Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs

Xu, Yuan; Zhu, Ting

doi:10.1126/science.abm0646

Cited by 33 publications

(23 citation statements)

References 56 publications

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“…If the ambidexterity of this protein family is evolutionarily significant, it transforms our understanding of the history of homochirality, as ambidextrous domains would have unique properties in a complex ecosystem where both chiral forms of dsDNA or protein are in competition. Ultimately, for questions of this nature to be answered, other ancient protein folds will have to be resurrected and their functional ambidexterity assayed experimentally, a goal that is increasingly within reach 46 .…”

Section: Discussionmentioning

confidence: 99%

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain

Weil-Ktorza

Fridmann-Sirkis

Despotović

et al. 2023

Preprint

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Homochirality of biopolymers emerged early in the history of life on Earth, nearly 4 billion years ago. Whether the establishment of homochirality was the result of abiotic physical and chemical processes, or biological selection, remains unknown. However, given that significant events in protein evolution predate the last universal common ancestor, the history of homochirality may have been written into some of the oldest protein folds. To test this hypothesis, the evolutionary trajectory of the ancient and ubiquitous helix-hairpin-helix (HhH) protein family was analyzed for functional robustness to total chiral inversion of just one binding partner. Against expectations, functional 'ambidexterity' was observed across the entire trajectory, from phase separation of HhH peptides with RNA to dsDNA binding of the duplicated (HhH)2-Fold. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed significant overlap between the binding modes of a natural and a mirror-image protein to natural dsDNA. These data suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed, and that the HhH protein family is an intriguing exception to the dogma of reciprocal chiral substrate specificity proposed by Milton and Kent (Milton et al. Science 1992).

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Section: Discussionmentioning

confidence: 99%

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain

Weil-Ktorza

Fridmann-Sirkis

Despotović

et al. 2023

Preprint

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“…2). Taken together, these syntheses represent a major fraction of the total number of D-proteins reported in the past five years (12 out of 36 reported targets [2][3][4]11,12,[16][17][18][23][24][25][26][27][28][29][30][31][31][32][33][34][35][36][37] ). We aimed to apply a recently reported screening platform based on phage display to generate high-affinity conformationally constrained ("stapled") α-helical peptides, termed Helicons, in a single MIDP screening pass 38 .…”

Section: Introductionmentioning

confidence: 90%

“…Currently, these mirror-image biomolecules can only be accessed by total chemical synthesis; the molecular cloning techniques ubiquitous in modern biology necessitate a large repertoire of components that have not yet been produced in their mirror-image forms. Recent work 3,4 has underlined the critical need for reliable access to mirror-image proteins to enable the development of potential therapeutics and to further explore biotechnological applications of mirror-image systems.…”

Section: Introductionmentioning

confidence: 99%

Single-Shot Flow Synthesis of D-Proteins for Mirror-Image Phage Display

Callahan

Gandhesiri

Travaline³

et al. 2023

Preprint

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Mirror-image biological systems have the potential for broad-reaching impact in health and diagnostics, but their study has been greatly limited by the lack of routine access to synthetic D-proteins. We demonstrate that automated fast flow peptide synthesis (AFPS) can reliably produce novel mirror-image protein targets without prior sequence engineering. We synthesized 12 D-proteins, along with their L-counterparts. All 24 synthetic proteins were folded into active structures in vitro, and characterized using biochemical and biophysical techniques. From these chiral protein pairs, we chose MDM2 and CHIP to carry forward into mirror-image phage display screens, and identified macrocyclic D-peptides that bind the recombinant targets. We report 6 mirror-image peptide ligands with unique binding modes: three to MDM2, and three to CHIP, each confirmed with X-ray co-crystal structures. Reliable production of mirror-image proteins with AFPS stands to enable not only the discovery of D-peptide drug leads, but to the study of mirror-image biological systems more broadly.

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“…Stabilizers can be added to protect the protein solution. , Nonetheless, the cold-chain requirement during transport and storage poses constraints on large-scale deployment, especially in low-resource communities. On the other hand, RNAs tend to degrade in solution due to two main factors: RNases and hydrolysis, mandating the aliquoting and ultralow storage of guide RNAs at −80 °C for long-term use. Lyophilization provides a solution to the storage and transport problem, since successful lyophilization prevents or minimizes degradation of both proteins and RNA species.…”

Section: Challenges and Potential Solutionsmentioning

confidence: 99%

CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications

et al. 2022

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The COVID-19 pandemic has challenged the conventional diagnostic field and revealed the need for decentralized Point of Care (POC) solutions. Although nucleic acid testing is considered to be the most sensitive and specific disease detection method, conventional testing platforms are expensive, confined to central laboratories, and are not deployable in low-resource settings. CRISPR-based diagnostics have emerged as promising tools capable of revolutionizing the field of molecular diagnostics. These platforms are inexpensive, simple, and do not require the use of special instrumentation, suggesting they could democratize access to disease diagnostics. However, there are several obstacles to the use of the current platforms for POC applications, including difficulties in sample processing and stability. In this review, we discuss key advancements in the field, with an emphasis on the challenges of sample processing, stability, multiplexing, amplification-free detection, signal interpretation, and process automation. We also discuss potential solutions for revolutionizing CRISPR-based diagnostics toward sample-to-answer diagnostic solutions for POC and home use.

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Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs

Cited by 33 publications

References 56 publications

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain

Single-Shot Flow Synthesis of D-Proteins for Mirror-Image Phage Display

CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications

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