Polynucleotide Assembly via Polymerase-Mediated Double-Stranded Conversion
Solution Overview
Problem
Current methods for synthesizing long double-stranded polynucleotides face challenges with repetitive motifs, high or low GC content, and secondary structures, leading to off-target annealing and reduced fidelity in assembling specific polynucleotide sequences.
Innovation Solution
An in vitro method involving the use of single-stranded polynucleotides with complementary overhangs, where primers bind to termini and are extended by polymerase to form double-stranded sequences, allowing for precise assembly through unique overhang pairing, reducing off-target annealing and improving fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If single-stranded oligonucleotides are annealed to assemble polynucleotide sequences, then the assembly process can proceed, but off-target annealing and secondary structure formation occur leading to reduced fidelity
Solution Approach 1:
The invention changes the physical state parameter of the polynucleotide fragments from single-stranded to double-stranded form before assembly. By converting SSPs to DSPs through polymerase-mediated extension, the method eliminates off-target annealing and secondary structure formation that plague single-stranded approaches, thereby improving assembly fidelity and reliability
Solution Approach 2:
The invention performs preliminary conversion of single-stranded polynucleotides to double-stranded polynucleotides before the assembly step. This preliminary action of creating DSPs with complementary overhangs ensures that subsequent annealing occurs only between intended complementary sequences, preventing off-target effects and improving assembly specificity
2Productivity
If traditional polymerase extension is used to fill gaps between fragments, then polynucleotide sequences can be assembled, but repetitive motifs and high/low GC contents cause off-target annealing
Solution Approach 1:
The invention changes the strand configuration parameter from single-stranded to double-stranded before assembly. By providing DSPs instead of SSPs, the method prevents off-target annealing caused by repetitive motifs and extreme GC contents, while maintaining high assembly efficiency through the pre-formed complementary overhangs that guide specific joining
Solution Approach 2:
The invention uses double-stranded polynucleotides with complementary overhangs as intermediaries between the final assembly steps. These DSPs act as stable, specific intermediates that prevent off-target interactions while maintaining the ability to assemble efficiently through their pre-configured complementary regions
3Adaptability or versatility
If secondary structures form in single-stranded sequences, then certain polynucleotide designs can be represented, but annealing is inhibited leading to reduced assembly fidelity
Solution Approach 1:
The invention changes the structural parameter of the polynucleotides from single-stranded to double-stranded form. By providing DSPs with complementary overhangs, the method prevents secondary structure formation that would otherwise inhibit annealing, while maintaining the ability to represent diverse sequence designs including those with repetitive motifs and extreme GC contents
4Ease of manufacture
If single-stranded oligonucleotides are used for assembly, then the process is simpler, but off-target annealing occurs reducing yield and fidelity
Solution Approach 1:
The invention changes the physical state parameter of the input polynucleotides from single-stranded to double-stranded. Although this requires an additional polymerase extension step, it eliminates off-target annealing and secondary structure formation, resulting in improved fidelity and yield that outweigh the additional procedural step
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the efficient and accurate assembly of polynucleotide sequences that are difficult to produce with existing techniques, such as highly repetitive sequences and large libraries, by converting single-stranded sequences to double-stranded before assembly, thereby reducing side products and increasing yield and fidelity.
Implementation Method 1
providing a first primer that binds at a terminus of the first SSP and a second primer that binds at a terminus of the second SSP and converting the first SSP to the first DSP and the second SSP to the second DSP through a polymerase-mediated extension
Implementation Method 2
assembling the target polynucleotide sequence by joining the first DSP to the second DSP via annealing of the complementary overhangs
Data Source
AI summary
Methods are provided for assembly of a target polynucleotide sequence comprising at least a first double stranded polynucleotide (DSP) and at least second DSP, and optionally further DSPs. The method comprises an assembly reaction comprising steps including providing a first single stranded polynucleotide (SSP) comprising the polynucleotide sequence of one strand of the first DSP, and a second SSP comprising the polynucleotide sequence of one strand of the second DSP and converting the SSPs to double stranded form via a primer and polymerase-mediated extension reaction. The DSPs comprise polynucleotide sequences that are complementary to other polynucleotide sequences within the assembly reaction such that the ordering and directionality of each of the first and second, and further DSPs is determined by unique overhang pairing. Nucleic acid libraries and methods of making such libraries are also provided.