Nucleopeptides in Biomedical Research and Biotechnology for Targeting DNA and RNA

Nucleopeptides in Biomedical Research and Biotechnology for Targeting DNA and RNA

Nucleopeptides are biomolecules made by joining nucleobases such as adenine, guanine, cytosine, or thymine to short peptide chains. Nucleopeptides integrate nucleic acid components, including nucleobases, nucleosides, or nucleotides, with amino acids or peptide backbones. By combining the structural self-assembly and enzymatic recognition of peptides with specific hydrogen-bonding and base-pairing capabilities of nucleobases, nucleopeptides bridge the chemical space between proteins and nucleic acids. Nucleopeptides self-assemble into nanofibrous hydrogels through Watson–Crick base pairing, π-π stacking, and hydrogen bonding, mimicking the extracellular matrix for biomedical uses. Nucleopeptides can be grouped into chiral nucleopeptides, achiral nucleopseudo-peptides, or peptidyl amino nucleoside conjugates.

Nucleopeptides are related to peptide-oligonucleotide conjugates (POC). POCs can be utilized as vehicles for oligonucleotide delivery in various medicinal applications, such as antimicrobial, antiviral, anticancer, or splice-switching therapies.

Examples of Nucleopeptides

The integration of nucleobases into peptides allows the creation of soft, DNA-binding biomaterials with self-assembly and DNA interaction properties, enabling the design of novel gene delivery platforms and DNA-targeted therapeutics. The nucleobases on the peptide side chains enable sequence-specific or base-pairing interactions with single-stranded DNA (ssDNA). ssDNA binding can modulate the self-assembly of nucleopeptides, altering fiber morphology or stability. When bound to ssDNA, nucleopeptides show reduced resistance to proteinase K, confirming direct interaction. Nucleopeptides can also interact with plasmid DNA and even deliver hairpin DNA into cells, suggesting their potential use for gene delivery applications. In water, nucleopeptides self-assemble, producing hydrogels or nanofibrous networks. DNA gel electrophoresis and live cell imaging allow observation of nucleopeptide binding to ssDNA, facilitating DNA delivery. Biostability tests confirm that DNA binding increases susceptibility to enzymatic degradation, supporting the binding claim.

Nucleopeptides can be designed to target specific DNA sequences for therapeutic delivery. By binding and delivering DNA, they may allow regulation of gene expression or DNA repair processes. Their ability to form gels and interact with DNA makes them candidates for supramolecular biomedical materials.

How were nucleopeptides discovered?

Azzam & Algranati (1973) observed that adding spermidine as a stabilizing agent for 70S monomers strongly inhibited subunit exchange. This result led the researchers to conclude that upon premature release of unfinished protein chains by the antibiotic, the ribosomes fall off mRNA as 70S particles. Kuyl-Yeheskiely et al, (1989) synthesized nucleopeptides H-Phe-Tyr(pGC)-NH2 and H-Phe-Ser(pGC)-Ala-OH via a phosphotriester approach by using the protecting groups di-n-butylaminomethylene, 2-nitrophenylsulfenyl, and the ester of 2-(hydroxymethyl)-9,10-anthraquinone to enable the preparation of nucleopeptide fragments containing 2'-deoxyguanosine and a free carboxylic acid group.

Dreef-Tromp et al. (1992) reported the solid-phase synthesis of an RNA nucleopeptide fragment from the nucleoprotein of poliovirus. The researchers prepared the naturally occurring RNA-nucleopeptide H-Ala-Tyr-[5’-pUUAAAAC-3']-NH2 using a solid-phase phosphite triester approach utilizing N-SiOMB/O-TBDMS-protected nucleotides, linked via a base-labile oxalyl ester anchor. Initial proton NMR analysis revealed that the attached peptidyl unit significantly impacts the conformational properties of the RNA portion. Robles et al. (1995) reported the solid-phase synthesis of a nucleopeptide from the linking site of adenovirus-2 nucleoprotein, -Ser(p5'CATCAT)-Gly-Asp-.

Debéthune et al. (2002) investigated how tyrosyl-DNA phosphodiesterase (Tdp1) processes synthetic nucleopeptides that mimic covalent topoisomerase I-DNA complexes. The results demonstrated that Tdp1 effectively cleaves short peptide-DNA adducts, providing key insights into cellular DNA repair mechanisms. Tdp1 processes nucleopeptides of up to 13 amino acid residues, but showed poor activity on larger structures such as a full 70 kDa topoisomerase I fragment linked to DNA.

