Biology subjects

Molina, R. S.

Publications and source records attributed to Molina, R. S..

7 recordsLinked to original sources

bioRxiv2025

Continuous hypermutation and evolution of luciferase variants

Several luciferases have been developed for imaging and biosensing, and the collection continues to grow as new applications are pursued. The current workflow for luciferase optimization, while successful, remains laborious and inefficient. Mutant libraries are generated in vitro and screened, "winning" mutants are picked by hand, and the isolated sequences are subjected to additional rounds of mutagenesis and screening. Here, we present a streamlined platform for luciferase engineering that removes the need for manual library generation during each cycle. We purposed an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of a well-known luciferase (GeNL). Short cycles of culturing and screening were sufficient to evolve the enzyme, with no repetitive manual library generation necessary. New GeNL variants were identified that exhibit improved light outputs with a non-cognate and inexpensive luciferin. We further characterized the novel luciferases in cell models. Collectively this work establishes OrthoRep and continuous hypermutation as a viable method to engineer luciferases, and sets the stage for more rapid development of bioluminescent reporters. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/669707v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3959dborg.highwire.dtl.DTLVardef@13a57c0org.highwire.dtl.DTLVardef@1572c04org.highwire.dtl.DTLVardef@abd59_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Simulating 500 million years of evolution with a language model

More than three billion years of evolution have produced an image of biology encoded into the space of natural proteins. Here we show that language models trained on tokens generated by evolution can act as evolutionary simulators to generate functional proteins that are far away from known proteins. We present ESM3, a frontier multimodal generative language model that reasons over the sequence, structure, and function of proteins. ESM3 can follow complex prompts combining its modalities and is highly responsive to biological alignment. We have prompted ESM3 to generate fluorescent proteins with a chain of thought. Among the generations that we synthesized, we found a bright fluorescent protein at far distance (58% identity) from known fluorescent proteins. Similarly distant natural fluorescent proteins are separated by over five hundred million years of evolution.

synthetic biology↗

bioRxiv2021

A genetically encoded fluorescent biosensor for extracellular L-lactate

To enable investigations of the emerging roles of cell-to-cell shuttling of L-lactate, we have developed an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. We demonstrate that this biosensor, designated eLACCO1.1, enables minimally invasive cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

molecular biology↗

bioRxiv2020

Far-red fluorescent genetically encoded calcium ion indicators

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca2+ ({Delta}F/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.

neuroscience↗

bioRxiv2020

High throughput instrument to screen fluorescent proteins under two-photon excitation

Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

neuroscience↗

bioRxiv2018

Understanding the Ca2+-dependent Fluorescence Change in Red Genetically Encoded Ca2+ Indicators

Genetically encoded Ca2+ indicators (GECIs) are widely used to illuminate dynamic Ca2+ signaling activity in living cells and tissues. Various fluorescence colors of GECIs are available, including red. Red GECIs are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a circularly permuted red fluorescent protein fused to a Ca2+ sensing domain, calmodulin and a calmodulin-binding peptide. A conformational change in the sensing domain upon binding Ca2+ causes a change in the fluorescence intensity of the fluorescent protein. Three factors could contribute to this change in fluorescence: 1) a shift in the protonation state of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient for one-photon excitation or the two-photon cross section for two-photon excitation. We conducted a systematic study of the photophysical properties of a select cohort of red GECIs in their Ca2+-free and Ca2+-saturated states to determine which factors are most important for the Ca2+-dependent change in fluorescence. In total, we analyzed nine red GECIs, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We found that these red GECIs could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases the magnitude of the change in fluorescence was different depending on one-photon excitation or two-photon excitation by up to a factor of two.

biophysics↗

bioRxiv2017

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein

Genetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

biophysics↗