Element Biosciences, Inc.

William Light

Junhua Zhao

Sinan Arslan

Michael Previte

Molly Min He

Samantha Snow

Matthew William Kellinger

Hua Yu

Michael Kim

Yu-Hsien Hwang-Fu

Marco Tjioe

Andrew Boddicker

Ramreddy Tippana

Chunhong Zhou

Ryan Kelley

Tyler Lopez

Michael Klein

Mark Ambroso

Virginia Saade

Tsung-Li Liu

The present disclosure provides compositions and related methods, e.g., for preparing immobilized nucleic acid nanostructures using compaction oligonucleotides. In some embodiments, rolling circle amplification reaction can be conducted with compaction oligonucleotides on-support or in-solution to generate concatemer molecules having multiple copies of a polynucleotide unit arranged in tandem. Each polynucleotide unit comprises a sequence-of-interest and at least one universal adaptor sequence that binds one end of a compaction oligonucleotide. The 5' and 3′ regions of the compaction oligonucleotide can hybridize to the concatemer to pull together distal portions of the concatemer causing compaction of the concatemer to form a nanostructure. Nanostructures having tighter size and shape compared to concatemers generated in the absence of the compaction oligonucleotides. The compact and stable characteristics of the nucleic acid nanostructures improves sequencing accuracy by increasing signal intensity and they retain their shape and size during multiple sequencing cycles.

Compositions and methods for preparing nucleic acid nanostructures using compaction oligonucleotides

The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors. The double-stranded splint adaptors () can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor () comprises a first splint strand (long splint strand ()) and a second splint strand (short splint strand ()), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor () having a double-stranded region and two flanking single-stranded regions. The second splint strand () carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence and/or an index sequence.

Double-stranded splint adaptors and methods of use

The present disclosure provides compositions comprising nucleic acid single-stranded splint strands, including kits, and methods that employ the single-stranded splint strands. The single-stranded splint strands can hybridize to portions of linear library molecules to form circularized library-splint complexes having a nick, where the nick can be ligated to form covalently closed circular molecules which can be subjected to downstream amplification and sequencing workflows.

Single-stranded splint strands and methods of use

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

Compositions and methods for pairwise sequencing

Provided herein are fluorescently-labeled nucleotide conjugates for nucleic acid analysis. Also provided are reagents used for forming binding complexes between a fluorescently-labeled nucleotide conjugate and a target nucleic acid sequence in the presence of one or more reagents disclosed herein. Binding complexes can be detected in the presence of the one or more reagents. For example, the one or more reagents may contain a photobleaching reducing agent configured to reduce photobleaching resulting from use of the fluorescently-labeled nucleotide conjugate to form the binding complex in a nucleic acid analysis. Such nucleic acid analysis may be used to identify sites of nucleobase binding or incorporation between the target nucleic acid sequence and one or more nucleotide moieties of the fluorescently-labeled nucleotide conjugate in a nucleic acid sequence reaction.

Methods and reagents for nucleic acid analysis

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Poway, CA, United States of America

William Light