Why is TNA coupling often more difficult

Threofuranosyl nucleotides are the building blocks of α-l-threose nucleic acids polymers (TNAs). The TNA moiety has one less atom in its backbone than the DNA backbone. A threofuranose moiety is part of the sugar backbone of threofuranosyl nucleotides. Unlike the ribose sugar in RNA and the deoxyribose sugar in DNA, threofuranose has four carbon atoms arranged in a furan ring, giving it a slightly different shape. TNA oligomers contain α-L-threofuranosyl nucleotide repeats connected by 2′-,3′-phosphodiester linkages.

Because TNA has a shorter 2′,3′-linked threose backbone than DNA or RNA, the geometry of the growing chain end differs.

During TNA synthesis, the incoming TNA unit can adopt a less favorable pre-catalytic base-pair geometry. As a result, TNA building blocks are more difficult to handle than standard DNA or RNA phosphoramidite units. TNAs form Watson–Crick pairs with TNA, DNA, and RNA, but their backbones are structurally different enough to make synthesis more difficult.

Changes to make to improve synthesis success

• Reduce consecutive TNA incorporations
• If you are inserting several TNA residues back-to-back, start with single-site or widely spaced TNA substitutions. Consecutive difficult couplings multiply losses very fast.
• Place TNA away from the most synthesis-sensitive region

A practical starting rule is to avoid an initial design with a dense TNA block at the end of the synthesis path.

Start with testing:

• One internal TNA
• One terminal TNA
• Two TNA residues separated by 2–4 natural bases

How to design a TNA-modified oligonucleotides?

There is not a universal “best TNA pattern,” so the safest design strategy is empirical testing.

Good starting design rules

For a first-pass TNA-modified oligo:

• keep the oligo short
• start with 1–3 TNA residues total
• avoid adjacent TNA residues
• place TNA at positions where you want nuclease resistance or to probe binding tolerance
• if targeting RNA, TNA is attractive because it can hybridize with RNA and has often been discussed as having a preference for RNA over DNA partners.

If your goal is the synthesis of antisense oligonucleotides for improved hybridization

A practical screening set is:

• Select the parent DNA/RNA oligonucleotides
• Place a single TNA at the 5′-end
• Place single TNA at the 3′-end
• Add a single internal TNA
• Or add two separated internal TNAs
• Or one 3′-terminal plus one internal TNA

The results will tell you quickly whether the chemistry or the biology is the coupling bottleneck.

If your goal is the synthesis of aptamers or binding ligands

Do not densely TNA-modify a known aptamer at random. TNA can support folding and function, but tertiary structure is sensitive. Start by modifying stem or support positions, not the most conserved binding loop, unless you are doing a dedicated selection campaign. TNA has been shown to support evolved functional structures, but that does not mean every DNA/RNA aptamer tolerates wholesale replacement.

How to start a synthesis of a TNA oligonucleotide

Us a simple starter design template

For a target sequence like:

5'-N₁N₂N₃N₄N₅N₆N₇N₈N₉N₁₀N₁₁N₁₂-3'

start with variants like:

• 5'-N₁N₂N₃N₄[TNA-N₅]N₆N₇N₈N₉N₁₀N₁₁N₁₂-3'
• 5'-[TNA-N₁]N₂N₃N₄N₅N₆N₇N₈N₉N₁₀N₁₁N₁₂-3'
• 5'-N₁N₂N₃N4N₅N₆N₇[TNA-N₈]N₉N₁₀N₁₁N₁₂-3'
• 5'-N₁N₂[TNA-N₃]N₄N₅N₆N₇[TNA-N₈]N₉N₁₀N₁₁N₁₂-3'

Avoid starting with:

• [TNA-N₁][TNA-N₂][TNA-N₃]...
• Or long contiguous TNA blocks
• Or very long oligonucleotides with many TNA sites before the single-site chemistry works

In summary

For TNA-modified oligos, design conservatively first:

• Use 1–2 isolated TNA substitutions
• Use a short sequence
• Do not us adjacent TNA residues