Robert Letsinger and the Evolution of Oligonucleotide Synthesis

Abstract

This retrospective summarizes a presentation given at the symposium "Bob Letsinger, PhD-100 Years of History" on September 28, 2021 as part of the 17th annual meeting of the Oligonucleotide Therapeutics Society (OTS). In it I look back at my encounters with Robert Lewis Letsinger (1921-2014) while at Northwestern University as Assistant Professor between 1995 and 2000.

Figure 1

Zoom picture of symposium speakers. Top row: Muthiah Manoharan (Alnylam Pharmaceuticals), Martin Egli (Vanderbilt University), Masad J. Damha (McGill University). Middle row: Marvin H. Caruthers (University of Colorado), Paul S. Miller (Johns Hopkins University), Margaret E. Schott (Northwestern University). Bottom row: Sergei M. Gryaznov (MAIA Biotechnology), Reed Letsinger (son). Missing: Chad A. Mirkin (NWU) and Kevin K. Ogilvie (former President of Acadia University and Senator for Annapolis Valley—Hants, Nova Scotia, Canada). Photo credit: Martin Egli.

Figure 2

Key steps in the development of oligonucleotide synthesis with selected contributions by Bob circled in red and a (very) brief history of chemically modified nucleic acids and the FDA approval of oligonucleotide therapeutics. See also: https://www.trilinkbiotech.com/a-short-history-of-oligonucleotide-synthesis. Adapted with permission from Egli, M.; Manoharan, M. Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics. Nucleic Acids Res. 2023, 51, 2529–2573. Copyright 2023 Oxford University Press.

Figure 3

Classification of synthetic oligonucleotides.

Figure 4

Key issues in the discovery and development of nucleic acid therapeutics.

Figure 5

Structures of (a) homo-DNA and (b) DNA and base pairing priorities in the two systems.

Figure 6

Conformational analysis of homo-DNA and the proposed (a) sc/sc and (b) ap/sc (α/ζ) backbone models. For the crystal structure of homo-DNA, please see ref (9).

Figure 7

Publication by Gryaznov and Letsinger in Nucleic Acids Research describing the synthesis and properties of oligonucleotides containing aminodeoxythymidine units. Adapted with permission from Gryzanov, S. M.; Letsinger, R. L. Synthesis and Properties of Oligonucleotides Containing Aminodeoxythymidine Units. Nucleic Acids Res. 1992, 20, 3403–3409. Copyright 1992 Oxford University Press.

Figure 8

Thermal stabilities of dodecamer duplexes with (a) DNA, (b) N3′ → P5′ phosphoramidate DNA, (c) P3′ → N5′ phosphoramidate DNA, and (d) RNA backbones. The T_m values were measured in 10 mM Tris·HCl pH 7.0 and 150 mM NaCl.

Figure 9

Ion coordination in the crystal structure of the N3′ → P5′ duplex [d(CnpGnpCnpGnpAnpAnpTnpTnpCnpGnpCnpG)]₂ established the orientations of the 3′-amino group lone pair and hydrogen. Adapted with permission from Egli, M; Gryaznov, S. M. Synthetic Oligonucleotides as RNA Mimetics: 2′-Modified RNAs and N3′ → P5′ Phosphoramidates. Cell. Mol. Life Sci. 2000, 57, 1440–1456. Copyright 2000 Springer Nature.

Figure 10

Crystal structure of (a) N3′ → P5′ phosphoramidate-DNA helped rationalize its stable self-pairing and cross-pairing with and mimicking of RNA as well as the inability of (b) P3′ → N5′ phosphoramidate-DNA to self-pair and cross-pair with RNA. Nitrogen lone pair and P–O5′ antibonding orbital are indicated by green and black lobes, respectively, and a red arrow indicates a steric conflict. Adapted with permission from Tereshko, V.; Gryaznov, S.; Egli, M. Consequences of Replacing the DNA 3′-Oxygen by an Amino Group: High-Resolution Crystal Structure of a Fully Modified N3′ → P5′ Phosphoramidate DNA Dodecamer Duplex. J. Am. Chem. Soc. 1998, 120, 269–283. Copyright 1998 American Chemical Society.

Figure 11

Origins of AP-RNA’s resistance to degradation by SVPD. (a) Gel images depicting time courses (in min) of the degradation of DNA, PS-DNA and AP-RNA. (b) Crystal structure of the complex between E. coli DNA Pol I Klenow 3′-exonuclease and an oligo-2′-deoxynucleotide carrying three AP-RNA residues at its 3′-terminal end.

Figure 12

Mechanism of 3′-exonuclease inhibition by AP-RNA. The absence of a second metal ion due to formation of a salt bridge between Asp424 and the 2′-O-AP substituent prevents cleavage of the phosphodiester bond (red cross). Adapted with permission from Egli, M; Gryaznov, S. M. Synthetic Oligonucleotides as RNA Mimetics: 2′-Modified RNAs and N3′ → P5′ Phosphoramidates. Cell. Mol. Life Sci. 2000, 57, 1440–1456. Copyright 2000 Springer Nature.

Figure 13

Stilbene–diamide conjugation: electron transfer and record thermal stability.

Figure 14

Stereo diagram depicting face-to-face and edge-to-face interactions between trans-stilbene and G:C base pairs in the crystal structure of stilbene–diether (Sd)-capped DNAs 5′-GTTTTG-Sd-CAAAAC-3′.^, Adapted with permission from Egli, M.; Tereshko, V.; Murshudov, G. N.; Sanishvili, R.; Liu, X.; Lewis, F. D. Face-to-Face and Edge-to-Face π–π Interactions in a Synthetic DNA with a Stilbenediether Linker. J. Am. Chem. Soc. 2003, 125, 10842–10849. Copyright 2003 American Chemical Society.

Figure 15

Tivoli Gardens in Copenhagen, Denmark. https://www.tivoli.dk/en. Photo credit: “One Day in Copenhagen Itinerary” by Gina: https://www.onedayinacity.com/one-day-in-copenhagen/.