Aqueous Compatible Post-Synthetic On-Column Conjugation of Nucleic Acids Using Amino-Modifiers

Abstract

Nucleic acid conjugation methodologies involve linking the nucleic acid sequence to other (bio)molecules covalently. This typically allows for nucleic acid property enhancement whether it be for therapeutic purposes, biosensing, etc. Here, we report a streamlined, aqueous compatible, on-column conjugation methodology using nucleic acids containing a site-specific amino-modifier. Both monophosphates and carboxylates were amenable to the conjugation strategy, allowing for the introduction of a variety of useful handles including azide, aryl, and hydrophobic groups in DNA. We find that an on-column approach is superior to post-synthetic template-directed synthesis, mainly with respect to product purification and recovery.

Keywords: Amide linkage; Nucleic acid conjugation; Phosphoramidate linkage; Solid-support conjugation.

Figure 1

Aqueous compatible on‐column functionalization to prepare oligonucleotides containing a phosphoramidate or amide linkages.

Scheme 1

Synthesis of activated 5′‐phosphoroimidazolide‐3′‐azido‐3′‐deoxythymidine 2 and −3′‐azido‐2′, 3′‐dideoxy‐5‐methylcytidine 5′‐O‐monophosphate 4. Reagents and conditions: (i) POCl₃ (4.0 eq.), Py, 0 °C, 10 min; (ii) 2‐methylimidazole (4 eq.), EDC⋅HCl (5 eq.), NEt₃ (10 eq.) DMF, 22 °C, 24 h; (iii) POCl₃ (4.0 eq.), NEt₃ (2.0 eq.), trimethylphosphate, 0 °C, 2 h.

Figure 2

Template‐directed synthesis (TDS) reaction of a DNA primer on an RNA template using (A) 2‐methylimidazole activated monomer 2. (i) Reaction conditions were DNA primer (1 μM), RNA template (2.5 μM), MgCl₂ (5 mM), monomer (40 mM), HEPES buffer (200 mM, pH 8.0). PAGE analysis illustrating reaction time‐course of (ii) templated and (iii) untemplated reaction. (B) EDC‐mediated condensation reaction using monophosphate 1. (i) Reaction conditions were DNA primer (5 μM), RNA template (10 μM), MgCl₂ (20 mM), 5′‐O‐monophosphate 1 (100 mM), EDC (400 mM) for 8 hours in HEPES buffer (200 mM, pH 8.0). (ii) PAGE analysis of a control experiment showcasing the effect of removing the template (−T), monomer (−M), magnesium (−Mg²⁺), and EDC (−EDC). “C” is for the primer‐strand only control. (iii) Same conditions as (ii) but at 3 °C instead of 22 °C.

Figure 3

On‐column EDC‐mediated conjugation using a 5′‐amino DNA construct. (A) (i) Reaction scheme of the on‐column amino‐modifier conjugation strategy using a 5′‐O‐monophosphate to generate a P5′→N5′ phosphoramidate linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐amino terminal and (b) the product strand containing the azido functional group. Values indicate retention times (min). (iii) Low‐resolution electrospray ionization mass spectrum (negative mode) of the purified product strand showcasing m/z signals in agreement with the expected m/z. (B) (i) The reaction scheme of the on‐column amino‐modifier conjugation strategy using a 3′‐O‐monophosphate generating a P3′→N5′ phosphoramidate linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐amino terminal and (b) the product strand containing the azido functional group. Values indicate retention times (min). (iii) Low‐resolution mass spectrum of product strand showcasing m/z signals in agreement with the expected m/z. The solid‐support was treated with 100 mM monophosphate, 100 mM N‐MeIm, 800 mM EDC in 250 mM HEPES pH 8.0 containing 25 % MeCN (v/v) and allowed to react for 8 h. The solid support was treated a second time with a freshly prepared coupling solution, after thorough washing of the solid‐support bound oligonucleotide.

Figure 4

On‐column EDC‐mediated conjugation using a 3′‐amino terminal oligonucleotide. (i) Reaction scheme of the on‐column amino‐modifier conjugation strategy using a 5′‐O‐monophosphate to generate a N3′→P5′ phosphoramidate linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐amino terminal and (b) the product strand containing the azido functional group. Values indicate retention times (min). (iii) Low‐resolution electrospray ionization mass spectrum (negative mode) of the purified product strand showcasing m/z signals in agreement with the expected m/z. On‐column EDC‐mediated conjugation using 3′‐amino DNA construct and 5′‐O‐monophosphate. The solid‐support was treated with a coupling solution of 100 mM monomer, 100 mM N‐MeIm, 800 mM EDC in 250 mM HEPES pH 8.0 containing 25 % MeCN (v/v) and allowed to react for 8 h. The solid support was treated a second time with a freshly prepared coupling solution, after thorough washing of the solid‐support bound oligonucleotide.

