Michal Hocek

Two approaches for the enzymatic synthesis of DNA oligonucleotides (ONs) bearing multiple site‐specific modifications were explored. The first approach based on repeated single nucleotide extension (SNI) and reverse transcription from RNA template with Bst 3.0 DNA polymerase followed by digestion and exchange of template efficiently produced an ON with single modification, but the second round gave mixtures of full‐length and truncated products. The second approach was based on repeated cycles of SNI and primer extension from a DNA template followed by λ exonuclease digestion and exchange of the template and it worked very well even for consecutive incorporation of three different base‐modified nucleotides at specific positions. This was demonstrated on the synthesis of a molecular beacon bearing Cy3 and Cy5 fluorophores for the FRET detection of target RNA. The method is an efficient single‐tube operation that does not require separation and purification of intermediates and gives clean ONs labeled at desired positions.

We designed and synthesized 2’‐deoxyribonucleoside triphosphates (dNTPs) bearing chlorohexyl and biotin modifications attached to position 7 of 7‐deazaadenine or to position 5 of cytosine. The modified dNTPs served as substrates for KOD XL DNA polymerase in the enzymatic construction of DNA bearing either one or a combination of the two modifications. The chlorohexyl‐linked DNA underwent covalent reactions with halotag protein, while the biotin‐linked DNA formed stable non‐covalent complexes with monomeric streptavidin. The dually modified DNA bearing both modifications at different distances apart from each other were used for sequence‐specific, spatially defined display of both halotag and streptavidin proteins. The study provides the foundation for display of proteins on DNA rulers.

We designed and synthesized 2’‐deoxyribonucleoside triphosphates (dNTPs) bearing chlorohexyl and biotin modifications attached to position 7 of 7‐deazaadenine or to position 5 of cytosine. The modified dNTPs served as substrates for KOD XL DNA polymerase in the enzymatic construction of DNA bearing either one or a combination of the two modifications. The chlorohexyl‐linked DNA underwent covalent reactions with halotag protein, while the biotin‐linked DNA formed stable non‐covalent complexes with monomeric streptavidin. The dually modified DNA bearing both modifications at different distances apart from each other were used for sequence‐specific, spatially defined display of both halotag and streptavidin proteins. The study provides the foundation for display of proteins on DNA rulers.

High‐throughput screening of the IMTM compound library for modulatory activity on adenosine receptors (ARs) identified two deazapurine hits based on 2,6‐diaryl‐7‐deazapurines. In this study, a series of 16 new 2,6‐diaryl‐7‐deazapurine nucleobases together with four N‐9 substituted derivatives were synthesized. The synthesis was based on regioselective Suzuki cross‐coupling into position 6 followed by a second Suzuki cross‐coupling at position 2 under harsher conditions. Both cross‐couplings were performed on N‐9 protected nucleobases. Biological activity profiling identified several potent antagonists of all four subtypes of ARs and four compounds as sub‐micromolar and very selective antagonists of A ₃ . Several final compounds showed potent in vitro cytotoxic activity on a small panel of cancer cell lines, but with no selectivity against nonmalignant fibroblasts BJ and MRC‐5.

Discovery of functional nucleic acids from randomized libraries typically relies on multiple, time-consuming iterative rounds of in vitro selection with low success rate. Here, we present a single-round selection strategy for rapid screening of multiple over-represented nucleobase-modified DNA libraries and various selection conditions, capable of identifying high-affinity modified aptamers. Double partition followed by amplification of eluted sequences, NGS analysis and clustering provides fast identification of aptamer candidates. Screening of modified DNA libraries containing modified adenine and uracil nucleotides bearing hydrophobic aromatic phenyl and indole moieties results in development of an aptamer binding human insulin receptor with sub-nanomolar affinity and exquisite specificity. Cryo-EM structure reveals the importance of each aromatic modification, either in stabilizing the secondary structure or facilitating interactions with the protein surface. This approach addresses the main drawbacks of aptamer selection and has potential for high-throughput screening and accelerating the development of next-generation aptamers for diagnostics or therapeutics.

CD73 generates immunosuppressive adenosine in the tumor microenvironment and is a promising target for cancer immunotherapy. We have designed and systematically studied diverse 2-substituted 7-deazapurine ribonucleoside 5'-O-bisphosphonates bearing a variety of (het)-aryl groups at position 6 and discovered their highly potent and selective CD73 inhibition activity. The most active compounds (with single-digit picomolar K _i) contained bicyclic (het)-aryl groups at position 6 in combination with chlorine at position 2. Further optimization of pharmacokinetic properties identified inhibitors with low clearance, long half-life, high solubility, and excellent selectivity over CD39 and NTPDase3. They effectively suppressed adenosine formation in MDA-MB-231 cells, rescued CD8⁺ T cell activation, and were nontoxic to human fibroblasts. Overall, their profile compares favorably with AB680, a CD73 inhibitor currently in phase I/II clinical trials.

Four nucleoside phosphoramidites bearing aliphatic or aromatic groups linked through flexible alkyl tether were synthesized and used for solid-phase oligonucleotide synthesis and for study of stability of duplexes.

Discovery of functional nucleic acid ligands and catalytic motifs from a vast sequence space of randomized libraries is mostly based on time-consuming and demanding iterative rounds of in vitro selection. Here, we present a streamlined method for rapid identification of nucleobase-modified aptamers in a single selection round by combining chemically modified over-represented libraries, consecutive partition steps, and sequence abundance distribution analysis coupled with clustering. This approach generates distinct clusters of sequences, avoiding the need for massive screening of aptamer candidates. We demonstrated the proof-of-principle and effectiveness of the single-round selection approach resulting in a human insulin receptor aptamer, moreover, this method offers the expedient ability to screen multiple targets, various selection conditions, or differently modified libraries in a parallelized manner. The modified aptamer composed of two hydrophobic indole- and phenyl-linked nucleotides and two natural nucleotides exhibited picomolar binding affinity and exquisite specificity, capable of distinguishing subtle amino acid variations. Additionally, we determined the structure of the aptamer-receptor complex using cryo-electron microscopy, confirming the crucial role of hydrophobic aromatic modifications for the interactions of the aptamer with the target protein. These results highlight the synergistic effect of the components involved and demonstrate the capability of this methodology to generate highly potent and specific modified aptamers without tedious iterative selection rounds and extensive sequence space, thus accelerating aptamer discovery for therapeutic and diagnostic applications.

A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.