of possible base pairs is doubled when xDNA is considered. xDNA contains expanded bases, in which a benzine ring has been added, which may pair with canon bases, resulting in four possible base-pairs. Another form of benzine added bases is yDNA, in which the base is widened by the benzine^[5].

Novel Base Pairs with special proprieties

A F-Z base: methylbenzimidazole does not form intermolecular hydrogen bonds with toluene F/F	An S-Pa base: purine thienyl/amine forms three intermolecular hydrogen bonds with pyrrole -/carbaldehyde

Some structures

Canon Bases	Adenine	Guanine	Cytosine	Uracil	Thymine	7-Methylguanine	5-Methylcytosine
Deaminated/Oxidated Bases	Chemical structure of uracil pseudoUracil	5,6-Dihydrouracil	Chemical structure of hypoxanthine Hypoxanthine	Chemical structure of xanthine Xanthine
Drugs	Aciclovir	Cordycepin	Didanosine	Chemical structure of Vidarabine Vidarabine	Chemical structure of Cytarabine Cytarabine	Emtricitabine
	Lamivudine	Chemical structure of Zalcitabine Zalcitabine	Chemical structure of Abacavir Abacavir	Stavudine	Zidovudine	Idoxuridine	Trifluridine
	Tenofovir disoproxil fumarate	Adefovir	Efavirenz	Nevirapine	Delavirdine	Chemical structure of Azathioprine Azathioprine	Mercaptopurine
Backbone Analogues	Chemical structure of Morpholino Morpholino	LNA	PNA	TNA (threose backbone in picture)	GNA (glycerine backbone in picture)<
Novel base Pairs	Isocytosine	Isoguanine	Chemical structure of dxA Size-expanded dxA	Chemical structure of dxT Size-expanded dxT	Chemical structure of dxC Size-expanded dxC	Chemical structure of dxG Size-expanded dxG
Chemical structure of dyC Size-widened dyC	Chemical structure of dyT Size-widened dyT	Chemical structure of Aminoallyl Uridine Aminoallyl nucleotide	Chemical structure of S 2-amino-6-(2-thienyl)purine (S)	Phosphoramidite

References

^ Petersson B et al. Crystal structure of a partly self-complementary peptide nucleic acid (PNA) oligomer showing a duplex-triplex network. J Am Chem Soc. 2005 Feb 9;127(5):1424-30.
^ Johnson SC et al. A third base pair for the polymerase chain reaction: inserting isoC and isoG. Nucleic Acids Res. 2004 Mar 29;32(6):1937-41.
^ Kimoto M et al. Fluorescent probing for RNA molecules by an unnatural base-pair system. Nucleic Acids Res. 2007;35(16):5360-9.
^ Zimmermann N et al. A second-generation copper(II)-mediated metallo-DNA-base pair. Bioorg Chem. 2004 Feb;32(1):13-25.
^ Liu H et al (ET Kool Lab)[1]. A four-base paired genetic helix with expanded size. Science. 2003 Oct 31;302(5646):868-71

@@ Line 60: / Line 60: @@
 Fluorophores find a [[Fluorescence#Biochemistry and medicine|variety of uses]] in medicine and biochemistry.
-=== natural non-canon bases ===
+===  non-canon bases ===
-In a cell, there are several noncanon bases present: CpG islands in DNA are often methylated, all eukaryotic mRNA are capped with a methyl-7-guanosine and several bases of the rRNAs are methylated. tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing in particular in/near the anticodon, inosine can base pair with C U and even with A, whereas thiouridine (with A) is more specific that uracil (with a purine), other common tRNA base modifications are pseudorindine (which gives its name to the TΨC loop), dihydrouridine (which does not stack as it is not aromatic), queosine, wyosine and so forth, nevertheless these are all modifications to normal bases and are not placed by a polymerase.
+In a cell, there are several noncanon bases present: CpG islands in DNA are often methylated, all eukaryotic mRNA are capped with a methyl-7-guanosine and several bases of the rRNAs are methylated. tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing in particular in/near the anticodon inosine can base pair with C U and even with A, whereas thiouridine (with A) is more specific that uracil (with a purine)  common tRNA base modifications are pseudorindine (which gives its name to the TΨC loop), dihydrouridine (which does not stack as it is not aromatic), queosine, wyosine and so forth  these are all modifications to normal bases and are not placed by a polymerase.
 === base pairing ===