Whereas activatable probes have greatly simplified the assays by eliminating the need to remove unbound probes, the development of new activatable probes is largely constrained by the scarce activation mechanisms (e.g., fluorescence resonance energy transfer (FRET)), the limited activation colors (e.g., existing FRET pairs), and the poor enhancement ratios (e.g., 10-to 60-fold for a typical molecular beacon). [2] NanoCluster Beacons (NCBs) [3] are a unique class of activatable probes as they provide a palette of activation colors from the same dark origin [4] (not via FRET) and achieve fluorescence enhancement ratios as high as 1500- [5] to 2400-fold. [6] The core of an NCB is a few-atom silver nanocluster [7] (e.g., Ag 8 , Ag 10 , or Ag 16 ) whose fluorescence can be tuned by its surrounding nucleobases. [7b,c,8] To create an NCB, a dark silver nanocluster (AgNC) is first synthesized in a C-rich DNA host (termed the NC probe), and a G-rich overhang (termed the activator) is brought into close proximity of the AgNC (via target-probe hybridization, Figure S1, Supporting Information) to activate its fluorescence (Figure 1A,B). [3-5,8a,d] NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange and red POTs, respectively. Based on these findings, a "zipper-bag" model is proposed that can explain how these hotspots facilitate the formation of distinct silver cluster chromophores and alter their chemical yields. Combining high-throughput screening with machine-learning algorithms, a pipeline is established to design bright and multicolor NCBs in silico.

Top-down characterization of histones, proteins that are critical participants in an array of DNA-dependent processes, offers the potential to examine the relationship between histone structure and mechanisms of genetic regulation. Mapping patterns of post-translational modifications (PTMs) of histones requires extensive backbone cleavages to bracket the sites of mass shifts corresponding to specific PTMs. Ultraviolet photodissociation (UVPD) causes substantial fragmentation of proteins, which is well-suited for PTM localization, but the resulting spectra are congested with fragment ions that may have overlapping isotopic distributions that confound deconvolution. Gas-phase proton transfer charge reduction (PTCR) decreases the charge states of highly charged ions, thus alleviating this congestion and facilitating the identification of additional sequence-determining and PTM-localizing fragment ions. By integrating UVPD with PTCR for histone proteoform analyses, sequence coverages up to 91% were achieved for calf thymus histone H4 containing acetylation marks at the N-terminus and Lys12 as well as a dimethylation at Arg3. UVPD-PTCR exhibited large gains in characterization for other histones, such as histone H2A, increasing the sequence coverage from 59 to 77% for monoacetylated H2A.

CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are a type of adaptive immune response in bacteria and archaea that utilize crRNA (CRISPR RNA)-guided effector complexes to target complementary RNA or DNA for destruction. The prototypical type III-A and III-B CRISPR-Cas systems utilize multi-subunit effector complexes composed of individual proteins to cleave ssRNA targets at 6-nt intervals, as well as non-specifically degrading ssDNA and activating cyclic oligoadenylate (cOA) synthesis. Recent studies have shown that type III systems can contain subunit fusions yet maintain canonical type III RNA-targeting capabilities. To understand how a multi-subunit fusion effector functions, we determine structures of a variant type III-D effector and biochemically characterize how it cleaves RNA targets. These findings provide insights into how multi-subunit fusion proteins are tethered together and assemble into an active and programmable RNA endonuclease, how the effector utilizes a novel mechanism for target RNA seeding, and the structural basis for the evolution of type III effector complexes. Furthermore, our results provide a blueprint for fusing subunits in class 1 effectors for design of user-defined effector complexes with disparate activities.

Protein-DNA interactions play crucial roles in DNA replication across all living organisms. Here, we apply a suite of mass spectrometry (MS) tools to characterize a protein-ssDNA complex, T4 gp32•ssDNA, with...

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed the polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here we report a high-throughput selection method that takes advantage of repurposed next-generation-sequencing (NGS) chips to screen the activation fluorescence of ~40,000 activator sequences. We find the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow and red POTs, respectively. Based on these findings, we propose a "zipper bag model" that explains how these hotspots lead to the creation of distinct silver cluster chromophores and contribute to the difference in chromophore chemical yields. Combining high-throughput screening with machine learning algorithms, we establish a pipeline to rationally design bright and multicolor NCBs.