What are Hexitol Nucleic Acids

Hexitol nucleic acids (HNAs) are synthetic DNA and RNA analogs in which the backbone contains a hexitol ring structure, a six-carbon moiety, in place of the standard five-carbon ribose or deoxyribose sugars. The structural change makes HNAs chemically stable and resistant to enzymatic degradation. HNA oligonucleotides are also able to hybridize with complementary DNA and RNA sequences. Oligonucleotides modified with hexitol nucleic acids contain phosphorylated 1,5-anhydro-D-arabino-2,3-dideoxyhexitol building blocks with a base moiety positioned in the 2-position. HNAs allow the design of therapeutic antisense oligonucleotides.

Chemical structure of HNAs

Applications and benefits of HNAs are:

Therapeutic Applications

Antisense and siRNA Technologies: HNAs can form stable duplexes with RNA, making them suitable for antisense therapies, where they can bind to and inhibit specific mRNAs to allow reducing the expression of harmful proteins.

Targeted Gene Silencing: Complementarily binding to mRNA of HNAs allows down-regulation of genes involved in disease processes.
Drug Delivery and Cellular Uptake: HNAs enable the design of scaffolds for drug delivery due to their stability and affinity for nucleic acids.

Diagnostic and Analytical Applications

Molecular Probes: Because of their strong binding affinity and selectivity, HNAs are used in molecular probes for detecting specific DNA or RNA sequences in diagnostic assays.

Hybridization Probes: HNAs are useful in fluorescence-based and electrochemical assays, where high sensitivity and specificity are crucial.

Biotechnological Applications

Primer Design in PCR: HNAs can be used as primers in PCR to improve the stability and specificity of hybridization, especially in challenging sequences.

Nucleic Acid Engineering: Due to their unique structure and binding properties, HNAs serve as a tool for studying nucleic acid interactions and engineering artificial nucleic acid systems.

RNA Aptamers and Ribozymes

Therapeutic Aptamers: HNAs can be used in the design of aptamers, which are structured oligonucleotides that bind specifically to target molecules. These aptamers are stable in biological conditions and can be used in various therapeutic contexts.

Catalytic Ribozymes: HNA-based ribozymes are explored for their potential in gene editing and catalysis due to their structural rigidity and resistance to nucleases.

Advantages of HNA over Traditional Nucleic Acids

Increased Stability: HNAs are more resistant to nucleases, allowing them to remain intact longer in biological environments.

Higher Binding Affinity: HNAs often have enhanced binding affinity to complementary nucleic acids, improving their performance in both therapeutic and diagnostic applications.

Reduced Off-Target Effects: Due to their structural differences from natural nucleic acids, HNAs can exhibit reduced off-target interactions in complex biological systems.

Enzymatic Synthesis

Vastman et al. (2001) showed that anhydrohexitol triphosphates could act as substrates for several DNA polymerases, and enzymes belonging to the B family appear more efficient. However, enzymatic synthesis could only incorporate two consecutive 1,5-anhydrohexitol nucleotides into a DNA primer-template complex. 

A brief History of HNAs

Verheggen et al. (1993) reported the synthesis of 1,5-anhydrohexitol nucleosides by alkylation of heterocyclic bases with tosylate or the alcohol group using Mitsunobu conditions. Evaluation of the resulting hexitol nucleosides for antiviral and cytostatic activities showed their high potential for antiviral activities.

A study performed by Hendrix et al. (1997) demonstrated that HNAs form highly selective and remarkably stable duplexes with RNA. The study unequivocally showed that HNA:RNA duplexes are exceptionally stable towards nuclease degradation, providing a reliable foundation for further research.

De Winter et al. (1998) studied the molecular associations between HNA and RNA and found that they are more stable than between HNA and DNA and natural nucleic acids (dsDNA, dsRNA, DNA/RNA). The (1)H NMR analysis of an HNA dimer confirmed the axial orientation of the base moiety with respect to the hexanol ring. Both complexes of RNA and HNA/DNA duplexes showed an A-type geometry and very similar hydrogen bonding patterns between base pairs. De Winter et al. suggested that minor groove solvation accounts for the relative stability of HNA/RNA versus HNA/DNA. The observed conformation avoids sterically unfavorable 1,3-diaxial repulsions.

Vastmans et al. (2002) reported that hexitol nucleic acids are rather well-tolerated as substrates for terminal deoxynucleotidyl transferase (TdT), even better than ribonucleotides, since up to 15 purine HNA nucleotides and four pyrimidine HNA nucleotides can be incorporated into DNA primers. However, these studies suggested that engineered TdT and PUP polymerases will be required for de novo XNA synthesis applications.

Declercq et al. (2002) reported the first high-resolution structure of a double helical nucleic acid with all sugars being hexitols. The atomic coordinates have been deposited in the Protein Data Bank (PDB ID codes 481D, 1D7Z) and in the Nucleic Acid Database (accession codes HD0001, HD0002).

