Oligonucleotide drugs (OND), also known as small nucleic acid drugs, are short-chain molecules composed of nucleotides (deoxyribonucleic acid or ribonucleic acid) with no more than 30 bases/base pairs. They achieve high selectivity by forming Watson-Crick base-pairing with DNA or RNA. Oligonucleotide therapies function through strategies such as gene silencing, splice modulation, and gene activation, effectively preventing the expression of many erroneous proteins.

Small nucleic acid drugs include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), aptamers, small activating RNA (saRNA), and peptide-oligonucleotide conjugates.

Peptide-Mediated Oligonucleotide Delivery
Small nucleic acids exhibit poor stability and low specificity, requiring various chemical modifications to enhance their stability and cellular absorption. Modifications to the phosphate backbone, ribose moiety, and the bases themselves have been widely employed to improve the drug-like properties of oligonucleotide drugs, enhancing delivery efficiency. These modifications significantly improve the pharmacokinetics, pharmacodynamics, and biodistribution of oligonucleotides.

Oligonucleotide Modification
The entry of small nucleic acid drugs into cells faces two main challenges: RNA exposed in the blood is susceptible to degradation by nucleases in plasma and tissues, and negatively charged RNA has difficulty crossing cell membranes. Breakthroughs in chemical modifications and delivery system technologies play a crucial role in the development of nucleic acid drugs. Specific ligand conjugation to nucleic acid drugs, such as peptide-oligonucleotide conjugates (POC), peptide-PMO (phosphorodiamidate morpholino oligomers), peptide-siRNA conjugates, peptide-PNA conjugates, and peptide-RNA conjugates, represents a potent strategy for achieving targeted delivery to specific cells.

Peptides as Delivery Vehicles
Peptides possess characteristics such as low molecular weight, low immunogenicity, high specificity, and renal excretion, combining the advantages of both large and small molecules. Peptides for drug delivery include cell-penetrating peptides (CPP), cell-targeting peptides (CTP), self-assembling peptides (SAP), and responsive peptides. By forming peptide-oligonucleotide conjugates (POC), they can deliver payload molecules specifically to target cells and tissues. Whether through covalent bonding or forming nano-complexes with payload molecules, peptide delivery carriers have significant advantages, allowing them to penetrate challenging tissues such as muscles, bone marrow, and the blood-brain barrier. For example, CPPs are short peptides capable of translocating small molecule cargo across cell membranes. POCs, as potential therapeutic drugs, have crucial functions and, due to their stability, can resist the action of intracellular enzymes in different cellular compartments. Thiol-maleimide coupling is a commonly used method for covalent bonding.

Methods of Peptide-Oligonucleotide Conjugation
The synthesis of peptide-oligonucleotide conjugates involves two main methods: one is the separate synthesis of peptides and oligonucleotide fragments followed by their conjugation in solution (more common). The other method involves the stepwise solid-phase synthesis of peptides (or oligonucleotides) on the same solid support, followed by the synthesis of oligonucleotide fragments (or peptides).

Post-Synthetic Conjugation Method
This method requires introducing a pair of chemically reactive groups into appropriate positions of the peptide and oligonucleotide fragments. Then, the peptide and oligonucleotide fragments are connected through chemoselective conjugation reactions, forming a covalent bond with the participation of corresponding reactive groups in each segment.

Thiol Group: Conjugation of peptides and oligonucleotides can occur through thiol-maleimide coupling or nucleophilic substitution with haloacetamides, commonly used in POC synthesis.

Carbonyl Group: Aldehydes can react with compounds containing aminoxy, 1,2-aminothiol groups (from cysteine), and hydrazine. For example, oligonucleotides with carbonyl groups can couple with peptides containing aminoxy groups.
Amino Group: Peptides with N-terminal cysteine residues and modified oligonucleotides connect through the formation of amide bonds.

Click Chemistry: Introduction of a clickable pair of reporter groups, such as alkyne-azide or tetrazine-diene, into peptide and oligonucleotide fragments.

Stepwise Solid-Phase Synthesis Method
This method eliminates the need for purification and cleavage of segments from the support until the assembly of the full-length conjugate is complete. The synthesis of peptide segments is carried out using Fmoc or Boc solid-phase chemistry, while the synthesis of oligonucleotide segments is conducted using standard phosphoramidite methods. In general, protecting groups for peptides and oligonucleotides in stepwise solid-phase synthesis should be compatible to allow for smooth deprotection and cleavage from the support at the end of synthesis, avoiding side reactions and residuals. To efficiently obtain POCs, it is necessary to select orthogonal combinations of protecting groups, conditions for deprotection and cleavage, solid supports, and linkers for connecting different segments.