Wei et al. (2005) reported that enzymatic DNA topoisomerases and site-specific recombinases create transient covalent nucleopeptide intermediates through a conserved active-site tyrosine, facilitating DNA cleavage and rejoining, and that these phosphotyrosyl-protein complexes are central to the mechanism of both type I topoisomerases and site-specific recombinases.

Grandas et al. (2007) reported synthesis protocols for the production of phosphor-diester-linked peptide-oligonucleotide conjugates, called nucleopeptides, and showed that nucleopeptides can be obtained by stepwise solid-phase synthesis. In this approach, a peptide is first assembled on a suitably derivatized solid matrix, and the oligonucleotide is subsequently elongated at the free hydroxyl group of the linking amino acid. Temporary acid-labile and permanent base-labile protecting groups are combined. However, careful choice of the protection scheme is required to prevent and minimize side reactions that may degrade the target molecule.

Walsh & Zhang (2011) explored how Nature builds complex peptidyl nucleoside antibiotic scaffolds from simple nucleoside and amino acid building blocks. The two scientists discussed current research on biosynthetic pathways for peptidyl nucleoside antibiotics, focusing on the chemical logic and enzymatic machinery for uridine transformation and coupling to peptides.

Li et al. (2011) created supramolecular nanofibers and hydrogelators by conjugating the nucleobases thymine, adenine, cytosine, and guanine to small peptides. Because of their Watson–Crick interactions, these nanofibers exhibit high biocompatibility and biostability and are regarded as promising new biomaterials.

Roviello et al. (2011) reported the structural classification, chemical synthesis strategies, and serum stability of synthetic nucleopeptides and peptidyl-nucleoside analogs. In addition, this study investigated the enzymatic stability of nucleopeptides in human serum and determined their half-life to be about 2 hours in the presence of 50% fresh human serum.

Du et al. (2012) reviewed how using enzymes to direct the self-assembly of adenosine nucleosides in water provides a new class of molecular nanofibers/hydrogels as functional soft materials.

Yuan et al. (2015) selected short peptide sequences from the interface of a heterodimer of proteins with known crystal structure for conjugation with nucleobases to form nucleopeptides. The resulting nucleopeptides self-assemble to form nanofibers, producing supramolecular hydrogels upon simple mixing of two distinct nucleopeptides in water, allowing the creation of soft biomaterials via a supramolecular hydrogelation triggered by mixing heterodimeric nucleopeptides, which have proteolytic resistance against proteinase K. 

Roviello et al. (2016) described two solid-phase synthetic routes to obtain a nucleo-oligolysine α-peptide containing all four natural nucleobases. The first strategy is based on oligomerizing the nucleobase-containing monomers. The second strategy has the advantage of avoiding solution synthesis of the monomeric building blocks, leading to the final nucleopeptide by direct solid-phase coupling of suitably protected nucleobases with the free amino groups on the growing peptide chain still anchored to the resin. Also in 2026, Roviello’s research group reported the solid-phase synthesis and RNA-binding activity of an arginine-containing nucleopeptide.

Du et al. (2017) reported the design and characterization of nucleopeptides that self-assemble in water and interact with single-stranded DNAs (ssDNAs). These nucleopeptides bind to ssDNAs. The interactions between nucleopeptides and ssDNAs decreased their proteolytic resistance against proteinase K. Further, it was found that nucleopeptides can interact with plasmid DNA and deliver hairpin DNA into cells.

Musumeci et al. (2018) described the synthesis and characterization of a thymine-bearing nucleoamino acid based on the L-diaminopropionic acid (L-Dap) and its solid-phase oligomerization to α-peptides (oligoDapT). The resulting products were characterized using mass spectrometry, spectroscopic techniques, and scanning electron microscopy (SEM) analysis. This analytical study suggested that Dap-based nucleopeptides are interesting nucleic acid-binding tools that need to be further explored for their ability to modulate DNA- and RNA-based biological processes.

Wang et al. (2018) reported that nucleopeptide assemblies selectively sequester ATP in complex conditions, such as in serum and the cytosol, illustrating that supramolecular assemblies can interact with small and essential biological molecules to control cell behaviors.

Baek et al. (2019) constructed a nucleo-tripeptide library to identify molecules that form hydrogels under physiological conditions. The research group used both experimental and computational approaches to study the resulting self-assembled structures. It demonstrated that nucleo-tripeptides can form nanofibrous hydrogels through Watson–Crick base pairing and π–π stacking interactions. Further, self-assembly conditions are mediated by nucleo-tripeptide hydrophobicity and amphiphilicity and therefore can be regulated by rational molecular design. Certain structures derived from specific conjugates of peptides and nucleobases form hydrogels under physiologic conditions, making them promising candidates for biomedical applications.