CRYSTAL STRUCTURES OF HEXITOL NUCLEIC ACIDS (HNAs) DUPLEXES

Selected References

De Bouvere B., Kerreinans L., Hendrix C., De Winter H., Schepers G., Van Aerschot A., Herdewijn P. Hexitol nucleic acids (HNA): Synthesis and properties. Nucleosides Nucleotides. 1997;16:973–976. [Nucleosides and Nucleotides]

De Winter, H., Lescrinier, E., Van Aerschot, A., and Herdewijn, P. 1998. Molecular dynamics simulation to investigate differences in minor groove hydration of HNA/RNA hybrids as compared to HNA/DNA complexes. J. Am. Chem. Soc. 120:5381-5394. [UA]

Declercq R, Van Aerschot A, Read RJ, Herdewijn P, Van Meervelt L. Crystal structure of double helical hexitol nucleic acids. J Am Chem Soc. 2002 Feb 13;124(6):928-33. [ACS]

Egli M, Pallan PS, Allerson CR, Prakash TP, Berdeja A, Yu J, Lee S, Watt A, Gaus H, Bhat B, Swayze EE, Seth PP. Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides. J Am Chem Soc. 2011 Oct 19;133(41):16642-9. [PMC]

Eremeeva E, Fikatas A, Margamuljana L, Abramov M, Schols D, Groaz E, Herdewijn P. Highly stable hexitol based XNA aptamers targeting the vascular endothelial growth factor. Nucleic Acids Res. 2019 Jun 4;47(10):4927-4939. [PMC]

Groaz, E., Herdewijn, P. (2023). Hexitol Nucleic Acid (HNA): From Chemical Design to Functional Genetic Polymer. In: Sugimoto, N. (eds) Handbook of Chemical Biology of Nucleic Acids. Springer, Singapore. [HCBNA]

Hendrix C., Rosemeyer H., De Bouvere B., Van Aerschot A., Seela F., Herdewijn P.. 1′,5′-Anhydrohexitol Oligonucleotides: Hybridisation and strand displacement with oligoribonucleotides, interaction with RNase H and HIV reverse transcriptase. Chemistry. 1997; 3:1513–1520. [CAEJ]

Istrate A, Johannsen S, Istrate A, Sigel RKO, Leumann CJ. NMR solution structure of tricyclo-DNA containing duplexes: insight into enhanced thermal stability and nuclease resistance. Nucleic Acids Res. 2019 May 21;47(9):4872-4882. [PMC]

Kozlov IA, De Bouvere B, Van Aerschot A, Herdewijn P, Orgel LE. Efficient transfer of information from hexitol nucleic acids to RNA during nonenzymatic oligomerization. J Am Chem Soc. 1999;121(25):5856-9. [ACS]

Novikova D, Sagaidak A, Vorona S, Tribulovich V. A Visual Compendium of Principal Modifications within the Nucleic Acid Sugar Phosphate Backbone. Molecules. 2024 Jun 26;29(13):3025. [PMC]

Pichon M, Hollenstein M. Controlled enzymatic synthesis of oligonucleotides. Commun Chem. 2024 Jun 18;7(1):138. [PMC]

Pochet S, Kaminski PA, Van Aerschot A, Herdewijn P, Marlière P. Replication of hexitol oligonucleotides as a prelude to the propagation of a third type of nucleic acid in vivo. C R Biol. 2003 Dec;326(12):1175-84. [dTTP]

Vandermeeren M., Préveral S., Janssens S., Geysen J., Saison-Behmoaras E., Van Aerschot A., Herdewijn P.. Biological activity of hexitol nucleic acids targeted at Ha-ras and intracellular adhesion molecule-1 mRNA. Biochem. Pharmacol. 2000; 59:655–663. [PubMed]

Vastmans K, Froeyen M, Kerremans L, Pochet S, Herdewijn P. Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides. Nucleic Acids Res. 2001 Aug 1;29(15):3154-63. [PMC]

Vastmans K, Rozenski J, Van Aerschot A, Herdewijn P. Recognition of HNA and 1,5-anhydrohexitol nucleotides by DNA metabolizing enzymes. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 2002;1597:115–122. doi: 10.1016/S0167-4838(02)00267-4. [PubMed]

Verheggen, I., Van Aerschot, A., Toppet, S. Snoeck, R., Janssen, G., Balzarini, J., De Clercq, E., and Herdewijn, P. 1993. Synthesis and antiherpes virus activity of 1,5-anhydrohexitol nucleosides. J. Med. Chem. 36:2033-2039. [PubMed]

Verheggen I, Van Aerschot A, Van Meervelt L, Rozenski J, Wiebe L, Snoeck R, Andrei G, Balzarini J, Claes P, De Clercq E, et al. Synthesis, biological evaluation, and structure analysis of a series of new 1,5-anhydrohexitol nucleosides. J Med Chem. 1995 Mar 3;38(5):826-35. [PubMed]

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