Figure 5

On‐column EDC‐mediated conjugation using a 5′‐amino DNA construct. (i) Reaction scheme of the on‐column amino‐modifier conjugation strategy using a 3′‐O‐monophosphate nucleoside to generate a P3′→N5′ phosphoramidate linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐amino terminal and (b) the product strand containing the azido functional group. The synthesis retained the DMTr. Values indicate retention times (min). (iii) Low‐resolution electrospray ionization mass spectrum (negative mode) of the purified product strand showcasing m/z signals in agreement with the expected m/z. On‐column EDC‐mediated conjugation using 3′‐amino DNA construct and 3′‐O‐monophosphate. The solid‐support was treated with a coupling solution of 100 mM monophosphate, 100 mM N‐MeIm, 800 mM EDC in 250 mM HEPES pH 8.0 containing 25 % MeCN (v/v) and allowed to react for 8 h. The solid support was treated a second time with a freshly prepared coupling solution, after thorough washing of the solid‐support bound oligonucleotide.

Figure 6

On‐column EDC‐mediated conjugation using 3′‐amino DNA construct and uridine 5′‐O‐monophosphate (UMP), follow by the simultaneous extension at both the 2′‐ and 3′‐OH. (i) Reaction scheme of the on‐column amino‐modifier conjugation strategy using a riboside 5′‐O‐monophosphate to generate a N3′→P5′ phosphoramidate linkage capable of subsequent branching. PG refers to the cyanoethyl group. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 3′‐amino terminal, (b) the product strand containing the uridine unit, and (c) the product strand resulting for subsequent extension of two thymidinyl units at the 2′‐ and 3′‐positions. Values indicate retention times (min). (iii) Low‐resolution electrospray ionization mass spectrum (negative mode) of the purified product strands showcasing m/z signals in agreement with the expected m/z. (a) After the coupling of the UMP unit and (b) after the branch extension.

Figure 7

On‐column EDC‐mediated conjugation using a 5′‐hexylamino‐containing DNA construct with monophosphate 1. (i) Reaction scheme of the on‐column amino‐modifier conjugation strategy using a 5′‐O‐monophosphate nucleoside to generate a phosphoramidate linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐hexylamino terminal and (b) the product strand containing the azido functional group. Values indicate retention times (min). (iii) Low‐resolution electrospray ionization mass spectrum (negative mode) of the purified product strand showcasing m/z signals in agreement with the expected m/z. The solid‐support was treated with a coupling solution of 100 mM monophosphate, 100 mM N‐MeIm, 800 mM EDC in 250 mM HEPES pH 8.0 containing 25 % MeCN (v/v) and allowed to react for 8 h. The solid support was treated a second time with a freshly prepared coupling solution, after thorough washing of the solid‐support bound oligonucleotide.

Figure 8

On‐column EDC‐mediated conjugation of amino‐containing DNA and carboxylates. (A) (i) Reaction scheme of the on‐column 5′‐amino‐modified DNA conjugation strategy using commercially available carboxylate handles such as potassium benzoate and Fmoc‐protected glycine (Fmoc‐Gly‐OH), generating an amide linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐amino terminal, (b) the crude reaction from the conjugation with potassium benzoate, and (c) the crude reaction from the conjugation with Fmoc‐Gly‐OH. Values indicate retention times (min). (iii) Low‐resolution mass spectrum of the purified product strands showcasing m/z signals in agreement with the expected m/z for (a) conjugation with potassium benzoate and (b) conjugation with Fmoc‐Gly‐OH. (B) Same as (A) but using a 3′‐amino modified DNA conjugated with oleic acid. Optimal conditions were as follows: 100 mM carboxylate, 100 mM N‐MeIm, 800 mM EDC in 100 mM HEPES pH 8.0 containing 50 % DMF (v/v). Treatment was repeated with fresh coupling solution after 8 h, after thorough washing of the solid‐support bound oligonucleotide.

Figure 9

On‐column EDC‐mediated conjugation of 5′‐amino‐modified DNA and DOTA. (A) (i) Reaction scheme of the on‐column conjugation strategy using commercially available protected DOTA, generating an amide linkage. (ii) SAX‐HPLC trace of (a) the crude starting material strand containing a 5′‐amino terminal, (b) the crude reaction from the conjugation reaction with DOTA. (iii) Low‐resolution electrospray ionization mass spectrum (negative mode) of the purified product strands showcasing m/z signals in agreement with the expected m/z for DOTA conjugate. Optimal conditions were as follows: 100 mM carboxylate, 100 mM N‐MeIm, 800 mM EDC in 100 mM HEPES pH 8.0 containing 50 % DMF (v/v). Treatment was repeated with fresh coupling solution after 8 h, after thorough washing of the solid‐support bound oligonucleotide. Note that the expected m/z includes a potassium adduct (as previously observed.