The Nucleotide Scaffold — Standalone Panel

Anatomy of a nucleotide — bases, sugar, backbone & termini

guanosine-5′-monophosphate centre

↓ one base + one sugar + phosphate assemble into a nucleotide ↓

Nucleobase

Ribose 2′

Phosphate backbone

5′ / 3′ termini

Modifications by domain & representative chemistries

Nucleobase modifications

base-pairing edge

Duplex stability ↑ Tm

2-amino dA 5-methyl dC C5-propynyl U/C G-clamp

mRNA · immune suppression

pseudouridine Ψ N1-methyl-Ψ 5-methylcytidine N6-mA

affinity · coding · innate immunity

2′-Ribose modifications

sugar · 2′ position

Nuclease resistance + stability

2′-OMe 2′-F 2′-MOE 2′-FANA 4′-thio-RNA

Conformational locking ↑ Tm

LNA cEt BNA ENA

RNase-H sparing · 3′-endo pucker

Phosphate modifications

internucleotide backbone

Nuclease resistance

phosphorothioate [PS] phosphorodithioate [PS2] mesyl phosphoroamidate [Ms]

Charge-neutral backbone

methylphosphonate PNA morpholino (PMO)

plasma stability · protein binding

5′ / 3′ terminus mods

chain ends

Exonuclease resistance

3′→3′ inverted dT 5′→5′ linkage 3× PS at termini

Attachment / detection handles

amino-C6 thiol-C6 DBCO azide biotin

5′-phosphate · 5′-vinylphosphonate

Abbreviations

LNA locked nucleic acid cEt constrained ethyl BNA BNA bridged nucleic acid ENA ethylene-bridged NA MOE 2′-O-methoxyethyl FANA 2′-fluoroarabinonucleic acid PS phosphorothioate PMO phosphorodiamidate morpholino PNA peptide nucleic acid DBCO dibenzocyclooctyne Ψ pseudouridine m1Ψ N1-methyl-pseudouridine Tm melting temperature RNase-H ribonuclease H

Modification goals & representative chemistries

Duplex stability

↑ Tm · tighter hybridization

Base

2-amino dA

5-methyl dC (5mC)

C5-propynyl U/C

N6-methyl adenosine

Sugar

LNA / cEt / BNA

2'-OMe · 2'-F

2'-MOE · ENA

mRNA / IVT

Pseudouridine (Ψ)

N1-methyl-Ψ

5-methylcytidine

Nuclease resistance

↓ exo/endonuclease cleavage

Phosphate

PS — most used

Phosphorodithioate

Mesyl phosphoroamidate

Sugar (2')

2'-MOE — best in vitro

2'-OMe · 2'-F

LNA · FANA

Termini / backbone

3'→3' inv. dT cap

5'→5' linkage

Morpholino (PMO)

PNA

Surface attachment

covalent & affinity tethering

Reactive handles (5′ / 3′)

Amino-C3/C6 (–NH₂)

Thiol-C3/C6 (–SH)

Carboxyl (–COOH)

Maleimide

NHS-ester

Click chemistry

Azide / DBCO (SPAAC)

TCO / tetrazine (iEDDA)

Alkyne / CuAAC

Affinity tethers

Biotin–streptavidin

Digoxigenin (DIG)

His-tag · NTA-Ni

Fluorescent detection

FISH · qPCR · molecular beacons

Terminal dyes

FAM (em 520 nm)

HEX · TET · JOE

TAMRA · ROX

AZ dyes series

CF dyes series

Atto dyes series

Cyanine series

Cy3 (em 570 nm)

Cy5 (em 670 nm)

Cy7 (NIR, 780 nm)

FRET / quenchers

BHQ-1 · BHQ-2

BBQ-650

Donor–acceptor pairs

Internal labels

dU-fluorophore

2-AP (intrinsic)

Pyrrolo-dC

Cellular delivery

↑ intracellular uptake

Hydrophobic conjugates

Cholesterol (3' conj.)

Tocopherol (α-VitE)

DHA · fatty acids

C22-2'O bases

Lipid Modified Oligos (LMO): C16, C18, C22, C24 etc.

Receptor targeting

GalNAc (ASGPR, liver)

Folate receptor

RGD peptide (αvβ3)

Aptamer conjugate

Nanoparticle / LNP

Ionisable lipid (LNP)

Cationic polymer

Cell-pen. peptide

Endosomal escape

pH-sensitive lipid

Fusogenic peptide

Photocleavable linker

siRNA / ASO therapeutics

RNAi · splice switching · steric block

siRNA modification pattern

Alternating 2'-OMe / 2'-F

PS at both termini (3×)

GalNAc-3' conjugate

5'-vinylphosphonate

ASO gapmers (RNase H)

LNA / cEt flanks

2'-MOE wings

Central DNA gap

Steric-block ASO

Full 2'-OMe / 2'-F

Morpholino (PMO)

PNA (splice-switch)

Reduce immunostimulation

2'-OMe at UG motifs

Pseudouridine substitution

Unmethylated CpG avoidance