Synthesis of specifically modified oligonucleotides for application in structural and functional analysis of RNA

Abstract

Nowadays, RNA synthesis has become an essential tool not only in the field of molecular biology and medicine, but also in areas like molecular diagnostics and material sciences. Beyond synthetic RNAs for antisense, aptamer, ribozyme, and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. This often requires labeling of the RNA with a suitable spectroscopic reporter group. Herein, we describe the synthesis of functionalized monomer building blocks that upon incorporation in RNA allow for selective reaction with a specific reporter or functional entity. In particular, we report on the synthesis of 5'-O-dimethoxytrityl-2'-O-tert-butyldimethylsilyl protected 3'-O-phosphoramidites of nucleosides that carry amino linkers of different lengths and flexibility at the heterocyclic base, their incorporation in a variety of RNAs, and postsynthetic conjugation with fluorescent dyes and nitroxide spin labels. Further, we show the synthesis of a flavine mononucleotide-N-hydroxy-succinimidyl ester and its conjugation to amino functionalized RNA.

Figure 1

Structures of the commercially available building blocks used in our RNA functionalization studies.

Figure 2

Amino-modified monomer building blocks used for RNA synthesis and functionalization.

Figure 3

Amino linkers used for preparation of amino-modified nucleosides.

Figure 4

Synthesis of the guanosine-phosphoramidite. (i) 8-bromoguanosine 15, 2.0 eq. L3, 0.1 eq. Pd(PPh₃)₄, 0.2 eq. CuI, 1.2 eq. DIPEA, 40°C, 20 h (80%); (ii) 16, pyridine, 8.2 eq. TMS-Cl, 0°C, 2 h, 2.2 eq. isobutyric anhydride, rt, 2 h, NH₃ up to 2.5 M, 0°C; (iii) 17, pyridine,1.5 eq. DMT-Cl, 0°C, 2.5 h; (iv) 18, THF, 3.7 eq. pyridine,  1.5 eq. AgNO₃, 15 min, 1.7 eq. TBDMS-Cl, rt, 8.5 h; (v) 19, DCM, 4 eq. DIPEA, 1.5 eq. 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, rt, 2.5 h.

Figure 5

Postsynthetic labeling of amino-modified oligonucleotides: (i) Chemical synthesis of amino-modified RNA; (ii) Deprotection; (iii) Postsynthetic labeling with fluorescent dyes.

Figure 6

Activated derivatives of fluorescent dyes for postsynthetic labeling of RNA.

Figure 7

MALDI-TOF mass spectra of the amino-modified oligonucleotide 2-L3-A (a) and its corresponding conjugate with ATTO647N (b). For the sequence of 2-L3-A, compare Table 1.

Figure 8

Hybridization of Alexa488-labeled D1L3 and Cy5-labeled A1L3: 3 μM Alexa488-labeled D1L3: 3 μM Cy5-labeled A1L3, 10 mM MgCl₂, 50 mM Tris buffer, pH 7.4, total volume 100 μL, 78°C 3 min, slowly cooling to rt. The hybrid was analyzed by 15% native PAA-gel: (left) illuminated at 254 nm, (right) illuminated at 365 nm.

Figure 9

Synthesis of protected riboflavine derivatives: (i) DMT-Cl, DMAP, pyridine, rt, overnight (53%); (ii) acetic anhydride, pyridine, 0°C → rt, overnight (90%); (iii) ZnBr₂ in nitromethane, 2 min (83%).

Figure 10

Synthesis of the fully protected linker unit: (i) Na, tert-butyl acrylate, THF, rt, 1 d (34%); (ii) 2-Cyanoethyl-N,N-diisopropropylchlorophosphoramidite, DIPEA, DCM, rt, 1.5 h (quant.); (iii) (a) BMT, MeCN, rt, 1 h; (b) 0.2 M I₂ in THF/pyridine/H₂O (2 : 1 : 1) (32%).

Figure 11

(a) Structure of the two nucleotide analogs used for the incorporation of an aliphatic amino group into the aptazyme RNA. (b) Two-stranded (left) and three-stranded (right) aptazyme. The modifications AMU and APU are located at position U12 (red) and U14 (green), respectively.

Figure 12

(a) Formation of the N-hydroxysuccinimidyl (NHS) ester. (b) PAGE analysis of the coupling reaction between amino-modified RNA HPAT1-BHI (for sequence see Table 3) and the FMN derivative. Visualization is at UV 254 nm (A) and 365 nm (B).

Figure 13

Strategy for postsynthetic introduction of spin labels to 4-thiouridine containing RNA.

Figure 14

Labeling of the 2′-amino group of a model RNA.