Avitabile et al. (2019) reported the structural and spectroscopic characterization of aggregates formed by nucleobases and peptide nucleic acid (PNA)-peptide conjugates. At high concentration, all studied nucleobases formed aggregates characterized by previously unreported fluorescence properties. The conjugation of bases via PNAs to the dipeptide Phe-Phe leads to the formation of novel hybrid assemblies, which an amyloid-like association of the monomers characterizes. These compounds shared the same basic cross-β motif; however, the nature and number of PNA units affected both the level of structural order and the intrinsic fluorescence of the self-assembled nanostructure.

Boback et al. (2020) reported the design and characterization of guanosine-containing self-assembling nucleopeptides that form nanosheets and nanofibers and proposed that the peptide component of the nucleopeptide drives the assembly into β-sheet structures with hydrogen-bonded guanosine forming additional secondary structures cooperatively within the peptide framework. The researchers synthesized and assembled a tetrapeptide (Gly-Lys-Phe-Phe) modified on the N-terminus with guanosine. Based on their analytical results, they proposed that the gs-GKFF-OH nucleopeptide accommodates individual G-quartets within the peptide framework.

Scognamiglio et al. (2021) reviewed the characteristics of nucleopeptides and pseudopeptides (PNAs) for diagnostic and therapeutic applications. The researchers described the properties of nucleopeptides, PNAs, and related supramolecular systems, as well as relevant applications of these systems. The researchers suggested that nucleopeptide assemblies formed by peptides carrying both single- and oligo-nucleobase may be utilized as components in the design of novel, valuable bioinspired materials useful as biomedical and nanomaterials.

Eruera et al. (2021) reviewed the nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, the nucleotidylylation process itself, and the roles that these modifications have in the viral life cycle. Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses. During the nucleotidylylation reaction, the norovirus protease-polymerase (ProPol) catalyzes a nucleophilic attack by the hydroxyl of Y26 of viral protein genome-linked protein (VPg) on the alpha phosphate of GTP, producing a phosphodiester bond that covalently links GMP to Y26 of VPg.

Also in 2021, Klabenkova et al. reviewed the chemistry of peptide-oligonucleotide conjugates. POCs represent a successful approach to increase cellular uptake, tissue delivery, bioavailability, and overall efficiency of therapeutic nucleic acids, for example, for antisense oligonucleotides and small interfering RNAs. 

Palumbo et al. (2022) reported synthesis methods for willardiine analogs and also discussed their neuropharmacological effects. Willardiine is a natural nucleobase amino acid containing uracil that acts as a partial agonist for ionotropic glutamate receptors (AMPA and kainate). Willardiine is part of the uracilylalanine family found in higher plants. This compound can serve as a building block for the synthesis of nucleopeptides-DNA chimeras.

Scognamiglio et al. (2023) studied the self-assembly properties of different nucleobase-containing molecules, focusing on their gel-forming properties. This study focused on design principles for enhancing the stability of natural nucleic acids needed for the development of advanced nucleobase-containing gel-based biomaterials.

Mucha et al. (2025) synthesized an Fmoc-protected nucleobase amino acid monomer (Fmoc-1,4-TzlNBAA) with adenine attached to the side chain of L-homoazidoalanine (Aha) through a 1,4-linked 1,2,3-triazole in combination with a Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) of Fmoc-Aha and N9-propargyladenine. Furthermore, a homotrinucleopeptide (HalTzlAAA) containing three 1,4-TzlNBAA residues was synthesized using different solid-phase peptide synthesis (SPPS) approaches.

Circular dichroism (CD) and fluorescence spectroscopy confirmed the binding of HalTzlAAA to U-rich motifs of the transactivation responsive element (TAR UUU RNA HIV-1) bulge and the anticodon stem–loop domain of human tRNALys3 (ASLLys3). 5′-(FAM(6))-labeled TAR UUU and hASLLys3 allowed fluorescence anisotropy binding studies. 

Giraud et al. (2026) presented a series of six nucleo­peptides derived from two distinct peptide sequences, Phe-Glu-Phe-Glu and Phe-Lys-Phe-Lys, which are negatively and positively charged at physiological pH, making them complementary in terms of electrostatic interactions. The peptides were functionalized with one of the four DNA nucleobases, introduced via a peptide nucleic acid (PNA) moiety. This study described the design of novel supramolecular hydrogels derived from nucleopeptides, enabling new design strategies for producing innovative hydrogels with finely tuned properties.

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