Biversal nucleotides can pair with more than one standard DNA or RNA base. The "biversal" pairing arises from their ability to exist in two tautomeric forms, structural isomers that can readily interconvert and have different hydrogen bonding patterns, each forming a distinct pattern of hydrogen bond donors and acceptors. This dual hydrogen bonding capability allows a single biversal nucleotide to pair with two different natural bases, unlike the standard one-to-one pairing (A with T/U, and G with C).
Scientific developments in the life sciences (physics, chemistry, biochemistry, molecular biology) in the 1920s have resulted in the emergence of synthetic biology. Key developments are: 1950s DNA double-helix structure, after 2000, sequencing of the genomes of organisms, and the merging of life science, physical sciences, and engineering, resulting in the development of synthetic biology. Synthetic biology (SynBio) includes biochemistry, microbiology, molecular biology, systematic biology, and non-life-science branches like computer and engineering sciences.
To increase the capabilities of synthetic biology, Yang et al. (2006) developed an artificially expanded genetic information system (AEGIS) utilizing a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1’-β-D-2’-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1’-β-D-2’-deosyribofuranosyl)-imidazo[1,2-α]-1,3,5-triazin-4(8H)-one (dP).
Benners group developed this concept further with the introduction of biversial nucleic acids. Biversal nucleobases exist in two tautomeric forms providing alternate hydrogen bonding patterns. Synthetic biversal nucleotides are designed to expand their base-pairing abilities, usually allowing them to pair with more than one type of standard nucleotide. When used in biotechnology and synthetic biology to create new functions or structures in DNA or RNA sequences, these modified nucleotides can expand the genetic alphabet. Automated solid phase phosphoramidite-based oligonucleotide synthesis allows the addition of biversal phosphoramidites to a growing oligonucleotide chain at a specific position.
Firebird Biomolecular Sciences has developed two main types of biversal nucleotides: [1 ] Pyrimidine Biversal: These can pair with either guanine (G) or adenine (A). [2] Purine Biversal: These can pair with either thymine (T) or cytosine (C). Biversal phosphoramidites are available at Firebird Biomolecular Sciences, LLC (https://www.firebirdbio.com/FirebirdProducts.aspx?fbp=30).
The concept of tautomerism explains the low barrier to interconversion between isomers. This phenomenon, where different compounds having the same molecular formula are known as isomers or isomeric compounds, is crucial in understanding the behavior of a tautomer. A tautomer is a constitutional isomer that can undergo rapid interconversion. For instance, under most common circumstances, carbon compounds and their corresponding enols are in rapid equilibrium. For example, carbonyl compounds with α-hydrogens are in equilibrium with small amounts of their enol isomers. The following image illustrates the enolization of esters.
Canonical nucleotides in DNA include adenine (A), thymine (T), cytosine (C), and guanine (G), with specific base-pairing rules (A pairs with T, C pairs with G). Designed biversal nucleotides are more flexible and can pair with multiple nucleotides rather than being restricted to one. This ability allows the creation of more robust and diverse genetic systems for studying nucleic acid interactions and functions in new environments or applications.
| Tautomeric forms of the Pyrimidine biversal. | Tautomeric forms of the Purine biversal. |
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Biversal nucleotides are primarily applied in synthetic biology, biotechnology, and molecular biology to expand the capabilities of genetic systems beyond what is possible with natural nucleotides. Biversal nucleotides enable the creation of artificial genetic systems beyond the four standard bases (A, T, C, G). By incorporating nucleotides that can pair with multiple bases, researchers can expand the genetic code to create artificial organisms with expanded genetic systems that have the potential to produce novel proteins or biomolecules that are not possible with natural systems. Some biversal phosphoramidites, such as dI-phosphoramidites, can act as universal bases by hybridizing with any of the four natural DNA bases (A, T, G, C).
Microbial studies routinely use universal primers. Usually, these studies extract total DNA from bacterial samples, followed by PCR amplification of small-subunit ribosomal RNA genes. For example, designing degenerate primers with varying options of nucleotides at several positions in the internal primer sequence allows improved amplification of related sequences of 16S rRNA genes from different microorganisms. Using inosine can achieve a fourfold degeneracy for a given location. Using inosine at the 3’-terminal ends of 16S ribosomal RNA (rRNA) gene universal primers allowed studying complex microbial communities by PCR. Biversal nucleotides can be incorporated into oligonucleotides as phosphoramidites, for example, to introduce new codons, possibly enabling the development of organisms that can synthesize proteins with non-standard amino acids, allowing expanding the diversity of protein functions.
In molecular biology, biversal nucleotides can be used to improve the robustness of PCR reactions. Standard PCR often struggles when there are mutations or variations in the DNA sequence. Biversal nucleotides, which can pair with more than one base, can help overcome this issue by allowing for amplification of sequences with mutations or polymorphisms, allowing scientists to more easily introduce or study mutations by accommodating variable base-pairing during replication. Also, by using biversal nucleotides in diagnostic tools, researchers can detect a wider range of genetic variations in samples.
To conclude: The unique properties of biversal nucleotides make them valuable tools for detecting divergent sequences in pathogens, PCR amplification, SNP detection, expanding the genetic code, and stabilizing DNA and RNA structures.
Reference
Inosine and fluoroinosine https://www.biosyn.com/tew/2%E2%80%99-Fluoroinosine,-a-Replacement-for-Inosine.aspx
Loudon, M.; Organic Chemistry. 5th edition. Roberts and Company Publishers. 2009, pp. 58 and 1054.
Tautomerism: Tautomerization defined; Tautomer
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