Branched chemically modified poly(A) tails enhance the translation capacity of mRNA.

Although messenger RNA (mRNA) has proved effective as a vaccine, its potential as a general therapeutic modality is limited by its instability and low translation capacity. To increase the duration and level of protein expression from mRNA, we designed and synthesized topologically and chemically modified mRNAs with multiple synthetic poly(A) tails. Here we demonstrate that the optimized multitailed mRNA yielded ~4.7-19.5-fold higher luminescence signals than the control mRNA from 24 to 72 h post transfection in cellulo and 14 days detectable signal versus <7 days signal from the control in vivo. We further achieve efficient multiplexed genome editing of the clinically relevant genes Pcsk9 and Angptl3 in mouse liver at a minimal mRNA dosage. Taken together, these results provide a generalizable approach to synthesize capped branched mRNA with markedly enhanced translation capacity.

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

Generating experimentally unrelated target molecule-binding highly functionalized nucleic-acid polymers using machine learning.

In vitro selection queries large combinatorial libraries for sequence-defined polymers with target binding and reaction catalysis activity. While the total sequence space of these libraries can extend beyond 1022 sequences, practical considerations limit starting sequences to ≤~1015 distinct molecules. Selection-induced sequence convergence and limited sequencing depth further constrain experimentally observable sequence space. To address these limitations, we integrate experimental and machine learning approaches to explore regions of sequence space unrelated to experimentally derived variants. We perform in vitro selections to discover highly side-chain-functionalized nucleic acid polymers (HFNAPs) with potent affinities for a target small molecule (daunomycin KD = 5-65 nM). We then use the selection data to train a conditional variational autoencoder (CVAE) machine learning model to generate diverse and unique HFNAP sequences with high daunomycin affinities (KD = 9-26 nM), even though they are unrelated in sequence to experimental polymers. Coupling in vitro selection with a machine learning model thus enables direct generation of active variants, demonstrating a new approach to the discovery of functional biopolymers.

Engineered pegRNAs improve prime editing efficiency.

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3' terminus of pegRNAs that enhance their stability and prevent degradation of the 3' extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3-4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3' structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3' motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

Xanthone derivatives could be potential antibiotics: virtual screening for the inhibitors of enzyme I of bacterial phosphoenolpyruvate-dependent phosphotransferase system.

The phosphoenolpyruvate phosphotransferase system (PTS) is ubiquitous in eubacteria and absent from eukaryotes. The system consists of two phosphoryl carriers, enzyme I (EI) and the histidine-containing phosphoryl carrier protein (HPr), and several PTS transporters, catalyzing the concomitant uptake and phosphorylation of several carbohydrates. Since a deficiency of EI in bacterial mutants lead to severe growth defects, EI could be a drug target to develop antimicrobial agents. We used the 3D structure PDB 1ZYM of Escherichia coli EI as the target to virtually screen the potential tight binders from NPPEDIA (Natural Product Encyclopedia), ZINC and Super Natural databases. These databases were screened using the docking tools of Discovery Studio 2.0 and the Integrated Drug Design System IDDS. Among the many interesting hits, xanthone derivatives with reasonably high Dock scores received more attentions. Two of the xanthone derivatives were obtained to examine their capabilities to inhibit cell growth of both Gram-positive and Gram-negative bacterial strains. The results indicate that they may exert the inhibition effects by blocking the EI activities. We have demonstrated for the first time that the xanthone derivatives have high potential to be developed as future antibiotics.

Benefits and efficacy of tonsillectomy for recurrent tonsillitis in adults.

CONCLUSIONS: Tonsillectomy is an effective procedure that provides medical and social benefits for adult patients with recurrent tonsillitis. OBJECTIVE: To evaluate the benefits, impact and overall efficacy of tonsillectomy for recurrent tonsillitis in adults. PATIENTS AND METHODS: Analysis of medical records with postoperative survey analysis of patients who underwent tonsillectomy in 1 work year with at least 1 year of follow-up. RESULTS: A total of 105 patients returned completed surveys. Their mean age was 26.25 years. The mean frequency of tonsillitis per year decreased from 8.37 (SD=3.75) to 1.43 (SD=1.86), p<0.005. The mean duration in days of each episode of tonsillitis decreased from 5.91 (SD=2.67) to 1.23 (SD=1.42), p<0.005. The mean number of work days missed decreased from 8.92 (SD=6.85) to 0.65 (SD=1.49), p<0.005. The mean pain score (1-10) was 6.88 (SD=5.75). An average of 14.17 (SD=5.75) days of medical leave was required postoperatively. In all, 37.1% of patients noticed postoperative voice changes; 92.4% would still choose tonsillectomy again for the same condition and 98.1% would recommend tonsillectomy to family or friends if they had recurrent tonsillitis.